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Etk/Bmx activation modulates barrier function in epithelial cells

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Etk/Bmx activation modulates barrier function in epithelial cells

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ETK/BMX ACTIVATION MODULATES BARRIER FUNCTION IN
EPITHELIAL CELLS
Copyright 2001
by
Hung-Kang Chang
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
December 2001
Hung-Kang Chang
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UNIVERSITY OF SOUTHERN CALIFORNIA
The Graduate School
University Park
LOS ANGELES, CALIFORNIA 90089-1695
This thesis, w ritten b y
Under the direction o f h .lT h e sis
Com m ittee, and approved b y a ll its members,
has been presen ted to an d accepted b y The
Graduate School, in p a rtia l fulfillm ent o f
requirem ents fo r the degree o f
Dean o f Graduate Studies
D ate December 17, 2001
THESIS COMMITTEE
• ...........
Chairperson
T
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ACKNOWLEDGEMENTS
Dr. David Ann’s advisement has been the major engine in the completion of this
thesis. Dr. Kwang-Jin Kim and Dr. Wei-Chiang Shen also afford me great support.
The greatest gratitude is for my thesis committee members. I would also like to
express my appreciation for Dr. Helen Lin, Dr. Xin Wen, Dr. Mark D. Zenter, Cindy
Chou, and Carlos Clavijo. They have been my warmest lab mates and the most
invaluable technical support. I like to thank my parents. If not for them, I could never
have the best learning experience in the School of Pharmacy at USC. Lastly, but not
the least, I want to thank my wife, Janet. She is the ultimate nursery of my works, as
well as of myself.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS........................................................................................ii
LIST OF TABLES/FIGURES.....................................................................................vi
ABSTRACT.................................................................................................................. vii
CHAPTER 1 INTRODUCTION 1
1.1 Protein tyrosine kinases............................................................................................1
1.1.1 The protein domains of Btk/Tec family of NRTKs..................................4
1.1.1.1 Pleckstrin homology domain (PH domain).............................. 4
1.1.1.2 Tec homology domain (TH domain).........................................5
1.1.1.3 Src homology 3 domain (SH3 domain).....................................6
1.1.1.4 Src homology 2 domain (SH2 domain).....................................6
1.1.1.5 Protein kinase domain (PK domain).......................................... 7
1.1.2 The activation of Btk/Tec kinases.............................................................7
1.1.2.1 The first step ofBtk/Tec kinases activation..............................8
1.1.2.2 The second step ofBtk/Tec kinases activation........................11
1.1.2.3 The third step ofBtk/Tec kinase activation.............................11
1.1.3 A novel two-step mechanism of activation for Itk.................................12
1.1.4 Upstream activators ofBtk/Tec kinases..................................................13
1.1.5 Btk/Tec kinases can modulate intracellular Ca2 + concentration
and the activity of Ca2 + -dependent proteins (PKC isoforms)................ 14
1.1.6 Btk/Tec kinases can modulate the activity of MAP kinases...................15
1.1.7 Btk/Tec kinases can modulate gene expression......................................16
iii
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1.1.8 Btk/Tec kinases can modulate apoptosis................................................18
1.1.9 Btk/Tec kinases can modulate cytoskeletal organization.....................19
1.2 Tight junction...........................................................................................................20
1.2.1 The molecular structure of tight junction............................................... 21
1.2.1.1 Occludin....................................................................................22
1.2.1.2 Claudins.....................................................................................23
1.2.1.3 The organization of claudins and occludin
in the TJ fibrils......................................................................... 25
1.2.1.4 Zonula Occludens (ZO)............................................................26
1.2.1.5 Symplekin..................................................................................27
1.2.1.6 Cingulin.....................................................................................28
1.2.1.7 Actin cytoskeleton....................................................................28
1.2.1.8 7H6............................................................................................ 29
1.2.1.9 Rab family, Sec 6/8, VAP-33.................................................. 29
1.2.1.10 G-proteins............................................................................... 30
1.2.2 The biogenesis of TJ.................................................................................31
1.2.2.1 Ca2 + -switch model.................................................................... 31
1.2.2.2 ATP depletion-repletion model............................................... 34
1.2.2.3 Common scenarios in both models...........................................35
1.2.3 Barrier function.........................................................................................36
1.2.4 Fence function.......................................................................................... 38
1.3 Pa-4AEtk:ER cell line.............................................................................................. 39
iv
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CHAPTER 2 MATERIAL AND METHOD..........................................................41
2.1 Reagents.................................................................................................................. 41
2.2 Cell line....................................................................................................................41
2.3 Measurement of transepithelial electrical resistance (TER).................................42
CHAPTER 3 RESULT.............................................................................................. 44
3.1 Phenotypic manifestation of epithelial cells that express Etk........................... 44
3.2 Etk activation protects Pa-4 cells against
hypoxia-induced decrease of TER.........................................................................46
3.3 Etk-induced enhancement of TER in response to hypoxia
involves regulation of the actin cytoskeleton....................................................... 50
3.4 Increased TER following Etk activation is dependent on
tyrosine kinase activity........................................................................................... 52
CHAPTER 4 DISCUSSION..................................................................................... 55
REFERENCES............................................................................................................62
v
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LIST OF TABLES/FIGURES
Table I Classifications of non-receptor tyrosine kinases (NRTKs)..............................2
Table II Expression Profiles ofBtk/Tec family of NRTKs.......................................... 3
Table III Claudin family................................................................................................39
Fig. 1. Etk activation increases the TER of Pa-4 and MDCK cell monolayers.........45
Fig. 2 Etk activation stably sustains TER of Pa-4 and Pa-4AEtk:ER
cell monolayers....................................................................................................46
Fig. 3. Relative changes in TER and Ieq of Pa-4 and Pa-4AEtk:ER
cell monolayers under hypoxic conditions.......................................................48
Table 1. TER of Pa-4 and Pa-4AEtk:ER cell monolayers was able to
recover from a 24-h hypoxia..........................................................................49
Fig. 4. Differential effect of latrunculin B on TER of
Pa-4 and Pa-4AEtk:ER cell monolayers...........................................................51
Fig. 5. Increased TER observed with Etk activation is diminished by pretreatment
with 1 pM (A) and 50 pM (B) genistein..........................................................53
Table 2. TER of Pa-4 and Pa-4AEtk:ER cell monolayers was
able to recover from a 24-h treatment of Genistein...................................... 54
Fig. 6 A putative model for protection against the injurious effect of prolonged
hypoxia on TER by Etk activation......................................................................61
vi
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ABSTRACT
Etk/Bmx is a member of the Btk/Tec family of cytoplasmic non-receptor
tyrosine kinases. Etk activation in Etk-stably transfected epithelial Pa-4 cells resulted
in a consistently increased transepithelial electrical resistance (TER). After twenty
four hours o f hypoxic (1% O2 ) exposure, the TER and equivalent active ion transport
rate (/eq) were reduced to less than 5% of the normoxia control in Pa-4 cells,
whereas, both TER and Ieq were maintained at comparable and 60% level,
respectively, relative to their normoxic controls. Etk activation also protected cells
from Latrunculin B (48 nM)-induced actin cytoskeleton depolymerization and TER
decrease. Furthermore, TER of Etk-activated cells was more sensitive to Genistein
treatment (1, 50 |iM). Based on these findings, we propose that Etk may be a novel
regulator of epithelial junctions during physiological and pathophysiological
conditions.
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CHAPTER 1 INTRODUCTION
1.1 Protein tyrosine kinases
Protein tyrosine kinases (PTKs), a large multigene family, can generally be
classified as: (1) plasma membrane-bound receptor tyrosine kinases (RTKs), which
typically contain extracellular ligand binding domain, a-helical transmembrane
domain, and intracellular tyrosine kinase domain; (2) non-receptor tyrosine kinases
(NRTKs), also referred to as cytoplasmic tyrosine kinases or Src-like kinases.
Currently, the already sequenced human genome contains 90 tyrosine kinase genes in
which 58 belong to receptor-type, as identified by possessing a consensus
transmembrane domain in their amino acid sequences; the rest 32 belong to non
receptor type. Based on the kinase domain sequence, the 58 RTKs and the 32
NRTKs can be further divided into 20 and 10 families respectively. Five tyrosine
kinase pseudogenes are also identified by the lack of introns, the truncation of the
coding regions compared to other members of the family, the presence of in-frame
termination codons, and the lack of evidence of expression (1). Table I shows the
classification of NRTKs. Etk/Bmx belongs to the Btk/Tec family.
RTKs are activated upon binding to signaling molecules through the
extracellular domain, and transduce this signal into the cell by activation of the
intracellular tyrosine kinase domain. NRTKs, which lack extracellular binding
domain, are found to be associated with membrane receptor that do not have
1
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intracellular kinase domains, and transduce the signal into the cell. Table II illustrates
the molecular sizes, expression profiles, and chromosome localization of Btk/Tec
family NRTKs. Etk/Bmx is unique in Btk/Tec family of NRTKs that it is not only
expressed in hematopoietic but also in non-hematopoietic cells, such as epithelial and
endothelial cells.
The mechanisms of regulations and functions ofBtk/Tec kinases have not been
completely determined. Because the protein domains within the family are
conserved, the following discussions of Etk/Bmx will not be separated from the other
members of this family, and an analogy may be inspired by the other Btk/Tec family
kinases.
\
Table. I Classifications of non-receptor tyrosine kinases (NRTKs)
ABL family — ABL1 /ABL
— ARG/ABL2/ABLL
ACK family — ACKl/ACK2(b)/Cdgip(m)
— TNK1
CSK family — CSK/CYL
— MATK/CTK/HYL/CHK/LSK/Ntk(m)
FAK family — PTK2/Fadk(m)
— PYK2/PTK2B/C AKp/RAFTK/FAK2/PKB
FES family — FER/TYK3/Fert 1 /2(m)
— FES/FPS
FRK family — BRK/PTK6/ Sik(m)
— FRK/RAK/Bsk(m)/IYK(m)
— SRMS/SRM
JAK family — JAK1
— JAK2
— JAK3/L-JAK
— TYK2/JTK1
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Table I - Continued
SRC-A family — FGR/SRC2
— FYN/SLK/S YN
— SRC
— YES1
SRC-B family — BLK
— HCK/JTK9/Bmk(m)/HCTK
— LCK/Tck(m)
— LYN
TEC family — BMX/ETK/PSCTK2
— BTK/ATK/PSCTK1/AGMX1/IMD1
— ITK/EMT/T sk(m)/P SCTK2
— TEC/PSCTK4
— TXK/PSCTK5/BTKL/Rlk(m)
— Tec29 (d)
SYK family — SYK
— ZAP70/SRK/STD
Pseudo genes — FERps, RYKps, TYR03ps, VEGFRlps, YESps.
m -Mus musculus
d-Drosophila melanogaster
(Adapted from Robinson, et al. Oncogene 19: 5548-5557, 2000)
Table II Expression Profiles ofBtk/Tec family of NRTKs
Protein
Protein
Size
(kDa)
Chromosome
Localization
(human)
Expression Profile
Etk/Bmx 80 Xp22.2
Bone marrow, lung, testis, colon, heart,
macrophage, neutrophil
Btk 77 Xq21.33-q22
Bone marrow, spleen, lymph node, fetal
liver, B, myeloid, erythroid, mast cells,
and megakaryocyte
Itk 72 5q31-q32
Thymus, spleen, lymph node, T, NK, and
mast cells
Tec
72/70/66/
58
4pl2
Bone marrow, spleen, thymus, T, B,
myeloid, and hepatocarcinoma cells
Txk 52/58 4pl2
Thymus, spleen, lymph node, tonsil, testis,
T, and myeloid cells
3
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1.1.1 The protein domains ofBtk/Tec family of NRTKs
N-
PH TH SH3 SH2 PK
Btk motif Proline-Rich (PR)
motif
The Btk/Tec family of NRTKs lack a myristoylation or a palmitoylation site
found in Src family kinases for membrane targeting, instead, a PH domain is present
on the N-termini for the same purpose (Rlk in mouse and Tec29 in Drosophila
melanogaster are exceptions and do not have PH domain). Btk/Tec family also lacks
a tyrosine phosphorylation site on the C-terminal tail that is found in Src family
kinases and is known to negatively regulate the kinase activity upon phosphorylation.
l .l .l .l Pleckstrin homology domain (PH domain)
The core structure of the PH domain is an orthogonal antiparallel P-sandwich,
composed of 3 and 4 P-strands respectively. A a-helix caps the C-terminal comer of
the PH domain. Several positively charged (basic) amino acid residues cluster to one
face of the PH domain and are thought to constitute the binding site for
phosphatidylinositides (Ptdlns) on the biological membranes with in vitro Kd as low
as 40 nM (2, 3). Since PI3-kinase can phosphorylate the inositol ring of the
phospholipid molecule, therefore it is thought to be a physiological mediator that can
4
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recruit PH domain-carrying proteins to the plasma membrane. Many mutations
associated with X-linked agammaglobulinemia (XLA), a human inherited
immunodeficiency disease, are located on or near the positively charged face of the
Btk PH domain, responsible for binding to PtdIns(3,4,5)P3 (4). Similarly, X-linked
immunodeficiency (xid) mice are also reported to carry a R28C point mutation on
the PH domain of Btk. In addition to phospholipids, the PH domain of Btk/Tec
kinases can also interact with PKC isoforms and G-protein subunits (Ga, Gaq, and
Gal 2), Stat3, F-actin, Fas, and FAK (5).
1.1.1.2 Tec homology domain (TH domain)
The TH domain is composed of two motifs, the Btk motif and the proline-rich
(PR) motif. The Btk motif is a globular structure right next to the PH domain, and
contains conserved cystein and histidine residues forming a 2inc-binding finger. The
PR motif adopts a dihedral angle polyproline type II (PPII) helix (PXPXXP), and is
linked to the SH3 domain via a so-called Tec-loop. The PR motif of Itk is reported to
be capable of binding to its own SH3 domain (6). However, Etk/Bmx does not have
a TH domain; two 22-amino-acid repeats (PSSSTTLAQVDNESKKNYGSQP) are
found in stead, and thus the intramolecular interaction is still questionable for
Etk/Bmx (7). Whether similar intramolecular interaction exists via the
aforementioned repeats requires further investigation.
5
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1.1.1.3 Src homology 3 domain (SH3 domain)
The SH3 domain is composed of two small P-sheets packed against each other
with approximately right angle. Intriguingly, as mentioned earlier, the SH3 domain
of Itk was shown to be able to bind to its intramolecular PR motif in the TH domain,
and this interaction is predicted to render the kinase inactive (8). Furthermore, a 21
amino acids deletion in the Btk SH3 domain is found in the B cells of XLA patients.
The modeled structure of this Btk mutant shows a loss of two C-terminal P-strands
that contain several critical residues forming the SH3 ligand binding pocket (9).
1.1.1.4 Src homology 2 domain (SH2 domain)
The three-dimensional crystal structure of SH2 domain is not yet available,
however,, the binding properties of the recombinant SH2 domain of Itk suggests that
it could stabilize the intramolecular binding between the SH3 domain and the PR
motif (8). SH2 domain can also bind to specific sequences containing phospho-
tyrosine residues. Mutation in this domain may be associated with X-linked
agammaglobulinemia (XLA) and other B cell defects (10).
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1.1.1.5 Protein kinase domain (PK domain)
Protein kinase domain, also called Src homology 1 domain (SHI domain), is the
most conserved domain among Btk/Tec family of NRTKs (7). It consists of a
consensus ATP binding motif (GXGXXG), corresponding to amino acid residues
424-429 in Etk/Bmx, and a tyrosine phosphorylation site (Tyr566 in Etk/Bmx) in the
so-called activation loop. PK domain is the catalytic site of all Btk/Tec family
kinases that can phosphorylate tyrosine residues on the substrates. Phosphorylation
of a tyrosine residue in this kinase domain (Tyr551 of Btk; Tyr566 of Etk/Bmx) by
Src family kinases is required to activate Btk/Tec family kinases (11,12).
1.1.2 The activation ofBtk/Tec kinases
Through the study of Itk, it is presumed that Btk/Tec kinases, under resting
state, assume a closed inactive intramolecular folding mediated by “PH — kinase
domain” and/or “PR motif — SH3 domain” interactions. Therefore, any molecules
(whether protein or non-protein) that can disrupt this inhibitory folding are potential
activators of Btk/Tec kinases. Several lines o f evidences suggest a three-step
mechanism of activation, and the membrane recruitment seems to be the first step of
Btk/Tec kinases activation. The palmitoylation site of Txk and the PH domain of the
other Btk/Tec family members act like membrane anchors to mediate signal-induced
plasma membrane recruitment.
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1.1.2.1 The first step ofBtk/Tec kinases activation
(1) PI3 kinase-induced membrane localization
Deletion o f the PH domain constitutively activates Etk/Bmx in prostate cancer
cells, rat parotid gland epithelial cells, and lung epithelial cells (7,13), suggesting the
involvement of PH domain in the inhibitory intramolecular folding. Upon antigen
binding and antigen receptor activation, the activated PI3 kinase can phosphorylate
PtdIns(4,5)P2 to yield PtdIns(3,4,5)P3, which can then attract PH domain-bearing
Btk/Tec kinases to the plasma membrane and activate them presumably by
disrupting the inhibitory folding. Co-expressions of Btk and the catalytic subunit of
PI3 kinase induce both the tyrosine phosphorylation and kinase activation of Btk (4).
Growth factors can induce the translocation of a PH domain of Btk to the plasma
membrane in a PI3 kinase-dependent manner (14). The SH2 domain of Btk/Tec
kinases can interact with the adapter subunits of PI3 kinase (15). Inositol
polyphosphate phosphatase (SHIP) that can dephosphorylate PtdIns(3,4,5)P3 is able
to down regulate the activity of Btk (4). Thus other enzymes possessing inositol
polyphosphate phosphatase activities, such as PTEN and SHIP-2, are potential
regulators of Btk/Tec kinases. In a prostate cancer cell line, LNCaP, Etk/Bmx is
activated by PI3K in response to IL-6 treatment (7). All these observations indicate
the inter-molecular interactions between the PH domains ofBtk/Tec family NRTKs
and Ptdlns(3,4,5)p3 as the first step ofBtk/Tec kinases activation. However, because
8
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Etk/Bmx possesses two 22-amino-acid repeats, instead of the TH domain, thus the
existence of the aforementioned mechanism requires further investigation.
Surprisingly, Itk was shown to constitutively associate with the plasma membrane
(16), indicating that membrane localization may not be sufficient for the activation of
some enzymes. The alternative mechanism of activation for Itk will be discussed
later.
(2) Protein-protein interaction
Protein binding partners that can disrupt the inhibitory intramolecular
interaction between SH3 domain and PR motif (and possibly PH domain and kinase
domain) may also activate Btk/Tec family NRTKs. Gaq subunit o f G-protein is
proposed to activate Btk via this mechanism. That the interaction with G«q involves
the TH domain of Btk suggests the mechanism of activation might be protein-protein
interaction, which unfolds the inactive conformation of Btk. (17). The binding of Btk
to the subunits of G-protein can increase the kinase activity (17-19). In addition,
another report also shows direct association of Gpy with Btk (19). The kinase-dead
Etk/Bmx is shown to inhibit Ga12- and Ga13-induced activation of serum response
factor (SRF).
PTPD1 is a protein tyrosine phosphatase with an ezrin domain (20). Recently
PTPD1 is found to associate with the PH domain of Etk/Bmx and activate it, as
evidenced by the activation of STAT3 by PTPD1-induced Etk/Bmx activation.
9
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Intriguingly, Etk/Bmx does not seem to be dephosphorylated. It is postulated that
PTPD1 might activate Etk/Bmx by interacting with the PH domain and relieving the
inhibitory intramolecular folding between the PH domain and the kinase domain
(21).
BRDG1 (B cell receptor downstream signaling 1) is a docking protein for B cell
receptor, and contains a PH domain and a PR-motif. BRDG1 is found to be a binding
partner for the kinase domain of Tec, but not Btk or Etk/Bmx (22). In addition, co
expression of BRDG1 and Tec can enhance Tec activity in a TH domain-dependent
manner. Since the TH domain of Itk has been shown to interact intramolecularly with
its own SH3 domain, thus the binding of BRDG1 to the TH domain of Tec may
relieve Tec from its inactive state and activate it. It is also likely that other Btk/Tec
kinases may be activated by BRDGl-like proteins.
There are still other proteins also being reported to interact with Btk/Tec
kinases. WASP, EWS, and Sam-68 proteins are found to bind to the SH3 domain of
Btk/Tec kinases, however their role in regulating this signal transduction pathway is
still not clear (23).
Other evidences that support this mechanism include: the mutations in the SH3
domain of several proto-oncogenes such as c-Src and c-Abl increase their
transforming activity, indicating that the SH3 domain negatively regulates the kinase
activity. Similarly, mutation of Tyr223 or deletion of SH3 domain increases the
transforming activity of Btk mutant (E41K) without affecting its kinase activity (5).
10
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1.1.2.2 The second step ofBtk/Tec kinases activation
After membrane localization, two steps of phosphorylation seem to be required
for the activation of Btk/Tec family (except for Itk). In Btk, Tyr551 in the kinase
domain activation loop (Tyr566 in Etk/Bmx) is phosphorylated by Src family
kinases, Lyn, Fyn, Blk, and members o f Jak family kinases (15). Src family kinases
are shown to bind the TH domain ofBtk/Tec kinases via their SH3 domain.
1.1.2.3 The third step ofBtk/Tec kinases activation
The phosphorylation of Y551 then leads to autophosphorylation of Y223 in the
SH3 domain of Btk (4). This autophosphorylation is thought to further prevent
refolding back to the inactive conformation. Deletion of the SH3 domain of Tec
increases its catalytic activity. However, deletion of the SH3 domain or a Y223F
mutation in Btk does not have apparent effect on its catalytic activity (23). Thus the
mechanism of activation might be more complicated than this two-step
phosphorylation, and/or may be slightly different among the members of Btk/Tec
kinases.
The tyrosine phosphorylation on the SH3 domain can profoundly affect the
binding of protein partners to Btk/Tec kinases (24,25). For example, phosphorylation
on Tyr233 significantly decreases the interaction between WASP and Btk, and Syk
preferentially binds to phosphorylated SH3 domain of Btk and Itk. Although Syk
11
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does not directly participate in the phosphorylation of Btk, however, it is still
essential for Btk activation, since in Syk' cells, Btk activity is significantly reduced
(26). Altogether, these observations indicate the significance of the SH3 domain
autophosphorylation ofBtk/Tec kinases in the regulation of their activities.
In addition to intramolecular interaction with the PR motif, SH3 domain may
negatively regulate Btk/Tec kinases by intermolecular interaction. Sab is identified
as a negative regulator of Btk. The binding of Sab to the SH3 domain of Btk inhibits
its kinase activity (27). This observation provides an alternative mechanism other
than inhibitory intramolecular folding, rendering the Btk/Tec kinases inactive under
resting state.
1.1.3 A novel two-step mechanism of activation for Itk
As mentioned earlier, Itk activation requires a novel mechanism. In Jurkat cells,
Itk is found to be constitutively associated with the plasma membrane. Two-step
activation is identified: first is the activation of Zap-70; second is the tyrosine
phosphorylation of LAT, which then is able to bind and activate Itk. Moreover, the
co-localization of Itk and LAT is found to be mainly in the glycolipid-enriched
membranes (16).
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1.1.4 Upstream activators ofBtk/Tec kinases
Btk/Tec family NRTKs can transduce signals from several different types of
cell surface receptors, such as antigen receptors, cytokine receptors, growth factor
receptors, and G-protein coupled receptors.
As previously mentioned, upon antigen binding to receptors, Src family,
Btk/Tec family kinases and Syk can be recruited and bind to the cytoplasmic IT AMs
(immunoreceptor tyrosine-based activation motif) o f antigen receptors. Src can
directly phosphorylate and activate Btk/Tec kinases. Furthermore, Syk is shown to
bind to the phosphorylated SH3 domain of Btk and Itk via its SH2 domain. Syk does
not involve in the phosphorylation of Btk, however, it is still essential for Btk
activation. In addition, PI3K signal is also required for Btk/Tec kinases activation as
previously described. Altogether, Syk, PI3K, Src family kinases concert to activate
Btk/Tec family kinases.
Among the cytokines, IL-2, IL-3, DL-6, GCSF, and erythropoietin can activate
Btk/Tec kinases. Although some o f the receptors for these cytokines involve Src
family kinases, another common signal transducer is Jak family kinases, Jakl, Jak2,
and Tyk2 (28). In B lymphocytes and hepatocytes, activated IL-6 receptors can form
complexes with Btk and Tec via Jak family kinases, and Jakl can directly
phosphorylate Btk (15). Intriguingly, in prostate epithelial cells, Etk/Bmx is not
associated with Jak family kinases, but is activated by EL-6 via PI3K (7). The
activation of Btk and Etk/Bmx by IL-3 in mast cells has been demonstrated and both
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involve PI3K (29). Similarly, JL-2 also activates Itk and Tec via PI3K. Again, PI3K
proves to be a common activator of Btk/Tec kinases.
1.1.5 Btk/Tec kinases can modulate intracellular Ca2 + concentration and the
activity of Ca2+ -dependent proteins (PKC isoforms)
SLPs (SH2 domain containing leukocyte adaptor protein), SLP-
65/BLNK/BASH and SLP-76 are tyrosine phosphorylated by Syk or ZAP-70 in
activated B and T cells. The tyrosine phosphorylation of SLPs allows the recruitment
of both Btk/Tec kinases and PLCy to come to the vicinity of each other, and then
PLCy can be phosphorylated and activated by Btk/Tec kinases. In B cells, following
the activation of B cell antigen receptors, the activated Btk participates in the
tyrosine phosphorylation of PLCr2 , whose activation results in the hydrolysis of
PtdIns(4,5)P2 and the generation of Ins(l,4,5)P3 and diacylglycerol (DAG). DAG
then induces the release of Ca2 + from Endoplasmic Reticulum (ER) and the
activation of PKC (30,31). The Btk-induced intramolecular Ca2 + store release is
thought to indirectly modulate the opening of calcium release activated channel
(CRAC) on the plasma membrane (32). Consistent with this notion, Ca2 + signal is
also found to be reduced in Itk-deficient T cells (33). Similarly, PKCg isoform has
been reported to inactivate STATs via Etk/Bmx (34).
Btk/Tec family kinases have also been found to associate with several PKC
isoforms and modulate their kinase activities (35-38). The binding of Btk to PKC
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is mapped to the region between residues 28 and 45, overlapping the
phosphatidylinositide binding site. Btk has been shown to phosphorylate and activate
PKC pi (26). Syk and ZAP-70 are important for normal immunological functions of
hematopoietic cells, and the loss of Syk can result in several hematopoietic defects
(39). Syk, ZAP-70, Src family and Btk/Tec family kinases have been found to
associate with IT AMs on the cytoplasmic domain of immuno-receptors.
Furthermore, Syk is found to bind preferentially to the phosphorylated SH3 domain
of Btk and Itk via its SH3 domain (24), and this is thought to be important for PKCpi
activation. Btk and PKC pi may form a negative feedback loop in BCR-mediated
signaling: Btk phosphorylates and activates PKC pi, which in turn phosphorylates
and down-regulates Btk activity. Etk/Bmx-activated PKC5 can inhibit STATs
activation possibly via PKC-dependent protein tyrosine phosphatase, SHP-1 (36).
1.1.6 Btk/Tec kinases can modulate the activity of MAP kinases
Btk/Tec kinases are found to be involved in the regulation of MAP kinases, i.e.
ERK, JNK, and p38 MAPK, via the interactions with vav (4,40). In the absence of
Btk, INK and p38 MAPK activation is impaired and the activation of ERK and Ca2 "
signal are not sustained. ERK activation is interrupted in Itk'A and Txk'/_ primary T
cells (41).
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1.1.7 Btk/Tec kinases can modulate gene expression
Btk/Tec family of NRTKs are also involved in the regulation of gene
expression. Tec has been shown to interact with a PR-motif within CD28 via SH3
domain, and regulate Dok-l/p62Dok phosphorylation and cytokine gene
transcription (42,43). In fact, Dok-1 has been shown to associate with Tec (but not
Btk or Itk). After the phosphorylation by Tec, Dok-1 can bind to the SH2 domain of
Tec, which in turn can further hyper-phosphorylate Dok-1. Since Dok-1 is originally
identified as a RasGAP-binding protein, therefore, Tec is postulated to be able to
regulate Ras signaling pathway, which regulates gene expression. Consistently, a
kinase dead Tec attenuates IL-2 expression upon T-cell activation. The activation of
Akt and PI3K by Tec has also been reported, and can lead to the activation of
transcription factors, Forkhead and NFK B (44,45). In a pro-B cell line, c-fos gene
transcription is induced by Tec. c-fos is a member of AP-1 transcription factor
family, which regulates genes expression, such as IL-2, IFNy , TNFa, and IL-4 (23).
Thus Tec is thought to be a general inducer of the transcription of several cytokine
genes.
Btk does not seem to regulate c-fos expression. However, studies in mast cells
show that Btk regulates the activity of an element derived from IL-2 promoter and
directs the binding of nuclear factor of activated T cells (NFAT) and AP-1 to these
promoters (46). Itk in T-cells is shown to affect the nulcear localization of NFAT as
well. Btk also regulates the phosphorylation and nuclear localization of TFII-I (47).
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Furthermore, TFII-I/BP-135 is shown to be directly and constitutively associated
with the PH-TH domain of Btk in resting B cells. Consistently, in the B cells of Xid
mice, a point mutation (R28C) in the PH domain causes the dissociation of TFII-I
from Btk and accumulation of it in the nucleus. TFH-I/BP-135 dissociates from Btk
upon anti-Ig antibody stimulation, with increased detection of TFII-I in the nucleus
(48). TFII-I is a basal transcription factor regulating the promoter activity of bcl2 or
bcl-Xi, , and c-fos. Thus although Btk does not seem to regulate the expression of c-
fo s directly, however, Btk can still affect its expression through TFII-I. To
summarize, these observations suggest that Btk may regulate nuclear localization of
the basal transcription factor, TFII-I, and thus the transcription of target genes.
Etk/Bmx is reported to be a potent activator of STAT1, 3, and 5 (Signal
Transducer and Activator of Transcription) in Cosl cells (36). At least for STAT3,
there is a physical interaction between the PH domain of Etk/Bmx and STAT3.
Intriguingly, the Etk/Bmx-induced activation of STATs is independent o f JAK, a
common activator of STATs, therefore providing an alternative source of activation
for STATs. Previously, STATs are reported to be activated by Src. With the
knowledge of STATs activation by Etk/Bmx, two distinct pathways appear to dictate
STATs activation: first Src activates p38 MAPK which induces serine
phosphorylation and activates STAT3 (49); second: Src phosphorylates Tyr566 and
activates Etk/Bmx which in turn phosphorylates and activates STAT3 (12). Since the
sites of phosphorylation on STAT3 by the two distinct pathways are different, thus a
synergism possibly exists for the activation o f STAT3 by these two pathways.
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1.1.8 Btk/Tec kinases can modulate apoptosis
Fas is a member of the tumor necrosis factor (TNF) receptor family which
modulates apoptosis in various cell types. Btk is shown to be constitutively
associated with Fas in wild-type DT-40 lymphoma B cells and human leukemia B
cell line NALM-6-UMI, and exerts negative regulation upon Fas-induced apoptosis
(50). The interaction between Btk and Fas involves both the PH and the kinase
domain of Btk, and appears to disrupt the ligation of FADD with Fas. This provides
a possible mechanism that Btk can inhibit Fas-induced apoptosis.
The observation that Etk/Bmx is the key effector of Src in the transformation of
hepatocytes and fibroblasts suggest their participation in anchorage-independent
growth (12,51). The role of Etk/Bmx in the neuroendocrine differentiation of
prostate cancer cells has also been documented by using a dominant negative mutant
(7). Overexpression o f Etk/Bmx protects prostate cancer cells from ionization and
Thapsigargin induced cell death (52). Intriguingly, over expression of Etk/Bmx
counteracts the anti-apoptotic effect to GM-CSF (29). How can Etk/Bmx, and the
other Btk/Tec kinases, be both pro-apoptotic and anti-apoptotic? There are several
possibilities: (1) a caspase-3 cleavage site is found in the SH3 domain of Etk/Bmx
that can be cleaved to yield a fragment containing only the SH2 and kinase domain
(53). Thus it is possible that the wild type Etk/Bmx may function in an anti-apoptotic
way, while the truncated form participates in cell apoptosis. (2) The Btk/Tec kinases
are involved in a plethora of signaling pathways, some are pro-apoptotic (e.g. p38
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MAPK), others are anti-apoptotic (e.g. Akt and NFk JB ), therefore the dual activity
might be achieved by the switch of balance between these two paradigms of
signaling events.
1.1.9 Btk/Tec kinases can modulate cytoskeletal organization
Cytoskeleton plays essential roles in cell division, cell shape, motility, and
chemotaxis. Btk is shown to colocalize with actin filament upon stimulation o f mast
cell by high affinity IgE receptor in a PH domain-dependent manner (37). The
binding site to F-actin is mapped to the basic residues of the 10 amino-acid stretch on
the PH domain of Btk. Since this region is highly conserved among all members of
Btk/Tec family kinases (except Txk, which does not have PH domain), thus the
actin-binding property may be common to other Btk/Tec kinases. In addition to actin
binding, the PH domain of Btk has been demonstrated to promote actin bundle
formation in vitro (37). Over-expression of Tec in NIH-3T3 cells resulted in increase
of actin polymerization, formation of stress fiber, and translocation of RhoA to
particular fractions (40). Etk/Bmx can also translocate to the plasma membrane,
colocalizing with F-actin exclusively in the membrane ruffles in fibronectin-
stimulated endothelial cells HUVEC (5).
Btk/Tec kinases may regulate Rho family GTPases that are important regulators
of the actin cytoskeletal organizations. Vav is a guanine nucleotide exchange factor
for Rho family small GTPases. By yeast two hybrid screen, it is found that Tec,
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thorough the SH2 domain, can complex with vav and phosphorylate it (15).
However, another group reported that Tec interacts with vav through TH domain
(54), thus there may be multiple interactions between Tec and vav.
In addition, focal adhesion kinase (FAK), a key mediator of integrin signaling in
response to extracellular matrix, has also been found to regulate Etk/Bmx.
Colocalization of Etk/Bmx and FAK is observed in membrane ruffles, enriched with
F-actin, and the interaction arises between the PH domain of Etk/Bmx and the FERM
domain of FAK. (2,37,40,55). These observations suggest that Btk/Tec family
kinases can translocate to the plasma membrane, coordinating with potential
downstream targets, Rho family small GTPases, and regulate the cytoskeletal
reorganization in response to stimuli.
1.2 Tight junction
One important role of epithelia and endothelia is the formation of a diffusion
barrier to separate the internal from the external environment and create a unique
intracellular biochemical milieu that is essential for the normal functioning of life. So
far, four specialized complexes are found in the junctions, tight junction (TJ),
adherens junction (AJ), gap junction, and desmosomes. Gap junction mediates
intercellular communication by allowing small molecules to pas through from one
cell to a neighboring cell (56). Desmosomes are “button-like” points of intercellular
contact that rivet cells together and provide anchoring sites for intermediate
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filaments (57). AJs form a continuous belt (the adhesion belt) to hold adjacent cells
together through a family of Ca2 + -dependent cell-cell adhesion molecule (cadherins),
which is also linked to actin cytoskeleton (58). TJ is the most apical structure in the
paracellular junction, and is important in terms of the following aspects: (1) It is
responsible for creating the aforementioned barrier and controlling the paracellular
permeability across the epithelial and endothelial layers (barrier function). (2) It
prevents the intermixing of apical and basolateral membrane components of
polarized epithelial and endothelial cells (fence function) (59,60). (3) It mediates
cell-cell adhesion. (4) Some molecular components of TJ share homology with
proteins that participate in tumor suppression, nuclear targeting, and cell
proliferation, suggesting other fundamental functions of TJ (61).
1.2.1 The molecular structure of tight junction
TJs encircle the cells at the most apical end of the lateral membrane, and in
freeze fracture replicas, shows a peculiar honey comb-like structure, consisting of a
meshwork of fibrils circumscribing the lateral membrane near the apical side (62). In
thin sections, they appear like zones of closely opposed plasma membranes (63).
Sometimes, depending on sample preparation, the neighboring plasma membranes
appear to be partially fused (64). The fibrils on one cell interact with fibrils on the
neighboring cell to close the paracellular space and define the barrier characteristic.
These fibrils are now known to be composed of at least two types of transmembrane
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proteins, occludin and claudin family. In addition, TJ also contains cingulin, ZO-1,
ZO-2, ZO-3, 7H6, and vinculin, etc. (65,66). Furthermore, TJ has been reported to
consist of lipids that have structural and physiological relevance (67).
1.2.1.1 Occludin
Occludin, a 65 kDa phosphoprotein, is a transmembrane protein found in TJs,
with 4 transmembrane domains, two extracellular loops that are composed mostly of
glycine and tyrosine, and both N- and C- termini in the cytosol (68). Several
evidences support this amino acid sequence-based prediction of topology. The end of
the C-terminal cytoplasmic domain can interact with ZO-1 (69). Both extracellular
loops become glycosylated if N-linked glycosylation sites are introduced (70). An
antibody raised against the extracellular loops has access to occludin on intact cells
(71). Occludin was originally thought to be the major sealing protein in TJ based on
the following observations. Occludin is adhesive, as demonstrated by its ability to
confer Ca2 + -indenpendent adhesion when transfected into occludin-null fibroblasts
(71). The addition of peptides corresponding to the extracellular loops decreases
transepithelial electrical resistance (TER), an indication of the integrity of the tight
junctional complexes (65). Mutation or overexpression of occludin affects TER and
paracellular permeability of some non-charged solutes (72). However, some cells
where occludin staining shows discontinuous distribution still retain barrier function,
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leading to the speculation that other unidentified protein(s) might facilitate junction
sealing and the subsequent discovery of claudin family.
Occludin migrates in SDS-PAGE as various bands with molecular weight from
62 to 82 kDa, exhibiting differential degrees of phosphorylation. Occludin is
phosphorylated on serine and threonine. The degree of phosphorylation can affect
cellular localization. The more phosphorylated occludin is found exclusively in the
TJ, whereas the less phosphorylated can be found in the TJ, basolateral membrane
and cytosol (73).
The carboxyl terminus of the cytoplasmic tail of occludin is the site of
association with ZO-1 as that deletion o f -150 amino acids in this region prevents
such interaction. The association of occludin with ZO-1 is important for the
localization in TJ, as that carboxyl terminal deletion perturbs TJ localization of
occludin (69).
1.2.1.2 Claudins
Claudins, -22 kDa, are much smaller than occludin, and also have two
transmembrane domains, two extracellular loops, with both amino- and carboxyl-
termini in the cytoplasm (although much shorter than occludin’s). To date, 20
claudin family members, with differential expression profiles, have been identified
(Table III). Claudins have intrinsic ability to polymerize into long fibrils (74). This is
in contrast to occludin, which can only form short fragments of strands. When
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occludin is transfected into claudin-expressing fibroblasts, it is recruited to the
claudin fibrils. Claudins exhibit even stronger adhesion than occludin (75). More and
more evidences suggest that claudins may be more important than occludin in
contributing the barrier function of TJs. When claudin-1 is over expressed in MDCK
cells, TER is increased several fold along with a corresponding decrease of
paracellular flux of FITC-labeled dextrans (76). Interestingly, it is also reported that
when occludin is oever expressed, TER and paracellular flux are both increased. This
suggests that in addition to sealing, occludin might have some other functions. Both
claudin-3 and -4 are sensitive to a cytotoxic enterotoxin, CPE, produced by
bacterium, Clostridium perfringens. CPE binds to claudin-3 and -4 through its
carboxyl-terminus, and exerts the cytotoxicity. The carboxyl terminus of CPE alone
is not cytotoxic. When MDCK cells (not expressing claudin-3) are exposed to the
noncytotoxic fragment of CPE, claudin-4 is removed from TJ with dramatic change
of the morphology of TJ fibrils: freeze fracture electron micrographs show
fragmented fibrils and incomplete network. The remaining network is presumably
CPE insensitive claudins and occludin. CPE-treated MDCK cells also exhibit
decreased TER (77). In claudin-11 knockout mice, TJ fibrils in Sertoli and central
nervous system myelin cells are completely lost (78,79). Together, claudins are
suggested to be the primary proteins in TJs conferring the barrier characteristic.
Furthermore, with the variety of expression patterns and permeation selectivity, it
can be expected that claudins may also be the main reason to account for the
differential permeability characteristics of TJs in various tissues.
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Mutations in claudin-16 (paracellin-1), are associated with defected TJ resulting
in a rare magnesium wasting syndrome called renal hypomagnesemia, along with
hypercalciuria and nephrocalcinosis (80). It is postulated that mutated claudin-16
may have formed the TJ that is unpermissive to magnesium since kidney is the main
organ responsible for magnesium resorption which only takes place through the
paracellular route driven by a positive electrical gradient. With the mutant claudin-16
in TJs, magnesium is not allowed to pass through the intercellular junction and lost
in the urine. Not only magnesium, claudin-16 is also selective for other divalent
cations, like Ca2 + . This is why renal hypomagnesemia patients also exhibit
hypercalciuria. This is a good example to demonstrate that not only forming a sealed
barrier, claudins may also be important for conferring a selectively permeable
diffusion pathway.
1.2.1.3 The organization of claudins and occludin in the TJ fibrils
Under freeze fracture micrographs, TJ fibrils appear to be of ~ 10 nm in
diameter, which is larger than any single molecule of claudin or occludin. Therefore
it is possible that the “monomer” of the fibrils actually is an oligomer containing
more than one occludins and/or claudins. Different compositions of different
claudins and occludins may define the various permeation selectivity in different
tissues (81). This idea is partially supported by the findings that paracellular
permeability of epithelia behaves like pores or channels with preference for cations
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and small molecules between 8-18A, and that claudin-1 and -3, and claudin-2 and -3
can interact across cells but not claudin-1 and -2 (82). Thus, of the 20 claudins, some
may interact with each other but some do not. How exactly a barrier and a selective
pore can be created at the same time when two TJ fibril oligomers from two
neighboring cells came in contact requires further study.
1.2.1.4 Zonula Occludens (ZO)
There are three isoforms of zonula occludens, ZO-1, ZO-2, and ZO-3, and they
all belong to the membrane-associated guanylate kinase (MAGUK) family,
characterized by having a SH3, a GUK (guanylate kinase; probably inactive), and up
to three PDZ (PSD-95/Dlg/ZO-l; postsynaptic density protein-95/Disc large/ Zonula
occludens-1) domains (83). Members of this family are often found in cell-cell
adhesion sites and may couple extracellular signals to cytoskeleton. All three
domains of ZOs are modules for protein-protein interactions, enabling the recruiting
and organization of TJ cytoplasmic protein scaffold (84). The SH3 domain is capable
of interacting with proline-rich motif and is important for interacting with signaling
molecules. The GUK domain is similar to ATP-dependent enzyme and can
potentially covert GMP to GDP (85), however, the activity of this kinase domain in
ZOs is still controversial. Occludin interacts with the GUK domain of ZOs. The PDZ
domain is found to interact with the cytoplasmic tails of several transmembrane
proteins, for example claudins bind to the first PDZ domain of ZOs. Furthermore,
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some PDZ domain containing proteins, such as p55 in red blood cells and human Dig
can bind directly to actin binding protein 4.1 (86). ZO-1 (zonula occludens-1) is a
cytoplasmic protein first identified in the tight junction (87). ZO-1 can localize to
sites other than the junction area, and can even be expressed in cells that never form
tight junctions (88-90). In epithelial cells, ZO-1 forms a heterotrimeric complex with
ZO-2 and pl30 (91,92). A fraction of ZO-1 accumulates in the nucleus in growing
epithelial cells (90). The C-terminal half of ZO-1 also contains an actin-binding site
(93,94). ZO-2 appears to be exclusively expressed in epithelial cell tight junctions
(95). ZO-1 and ZO-2 show homologies with a Drosophila tumor suppressor DlgA,
and postsynaptic densities, PSD-95/SAP-90 (95-98).
1.2.I.5 Symplekin
Symplekin, a 127-150 kDa membrane protein can be found in both the nuclei
and TJs of growing and confluent epithelial cells, but not in the endothelial junctions.
In cells that do not form TJs, symplekin and ZO-1 are exclusively found in the
nuclei. But, ZO-1, not like symplekin, can only be found in the nuclei of growing
cells. Furthermore, symplekin is suggested to be involved in the polyadenylation of
mRNA in the nuclei, suggesting the involvement of ZO-1 and symplekin in the
regulation of cell growth and differentiation (90, 99, 100).
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1.2.1.6 Cingulin
Cingulin, a 140~160 kDa phosphoprotein, can directly interact with ZO-1 (101),
and is composed of two peptides intertwined with each other to form a “coiled coil”
(102). Cingulin can be found in both AJs and TJs, and has structural similarity with
myosin, tempting to speculate that cingulin might also interact with actin and provide
an additional link of TJ complex to the regulation by contractile actomyosin ring.
1.2.1.7 Actin cytoskeleton
A ring of actin microfilament, containing myosin II, underlies the apical
junctional complex, and is linked to E-cadherin through p i30, a-, (3-, y-catenins,
vinculin, a-actinin, fodrin, and spectrin (103). Actin is also linked to TJ components,
such as ZO-1, ZO-2, ZO-3, occludin, cingulin, etc. Cytoskeleton possibly serves as a
structural and informative network between TJ and AJ. Contraction of this apical
actomyosin ring has been proposed to regulate paracellular permeability. Myosin
movement along the perijunctional ring is regulated by ATP and phosphorylation of
the regulatory light chain by Ca2 + -calmodulin-activated myosin light chain kinase
(MLK). Increased intracellular Ca2 + concentration can activate MLK, which in turn
phosphorylates myosin light chain, resulting in the contraction of perijunctional ring,
and the subsequent opening of TJs. Drugs that interfere with microfilaments and
microtubules, such as phalloidin, cytochalasin, and latrunculin, disintegrate the pre-
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established junctional complexes, and alter the morphology of junction fibrils (104).
Small GTPases, Rho, which is capable of regulating actin cytoskeletal, may also
regulate the permeability of TJ (105). Other small GTP binding proteins like Cdc42,
Rac, and focal adhesion kinase (FAK) that can regulate membrane ruffling and the
establishment of focal adhesion may be capable of regulating TJ as well.
1.2.1.8 7H6
7H6 is a 155 kDa protein found in TJs of both epithelial and endothelial cells
(106), and it could regulate paracellular permeability (107,108). But the function of
7H6 requires further investigation.
1.2.1.9 Rab family, Sec 6/8, VAP-33
Small GTPases such as the Rab family (e.g. Rabl3 and Rab3B), vesicle
associated membrane protein (VAMP)-associated protein-33 (VAP-33), and Sec 6/8
are also found to be associated with TJs and may be linked to vesicle transport
(109,110). Vesicle trafficking may occur through the apical junctional complex in an
early observation (111).
Sec 6/8 complex is a part of exocyst, which is required for specific vesicle
targeting to the bud tip of yeast (112). In addition, in streptolysin-O-permeablized
MDCK cells, antibodies against the mammalian homologue Sec 6/8 specifically
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inhibits the delivery of low density lipoprotein (LDL) receptor containing vesicle to
the basolateral membrane, while not affecting other proteins (113). Therefore Sec 6/8
in the TJs may be used as a marker for basolateral membrane targeting.
VAP-33 is found to be associated with the carboxyl-terminus of occludin. Over
expresion of VAP-33 leads to lateral relocation of occludin from TJ. VAP-33 may be
associated with the regulation of occludin localization in the TJs.
Rabs are generally thought to participate in vesicular transport. Rab 13
colocalizes with vesicular structures in fibroblasts (109). Rab8, which is involved in
basolateral transport, colocalizes with ZO-1 in MDCK cells (114). The function of
Rabs in TJs is not clear. The tyrosine protooncogene c-Yes and the Src substrate
p i20 are also found in the TJ region (115,116).
1.2.1.10 G-proteins
Several Ga subunits of heterotrimeric G-proteins have been found in the vicinity
of TJ, including Gai-2 , Gai2 , Gao , and Gas. The specific G-proteins involved in TJ
assembly and maintenance have not been identified. PKA may play a negative role
during TJ assembly. Stimulation of adenylyl cyclase with forskolin or the use of
cAMP analogue significantly reduces TER development in Ca2 + -switch experiment.
G «o is found to be localized in TJ and can be immunoprecipitated with ZO-1.
Stably expressed G ao or a constitutively activated G ao (Q205L) has no effect on TER
in confluent monolayer, however, the same treatment accelerates TJ biogenesis
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and facilitates the establishment of higher TER (117). Treatment with AIF3 , G-
protein activator, also affected TER, suggesting that G-proteins may also participate
in the steady-state regulation of TJ (118).
1.2.2 The biogenesis of TJ
Several signaling molecules are found to participate in the biogenesis of TJs,
such as kinases, Ca2 + , G-proteins, calmodulin, cyclic-adenosine-3’, 5’-
monophosphate (cAMP), phospholipase C (PLC). To date, two models are widely
used to study the biogenesis of TJs: Ca2 + -switch model and ATP depletion-repletion
model. The mechanisms of junction disassembly and reassembly appear to be
different in these two models (119).
1.2.2.1 Ca2 + -switch model
Ca2 + is essential for the functional assembly, maintenance, and regulation of TJ.
Depletion of Ca2 + can cause the opening of TJ, whereas repletion can induce the re
sealing of TJ (Ca2 + -switch) (120). When fully established epithelial monolayers, e.g.
MDCK, are incubated in the medium with low Ca2 + concentration, the cells become
rounded, losing apical and basolateral domains, allowing unregulated flux through
the paracellular pathway, and a decrease of transepithelial electrical resistance
(TER). ZO-1 is found in intracellular granules that may also contain catenins after
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Ca2 + depletion. Upon restoration to normal Ca2 + concentration, ZO-1 translocates to
the lateral membrane and becomes more tightly associated with actin cytoskeleton.
In the Ca2 + -switch model, TJ assembly appears to be induced by E-cadherin, a
component in the adherens junction (AJ). Extracellular Ca2 + is required for
homotypic interactions of E-cadherin and is likely to be the initial step of junctional
complex formation (58). After being translocated to the lateral membrane guided by
an addressing signal in its amino acid sequence, E-cadherin serves as an assembly
nucleus for further clustering of other TJ molecules. It is suggested that E-cadherin
might trigger the formation of TJ by activating downstream signaling cascades in the
cytoplasm. After the successful allocation and the establishment of homophilic
interaction between E-cadherins from neighboring cells, a putative contact receptor
is activated, followed by the activation of two different G-proteins controlling PLC,
calmodulin, PKC (118), and mitogen-activated protein kinase (MAPK) (121).
Ca2 + acts primarily on the extracellular side (122). Several lines of evidences
support this notion. An extracellular Ca2 + concentration of 0.1 mM is high enough to
trigger junction formation and cell polorization, yet insufficient to increase the
cytosolic Ca2 + level (to -14 ± 8 nM), which was reduced as a consequence of
preincubation in Ca2 + -depleted medium (20 ± 8 nM). La3 + blocks Ca2 + penetration,
but does not prevent the assembly of TJ and cell polarization. Cd2 + , which blocks
Ca2 + penetration and interacts with the extracellular part of E-cadherin, inhibits the
assembly of TJ (122). After Ca2 + activation, the E-cadherin molecule starts to
dimerize that will favor interaction with E-cadherin molecules in the neighboring
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cells (123). When the outermost part of E-cadherin is blocked by the specific
antibody, ECCD-1, TJ does not form.
Not only extracellular Ca2 + is essential to trigger the initial positioning of E-
cadherin, intracellular Ca2 + is also important for the succeeding assembly of TJ
components. Chelation of intracellular Ca2 + attenuates the formation of TJ, retards
the movement of ZO-1 from cytoplasmic sites to the plasma membrane (124).
Further investigation reveals that ER-Ca2 + stores play essential roles in TJ formation
(125). Thapsigargin (TG) inhibits endoplasmic reticulum (ER) Ca2 + -adenosine
triphosphatase (Ca2 + -ATPase) and depletes intracellular ER stores of Ca2+. Depletion
of ER-Ca2+ store disrupts the biogenesis of desmosomes and TJ without obviously
affecting E-cadherin and AJ. The sorting of ZO-1 and the desmosomal protein
desmoplakin I is disturbed in TG-treated cells in spite of normal intracellular Ca2 +
concentration.
The dependence of TJ formation on intact intracellular Ca2 + store is consistent
with the aforementioned observation that the activation of the classical signaling
pathway involving PLC, calmodulin, and PKC, is important following the initial E-
cadherin assembly and the activation of a putative contact receptor and G-proteins.
Both intact intracellular Ca2 + store and PKC are required for TJ formation. However,
PKC does not seem to be required for the stabilization of ZO-1 in the junctional
complex, whereas, intracellular Ca2 + store is required for the maintenance of ZO-1 in
the TJ. PKC inhibitors, l-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and
calphostin, inhibit the development of TER in MDCK cells after being switched
33
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from low Ca2 + to normal Ca2 + medium; it also inhibits the translocations of ZO-1 and
cingulin from an intracellular site to the lateral membrane (126). PKC agonist, 1,2-
dioctanoyl-glycerol (DiCg) (92), stimulates translocation of ZO-1 from the cytoplasm
to the membrane and promotes actin cytoskeletal reorganization in the presence of E-
cadherin antibody; it also increases the number of TJ fibrils and decreases
paracellular flux, however, only slightly increases TER (127). A PKC isoform,
PKQ, has been found in the TJ of epithelial cells (128), and by immunofluorescence,
PKQ has been found to translocate to the lateral membrane and colocalize with ZO-
1 . However, inhibition of PKC by calphostin has no effect on the association of ZO-1
with actin cytoskeleton, which requires Ca2 + (124). PKC-a may also regulate TJ
assembly as that cells stably expressing dominant negative PKC-a become resistant
to 12-0-tetradecanoylphorbol-13-acetate (TPA)-induced increase of paracellular flux
(129). Stimulation of cAMP/PKA promotes barrier function of thyroid cells
presumably via stabilization o f Ca2 + -dependent cell adhesion (130). Thus PKA may
also be involved in the maintenance of TJ.
1.2.2.2 ATP depletion-repletion model
The characterization of ATP depletion-repletion model is not so well as Ca2 + -
switch model. The ATP depletion-repletion model may have physiological relevance
with the restoration of TJ during ischemia-reperfusion. During ATP depletion, TER
also drops rapidly and reversibly in parallel with declining ATP level, but ZO-1
34
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remains in the subapical lateral membrane and becomes more tightly associated with
fodrin and other cytoskeletal proteins on the cytoplasmic face. ZO-1, ZO-2 and
cingulin shift into a Triton X-100-insoluble pool, consistent with increased
cytoskeletal interaction with TJ proteins. Analysis of immunoprecipitations from
ATP-depleted cells identified ZO-1 and fodrin (a spectrin analogue that links
proteins to cytoskeleton) within a large molecular weight complex (89,119). Upon
ATP repletion, ZO-1 interactions with cytoskeletal proteins decrease to normal level,
and TER returns to baseline. Therefore, the major difference between Ca2 + -switch
and ATP depletion-repletion models is the interaction of actin cytoskeleton with TJ
proteins. During ATP depletion, ZO-1 never dissociates from actin cytoskeleton, and
the whole TJ complex along with actin cytoskeleton moves inward, resulting in
dissociation of occludins and claudins from neighboring cells.
1.2.2.3 Common scenarios in both models
The targeting mechanism of occludins to the TJs is conserved among species
being studied so far. Transient and stable-transfection experiments show that chicken
occludin can be efficiently escorted to TJs in bovine, human, and canine epithelial
cells (69,131,132). The C-terminal cytoplasmic region of occludin is essential for
localization to the TJs (69). C-terminal region mutated occludins in transient
transfection experiments accumulate in the Golgi complex (70,133). The C-terminal
domain of occludin contains a basolateral sorting signal, since this particular domain
35
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alone can direct the basolateral localization of a reporter protein to the basolateral
membrane (70). It is suggested that the biogenesis of TJs occurs from the basolateral
membrane. Once on the basolateral membrane, the accumulation of occludins in TJs
seems to involve their extracellular loops. The addition of a peptide corresponding to
the second extracellular domain of occludin to A6 cells can result in the
disappearance of occludin from TJs (134). This is a very slow process, suggesting
that it is mediated by the inhibition of relocation of newly synthesized occludin to TJ
rather than internalization of already-integrated occludins. Deletion of the
extracellular loops results in lateral accumulation of occludins. How the extracellular
loops can mediate the targeting of occludin to TJs is not clear. It is postulated to be
mediated by the intercellular homophilic or heterophilic interactions of occludins
from the neighboring cells, since peptides derived from the extracellular loops of
occludins can interfere with the aggregation of occludins in TJs (71).
1.2.3 Barrier function
TJ is the limiting factor for paracellular permeability in epithelial and
endothelial layers. The tracer molecules can freely diffuse through the paracellular
pathway until the completion of TJs assembly. Occludin seems to be directly
involved in the formation of paracellular barrier. Chicken occludins stably expressed
in MDCK cells can increase transepithelial electrical resistance by 40 to 400%,
depending on the expression system (131,132). Transepithelial electrical resistance
36
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(TER) is an indication of the tightness of TJs, since the paracellular route has a
higher conductivity for the short electrical current than the transcellular route in
cultured epithelial monolayers (135,136). There is also a correlation between the
amount of occludin, both endogenous and transient expressed, and TER (137), and
TER decreases when occludin is depleted from TJs (134). In different tissues, TER
ranges from 10 (e.g. proximal renal tubule) to > 10,000 (e.g. urinary bladder) Q •
cm2 , indicating that TJ can tune up the tightness in accordance with the physiological
needs.
Rather than forming an absolute barrier, TJ exhibits the feature as a
semipermeable filter, with an equivalent pore radii of 30-40 A, allowing the passage
of certain solutes (138). The ion selectivity of paracellular permeability can be
altered by acidification or the addition of polyvalent cations, suggesting that these
“pores” contain dipoles with negative sides oriented towards the aqueous phase
(139,140). The TJ molecules can also change the degree of phosphorylation in
response to stimuli (65,66,118). The most dramatic example of the dynamic
regulation of TJ is that it can be opened up to the extent that allows the traverse of
macrophages to the inflammatory site, or the passage of spermatozoa from the
Sertoli cell to the lumen of seminiferous tube (66).
3 7
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1.2.4 Fence function
TJs are also involved in the maintenance of cell polarization by exerting a fence
function to restrain apical and basolateral compositions in their designated domains
(59,60). This fence function is not directly related to the “barrier function” since 10
minutes of ATP depletion are sufficient to decrease TER by 95% but do not have any
effect on the fence function or the ultrastructural appearance of TJs (141).
Breakdown o f the fence function due to longer times of ATP depletion correlates
with the fragmentation of the intramembrane strands (142). Disruption of the
continuous junctional distribution of endogenous and transfected occludin in MDCK
cells causes a breakdown of the fence function (131). Transfection of occludin with
truncated carboxyl terminus impairs the ability o f TJ to maintain a fluorescent lipid
marker in the apical membrane (131). These observations suggest that occludin is
involved in the fence function.
Etk/Bmx is not only expressed in hematopoietic cells, but also in epithelial and
endothelial cells. Btk/Tec family of non-receptor tyrosine kinases have been shown
to be able to translocate to the plasma membrane upon activation, colocalizing with
actin cytoskeleton. The biogenesis and regulation of epithelial and endothelial
junctions, the specialized protein complex conferring the barrier and fence
characteristic of epithelia and endothelia, are suggested to be closely regulated by
actomyosin ring. All these clues prompted us to explore the possible involvement of
Etk/Bmx in the regulation of the intercellular junctions.
38
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Table HI Claudin family
Claudin Disease Hum an Amino mRNA or Protein Tissue Distribution (m urine)
Locus Acids
Liver L ung Kidney Testis H eart Brain
1 211
---- ~ — ~ ~
2 230 ~ 0
----
0 0
3 C P E receptor 7qll 220
---- —
~ ~ 0
4 C PE receptor 7ql 1.23 209 0 ~
----
0 0
—
5 TM VCF? 22qll.2 218 ~
----
~ ~ ~ -
6 16pl3.3 220 0 0 0 0 0 0
7 17pl2 211 ~
— ---- —
0
8 21q22.1 225 0 -
----
~ 0
9 I6pl3.3 217
10 13q31-34 228 ~ ~
1 1 Brachm ann-de
lange?
3q26.2-3 207 0 0 0 0 ----
12 7q21 244
13 211
14 21q23.3 239
15 7q21.3 228
—
16 Renal
hypomagnesemia
3q
256 0
----
0 0 0
17 21q22.1 224
18 261
19
20 219
(Adapted from Mitic, L.L., et al., Am. J. Physiol. 279: G250-G254, 2000)
The length of tilde represents level of mRNA, 0 and blank indicate no detected
expression and lack of data, respectively.
1.3 Pa-4AEtk:ER cell line
To specifically study the signaling events downstream of Etk, a (3-estradiol (E2 )-
inducible Etk chimeric protein (AEtk:ER) was constructed as described (13). Briefly,
the hormone binding domain of human estrogen receptor (hER-HBD) was fused in-
frame to an amino-terminus truncated form of human Etk, PHAl-68Etk, to
39
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engineer AEtk:ER. It has been shown by Wen et al. that the fusion of hER-HBD to
the activated form of Etk renders the tyrosine kinase activity of this protein
cytokine/growth factor-independent, but E2 -dependent. Using E2 as an activator, the
kinase activity of the chimera, assessed by its tyrosine phosphorylation profile, is
rapidly turned on in the stably transfected Pa-4 cell line (Pa-4AEtk:ER). This
observation is consistent with the previously proposed model, in which E2 binding to
HBD stimulates the release of Hsp90 from the HBD o f Etk chimera. It then allows
the access of partner protein(s) to the AEtk:ER molecule for subsequent activation.
This model also explains the possible scenario by which E2 -bound AEtk:ER chimera
is able to homodimerize, which is sufficient to result in trans-Tyr5 6 6 phosphorylation
of Etk and the consequent activation.
4 0
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CHAPTER 2 MATERIAL AND METHOD
2.1 Reagents
The cell culture media, serum, and antibiotics were from Life Technologies
(Rockville, MD). Latrunculin and genistein are both from Calbiochem (San Diego,
CA).
2.2 Cell line
The rat parotid epithelial cell line Pa-4, also known as parotid C5 cells (143),
was plated on Primaria culture dishes (Falcon) in Dulbecco’s modified
Eagle’s/Ham’s F 12 (1:1) medium supplemented with 2.5% fetal calf serum, insulin
(5|ig/ml), transferring (5|ig/ml), epidermal growth factor (25ng/ml), hydrocortisone
(1.1 |iM), glutamate (5 mM), and kanamycin monosulfate (60 |ig/ml) and maintained
in a humidified atmosphere of 5% CO2 and 95% air at 35 °C. Pa-4AEtk:ER cells
were established by stably transfecting Pa-4 cells with an inducible Etk-estrogen
receptor (ER) chimeric construct (AEtk.ER) by a LipofectAMINE™ (Life
Technologies, Rockville, MD)-mediated method. The tyrosine kinase activity of
AEtk:ER cells can be further induced by the addition of 1 (iM estrogen receptor
agonist, (3-estradiol, to the culture media, as demonstrated by the
41
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autophosphorylation of Tyr-566 of Etk (13). Pa-4AEtk:ER cells were maintained
with Geneticin (G418, 600 pg/ml) and Dulbecco’s modified Eagle’s/Ham’s F I2 (1:1,
phenol red-free) medium supplemented with 2.5% charcoal-stripped fetal calf serum
plus the aforementioned ingredients. MDCKAEtk:ER cell clones were established
and screened as described previously (13).
2.3 Measurement of transepithelial electrical resistance (TER)
Epithelial cells were grown on permeable membranes (Transwell , Costar-
Coming, San Francisco, CA) that allow visual monitoring the growth of polarized
epithelial cells to confluence. Bioelectric parameters of cell monolayers were
monitored at predesignated time intervals with a MilliCell ERS screening device
(Millipore, Bedford, MA) that one can measure spontaneous potential difference
(SPD, expressed in the unit of mV, taking the apical aspect as reference) and TER
2
(expressed in kfl-cm ) with chopstick-style electrodes. Background potential
difference arising from the asymmetry of voltage sensing electrodes and electrical
resistance contributed by both bathing fluids and the filter membrane were measured
and averaged using the values observed at the beginning and end of each set of SPD
and TER measurements from two blank filters bathed with the same medium utilized
in cultivation of epithelial cells. These background SPD and electrical resistance
were subtracted from the raw data of SPD and TER, respectively. Using the
42
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corrected SPD and TER, equivalent active ion transport rate (/eq; alias, equivalent
short-circuit current) is estimated as SPD/TER, assuming the prevalence of Ohm’s
law for a given epithelial cell monolayer system.
Approximately 5 to 8 days after cell monolayers reached confluence, 1 |iM
estradiol (E2) was added approximately 4-16 hrs prior to the treatment with drug or
®
hypoxia. For hypoxic treatment, cells grown on Transwell were transferred to an
exposure chamber, flushed with 1% O2 balanced with 5% CO2 and 94% N2, and
sealed airtight. Measurements were obtained every 4-h during a total of 24 h
hypoxia followed by 8 h reoxygenation with 5% CO2 balanced with room air.
Latrunculin B or genistein were added to the bathing fluids of cells cultured on the
( § )
Transwell . SPD and TER of these monolayers were measured at the indicated time
intervals during drug treatment.
4 3
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CHAPTER 3 RESULT
3.1 Phenotypic manifestation of epithelial cells that express Etk
To investigate the role of Etk in the regulation of epithelial paracellular
junctions, both cells over-expressing AEtk:ER inducible construct (Pa-4AEtk:ER and
MDCKAEtk:ER) and parental cells (Pa-4 and MDCK cells) were grown to form
confluent monolayers on polyester Transwell® and their TER values were measured.
As shown in Fig. 1, TER in stably transfected and E2 -stimulated Pa-4AEtk:ER as
well as MDCKAEtk:ER epithelial cells consistently manifested a higher level than
that in corresponding parental cells. The mean TER values for Pa-4, and Pa-
4AEtk:ER cells were 2.06+0.05 and 2.75±0.10 kD-cm2 respectively, and the mean
TER values for MDCK and MDCKAEtk:ER cells were 38.91±2.32 and
241.85±15.95 fi-cm2 respectively. The mean of the corresponding equivalent active
ion transport rate (7eq) for E2 -stimulated parental and Pa-4AEtk:ER cell monolayers
were 1.85±0.08 and 1.08±0.09 |iA/cm2 , respectively. Since the measurements of
both potential difference (SPD) and TER in MDCK cells were too low to give
reproducible calculations of /eq, the calculated 7eq from MDCK and MDCKAEtk.ER
cells were not shown. The observed difference in TER between parental and Etk-
activated cells was persistent for at least 5 days throughout the period of
measurement (Fig. 2), suggesting the tightening of the paracellular seals upon Etk
44
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activation. The transepithelial electrical resistances of parental Pa-4 and MDCK
cells are quite different, in that Pa-4 cells exhibit intrinsically higher TER than do the
MDCK cells (Fig.l). Thus, Etk activation enhances TER in epithelial barrier of
either leaky or tight nature. The epithelial barrier to the diffusion of hydrophilic
solutes through the paracellular pathway is afforded by tight junctions. Permeation
across tight junctions is not static but is dynamically regulated under physiological
environment and under pathophysiological conditions, such as hypoxia. In
particular, epithelial cells have been reported to respond to hypoxic stress, rendering
the depletion of ATP and causing the loss of TER (144).
2.51
200 ™
E S
o
150 a
' 100 q
*■ - .< ?- X*- #
• F &
Fig. 1. Etk activation increases the TER of Pa-4 and MDCK cell monolayers.
Cells were grown on semipermeable polyester Transwell® membranes until a
confluent monolayer was established. E2 was added to the culture media at a final
concentration of 1 pM 4 h before TER measurements. Error bars represent the
standard error of the mean based on four independent measurements performed in
triplicates of E2 -treated parental and Etk chimera stably transfected cells,
respectively.
4 5
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□ Pa-4
□ Pa-4AEtkER
i t
Day 4 Day 5 Day 6 Cay 7
Day After Seeding
Day 8
Fig. 2 Etk activation stably sustains TER of Pa-4 and Pa-4AEtk:ER cell
monolayers. Cells were grown on semipermeable polyester Transwell® membranes
until a confluent monolayer was established. E2 was added to the culture media at a
final concentration of 1 p,M approximately 16 h before TER measurements. The
error bars represent standard error of the mean from at least four independent
measurements performed in triplicates of E2 -treated parental and Etk chimera stably
transfected cells, respectively.
3.2 Etk activation protects Pa-4 cells against hypoxia-induced decrease of TER
To probe the consequences of hypoxic stress in Pa-4AEtk:ER cells that possess
the property of an elevated TER as compared to the parental Pa-4 cells, we exposed
both E2-treated parental and Pa-4AEtk:ER cells to prolonged periods of hypoxia.
During the first 8-h of hypoxic treatment, TER increased about 30% and 20%,
normalized by the control in normoxia, above the baseline values (Oh) in Pa-4 and
Pa-4AEtk:ER cell monolayers, respectively. However, after 24-h of hypoxia, TER
46
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decreased drastically to approximately 5% of the baseline levels in Pa-4 cells,
whereas Pa-4AEtk:ER cells were able to maintain substantially higher TER that was
comparable to their normoxic controls (Fig. 3A). Even with the compromised TER,
both Pa-4 and Pa-4AEtk:ER cells were mostly viable after 24-h of hypoxia since
TER values in both cells started to recover 4-h post-hypoxia (Table 1). These data
showed that Etk activation sustains TER in epithelial cells under prolonged hypoxic
conditions. Since TJ integrity is disrupted by hypoxia and TER is mostly afforded
by TJ, thus the result suggests that Etk activation may prevent the hypoxia-induced
TJ disruption in epithelial cells.
Equivalent active ion transport rate (/eq) of Pa-4 and Pa-4AEtk:ER monolayers
was also determined. As shown in Fig. 3B, /eq in Pa-4 cells decreased to almost 0%
of the baseline values (Oh), normalized with the control in normoxia, after 24-h
hypoxia treatment, while about 60% of baseline /eq remained in Pa-4AEtk:ER cells
after the same period of hypoxic exposure. This suggests a beneficial effect of Etk
on active ion transport. As the measurement of TER is generally believed to be a
reliable gauge of the junctional tightness between epithelial cells, we conducted
further investigations utilizing TER measurement as a means to elucidate the role of
Etk activation in epithelial cell biology.
4 7
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(A ) 180 T-
160 ■
140 ■
3 120 "
80 •
40 -
20 ■
-20 ■
-40 -
Time (hr)
140 x-
120--
100 ji.
8 0 --
6 0-
40 -
20-
-20 ■
-40 ■
Time (hr)
Fig. 3. Relative changes in TER and Ieq of Pa-4 and Pa-4AEtk:ER cell
monolayers under hypoxic conditions. The Pa-4 and Pa-4AEtk:ER cells were
cultured on polyester Transwell® semipermeable membrane until confluent. Cells
were treated with E2 about 16h before hypoxia treatment. Cells were then cultured in
an air-lock chamber with 1% oxygen. TER (A) was measured and the corresponding
/eq (B) was calculated (see text for details) at 0, 4, 8, and 24 h after the on-set of
hypoxia treatment. The curves A (TER) and B (/eq) represent the percentage
changes normalized with that from normoxia control, which is designated as 100%.
Error bars represent the standard error of the mean based on four independent
measurements of E2 -treated parental and Etk stably transfected cells, respectively.
4 8
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H ypoxia (1%
O xygen)
Recovery
Time (h) 0 4 8 12 24 4
1 0.70 0.96 0.91 0.37 0.20 0.31
2 0.98 1.29 1.25 0.52 0.20 0.37
Pa-4 3 0.83 1.20 1.17 0.53 0.21 0.32
4 0.77 1.06 1.04 0.48 0.20 0.31
5 0.70 0.94 0.89 0.42 0.16 0.27
Mean 0.80 1.09 1.05 0.46 0.19 0.32
SS 0.05 0.09 0.10 0.02 0.00 0.01
SE 0.05 0.07 0.07 0.03 0.01 0.02
1 1.29 1.78 2.03 1.21 1.02 1.82
2 1.36 1.92 2.17 1.27 1.07 1.77
Pa-4AEtk:ER 3 1.11 1.68 2.04 1.25 1.00 1.73
4 1.03 1.59 1.99 1.28 1.02 1.72
5 1.15 1.73 2.03 1.31 1.07 1.77
Mean 1.19 1.74 2.05 1.26 1.03 1.76
SS 0.07 0.06 0.02 0.01 0.00 0.01
SE 0.06 0.05 0.03 0.02 0.01 0.02
SS: Sum of Squares
SE: Standard Error
Table 1. TER of Pa-4 and Pa-4AEtk:ER cell monolayers was able to recover
from a 24-h hypoxia. The Pa-4 and Pa-4AEtk:ER cells were cultured on polyester
Transwell® semipermeable membrane until confluent. Cells were treated with E2
approximately 16 h before hypoxia treatment, and then cultured in an air-lock
chamber containing 1% oxygen. TER was measured at 0, 4, 8, 12, 24, and 28 h after
the beginning of hypoxia treatment. The value of TER in the table is in kQ-cm2. The
result is a representative of at least four independent experiments.
4 9
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3.3 Etk-induced enhancement of TER in response to hypoxia involves regulation
of the actin cytoskeleton
One of the injurious effects of hypoxia on cells is the induction of actin
depolymerization (145,146). Since the ability of the tight junction to form a seal is
dependent on the actin filament organization (66), we investigated whether the
hypoxia-induced reduction in TER might be mimicked by disassembly of actin and
further, whether Etk activation could preserve TER under conditions of actin
filaments loss. Latrunculin B, an actin-depolymerizing agent (147), was utilized for
this purpose.
As shown in Fig. 4A, during 24-h treatment o f latrunculin B (48 nM), the TER
values of E2 -treated Pa-4 and Pa-4AEtk:ER cell monolayers decreased with time.
However, the measured TER values from E2-treated Pa-4AEtk:ER cell monolayers
were reproducibly and substantially higher than those of the parental cell monolayers
during the time of latrunculin B treatment. Similar observations were also made
when higher concentrations of latmnculin B (0.1 pM) was used. Furthermore, at 24-
h post-latrunculin B treatment, TER in Pa-4 cell monolayers exhibited more severe
decrease than Pa-4AEtk:ER monolayers. This observation is an extension of our
previous notion that proper actin filament organization is essential for the assembly
of functional barrier junctions. Moreover, Etk activation is capable of protecting Pa-
4 cells from actin filament depolymerizing agent like latmnculin B. The data
50
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presented in Figs. 3 and 4 together suggested that Etk activation might in some way
regulate the actin cytoskeletal elements involved in the formation of TJs and/or AJs
in response to pathophysiological perturbations from hypoxia and/or latrunculin B.
( A )
25
rT
20 ■
3
15 •
a
O ' 1.0 •
0.5 ■
0.0 ■
( B )
3.0 - I
a T
2.5 •
3 20 ■
a 1.5 ■
2
1.0 ■
H
0.5 -
0.0 -
48 nM □ Pa-4
□ Pa-4AEtk:ER
Tune (h)
□ Pa-4
□ Pa-4AEtk:ER
Time (h)
Fig. 4. Differential effect of latrunculin B on TER of Pa-4 and Pa-4AEtk:ER cell
monolayers. Pa-4 and Pa-4AEtk:ER cells were grown and treated with E2 as
described in Fig. 3. Latrunculin B (48 nM and 0.1 pM) was instilled into the culture
media 4 hrs prior to the first measurement. TER was measured at 4, 8,12, and 24 hrs
following the administration of latrunculin B. Error bars represent standard error
from the mean of four independent experiments, each contains triplicate samples.
51
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3.4 Increased TER following Etk activation is dependent on tyrosine kinase
activity
Etk is a non-receptor tyrosine kinase. To establish the mechanistic role of Etk
in the enhancement of TER in AEtk:ER-stably transfected cells, we utilized a widely
used tyrosine kinase inhibitor, genistein (Fig. 5). Treatment of genistein caused TER
decreases in Pa-4, Pa-4AEtk:ER, and E2-stimulated Pa-4AEtk:ER cell monolayers
over 24-h measurements. The rate and extent of genistein-induced TER decreases
were barely distinguishable between Pa-4 and Pa-4AEtk:ER monolayers without E2-
treatment. However, TER decrease in E2~activated Pa-4AEtk:ER monolayers in
response to genistein at both 1 pM and 50 pM was more pronounced than in those
without E2-activation. Moreover, genistein-elicited TER decreases in both Pa-4 and
Pa-4AEtk:ER cell monolayers, except for exposure to extremely high concentrations
(e.g. 200 pM) of genistein, were reversible after 24-h treatment (Table 2). Although
genistein is not a specific Etk inhibitor, our results demonstrated that the catalytic
activity of tyrosine kinase(s) is necessary to maintain TER in both Pa-4 and Pa-
4AEtk:ER cell monolayers. The observation that Pa-4AEtk:ER (+E2) cells are more
sensitive to genistein than the parental Pa-4 and Pa-4AEtk:ER (-E2) cells is also
consistent with our hypothesis that Etk activation enhances epithelial barrier function
and that Etk may be the critical tyrosine kinase involved in this signaling pathway.
52
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140
(A)
120
100
Pa-4
Pa-4AEtk:ER
Pa-4AEtk:ER + E 2
2
a
o
o
#
(B)
100 \y
H 80
Pa-4
Pa-4AEtk:ER
Pa-4AEtk:ER +E,
0 4 8 12
Time (h)
Fig. 5. Increased TER observed with Etk activation is diminished by
pretreatment with 1 pM (A) and 50 pM (B) genistein. Pa-4 and Pa-4AEtk:ER cell
monolayers were grown and treated with E2 as described in Fig. 3. TER was
measured at 4, 8, and 12 hrs following the administration of indicated concentrations
of genistein. The data represent percentage change as compared with TER obtained
at time 0, which is designated as 100%, normalized by the TER measured in
corresponding cells maintained in genistein-free cultures. Similar results were
obtained from 3 independent experiments; 1 representative result is shown. Values
are means ± SE calculated from triplicate data.
53
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Table 2
Tim e (h) Recovery
Genistein (m-M) 0 0.25 4 8 12 24 28
Control 0.78 1.005 1.12 1.45 0.955 0.29 0.56
1 0.91 1.125 1.41 1.74 1.185 0.33 0.68
Pa-4 50 0.72 0.835 0.84 0.86 0.685 0.35 0.62
100 0.71 0.805 0.62 0.25 0.125 0.31 0.55
200 0.73 0.785 0.29 0.16 0.035 0.02 0.04
Control 1.41 1.565 1.86 2.12 1.385 0.82 1.83
1 1.41 1.505 2 2.26 1.525 0.9 1.74
Pa-4AEtk:ER 50 1.34 1.505 1.26 0.58 0.645 0.83 1.37
100 1.26 1.485 0.4 0.06 0.065 0.57 0.91
200 1.12 1.035 0.11 0.06 0.035 0.03 0.03
Table 2. TER of Pa-4 and Pa-4AEtk:ER cell monolayers was able to recover
from a 24-h treatment of Genistein. Pa-4 and Pa-4AEtk:ER cell monolayers were
grown and treated with E2 as described in Fig. 3. TER was measured at 15 min, 4, 8,
12, 24, and 28 hrs following the administration of indicated concentrations of
genistein. The values in the table are TER in the unit of kQ-cm2. Similar results were
obtained from at least 4 independent experiments; 1 representative is shown.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
CHAPTER 4 DISCUSSION
The use of (3-estradiol (E2 ) inducible AEtk:ER chimera facilitates the study on
the signaling events specifically down-stream of Etk. It has been shown that
AEtk:ER can be rapidly activated upon the treatment of E2 , as assayed by its tyrosine
autophosphorylation in the in vitro kinase reaction (13). With proper control, it is
possible to avoid interference arising from using rather promiscuous extracellular
stimulators. AEtk.ER construct proves to be a useful approach to study the role of
Etk in cellular signaling.
Under normoxia, Etk activation increases the baseline level of TER in both Pa-4
and MDCK cell monolayers, suggesting a function in regulating the paracellular
junctions in epithelial cells (Fig. 1). This observation sheds a new light on Etk, as
that most of the studies up to date still focus on elucidating the signaling pathways of
Etk, and thus little is known about the physiological functions of this novel non
receptor tyrosine kinase. Genistein, a widely used tyrosine kinase inhibitor, was
employed to demonstrate the role of Etk tyrosine kinase activity in increasing TER
(Fig. 5). The result shows that genistein treatment induces the decrease of TER in
both Pa-4AEtk:ER and the parental cell monolayers, and furthermore, Pa-4AEtk:ER
cells are more sensitive to genistein. This suggests that tyrosine kinases and tyrosine
phosphorylation are important to the maintenance of TER and that Etk may well play
a very important role in regulating epithelial paracellular junctions.
55
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As TER has been generally accepted as a gauge of the integrity of paracellular
junctional complexes, the increase of TER upon Etk activation may imply an
enhancement of the functional assembly of junctional components. Although the
localization ofZO-1 and occludin, two important components of TJs, was not found
to be altered, however, Etk activation significantly increased occludin
phosphorylation as shown by protein mobility shift assay on the Western analysis
(148). Occludin has been a prime target for a number of signaling pathways involved
in the regulation of TJs, and the level of occludin tyrosine phosphorylation has been
reported to correlate with TER level (121,149). In these reports, increased occludin
tyrosine phosphorylation was shown to be associated with the reassembly of TJs
after ATP depletion and TJ restoration following “rescue” from Ras-mediated
transformation in MDCK cells. Nevertheless, it is still plausible that other
phosphorylation targets for Etk, in addition to occludin, may exist in the AJ and TJ
complexes. In addition, we had also shown that Etk activation elicited recruitment of
P-catenin and F-actin, two AJ components (F-actin is also associated with TJ), to the
cell periphery (148). Having known the inter-dependence between TJ and AJ, and
the importance of proper assembly of the junctional components, these evidences
suggest that Etk activation may increase TER by enhancing the functional assembly
of AJ and TJ.
It is worthwhile pointing out that, during the experiment, E2 treatment failed to
further increase the baseline TER of Pa-4AEtk:ER monolayers. According to the
56
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proposed model (150), this can be attributed to the leakage of Hsp90 inhibition on
AEtk:ER chimera in the absence of E2. The inability of E2 to further stimulate TER in
Pa-4AEtk:ER monolayers possibly reflects that the function of paracellular junctions
are so delicately regulated by Etk, and the decimal leakage is sufficient to manifest
such phenotype. Along with the evidences that several TJ and AJ components, such
as P-catenin, occludin, and F-actin, are regulated by Etk, it is rather unlikely that the
enhancement of TER in epithelial cells could be due to the over-expression of
AEtk:ER per se.
The literature on the role of tyrosine phosphorylation in TJ and AJ
assembly/disassembly is somewhat controversial and inconclusive. For example, as
epithelial cells reach confluence and undergo the process of contact inhibition,
tyrosine phosphorylation of catenins decreases. This observed decrease in tyrosine
phosphorylation in catenins is correlated with an increased association of tyrosine
phosphatase activity (151,152). Considerable circumstantial evidence also
implicates tyrosine phosphorylation in the disassembly of cadherin-mediated cell-cell
adhesion. Specifically, expression of constitutively activated v-Src oncoproteins,
which induce tyrosine phosphorylation of P- and pl20-catenins and E-cadherin,
leads to the loss or weakening of AJs (153,154). However, very little information is
available on the regulation of AJs by other tyrosine kinases such as Etk that may be
more involved in the modulation of cell function, rather than proliferation and
differentiation afforded by Src tyrosine kinase activation. As a matter of fact, we
have shown that although occludin and ZO-1 localization is not altered, however
57
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upon Etk activation, the phosphorylation of occludin is increased, and the functional
assembly of P-catenin and F-actin in the junctional area is increased in correlation
with TER increase (148).
The positioning of these junctions is coordinated and stabilized through an
association with a continuous band of bundled actin filaments, known as the
adhesion belt (155). However, it is unclear how the expression and function of each
TJ and/or AJ molecule(s) are regulated to confer the overall epithelial barrier
function. A profound Etk-induced reorganization of actin cytoskeleton into bundles
of peripherally localized filaments may influence TER, as has been previously
suggested (144,156). Because hypoxia has been known to induce actin cytoskeleton
depolymerization, therefore, actin filament depolymerizing agent, latrunculin B, is
used to mimic the hypoxia effect and to see whether Etk can prevent it. Etk
activation prevents latrunculin B- and hypoxia-induced TER decrease (Fig. 3, 4), and
the fragmentation of peripheral actin and P-catenin elicited by hypoxia (148). In
fact, we propose that reorganization and stabilization of actin filaments may be one
of the principal functions of Etk, whether or not additional direct regulation of AJ or
TJ components occurs. The proposal that Etk serves as a major regulator of actin
cytoskeleton is derived from the observations of (i) the dramatically improved barrier
function against hypoxic stress (Fig. 3); (ii) the observed reorganization of F-actin
and P-catenin by Etk (148); (iii) the blockage of effects on F-actin and P-catenin in
cells exposed to hypoxia (148) and (iv) the preservation of TER from latrunculin B-
treated Pa-4AEtk:ER cells (Fig. 4). Moreover, Btk, closely related to Etk, was
58
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shown to co-localize with actin filaments upon stimulation (14,37). In a separate
study, the activated Bmx-GFP was demonstrated to be localized in the membrane,
while the resting Bmx-GFP is restricted to the cytoplasm (29) further supported our
notion.
Several studies have explored the relationship between hypoxia and
disassembly of actin filaments. For instance, hypoxia induces dephosphorylation and
activation of ADF/cofilin, an actin regulatory protein that mediates cellular actin
dynamics (146). Cofilin dephosphorylation is associated with acceleration of actin
exchange on filament polymerization as well as loss of F-actin. Enhancing or
preserving actin depolymerization factor (ADF)/cofilin phosphorylation is therefore
one way of preserving cellular F-actin. Recent work has implicated two LIM kinases
in regulation of ADF/cofilin phosphorylation and actin dynamics: LIM-kinase 1 via a
Rac-mediated pathway (157,158) and LIM-kinase 2 via a Rho and/or Cdc42-
mediated pathway (159). If Etk-induced protection from hypoxic injury involves
prevention of ADF/cofilin-induced actin disassembly, this could occur either by
direct activation of LIM-kinases or indirectly through actions on Rho-, Rac- or
Cdc42-based signaling pathways. Etk may also act to alter effectors of actin
filaments assembly other than ADF/cofilin. The membrane association of activated
Etk also implicates the possibility that Etk is involved in Rho/Rac/Cdc42-mediated
signaling pathways.
5 9
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Based on our results, we hypothesize that the Etk activation in Pa-4 and also
possibly in MDCK cells may “prime” these cells against hypoxic injury. In
particular, Etk activation may upregulate the ATP-producing pathway(s) by
improving their stoichiometric efficiency via phosphorylation/activation modalities
or by inducing the expression o f genes for ATP-supplying glycolytic enzymes.
Alternatively, Etk activation may repress the activity or expression of the less
required enzymes or pathways that consume ATP. These working hypotheses are
consistent with our observations that (i) /eq was sustained under prolonged hypoxic
conditions in cells with activated Etk (Fig. 3B); (ii) the injurious effect on TER
elicited by latrunculin B was rapidly ameliorated by Etk activation (Fig. 4); and (iii)
the organization of actin-based cytoskeleton and the assembly of TJ were altered,
concomitantly with the augmentation of sealing function of TJs in the stimulated Pa-
4AEtk:ER cells (148).
In summary, this study provides the first evidence demonstrating that activated
Etk enhances TER under resting conditions, and sustains TER as well as maintains
barrier function under prolonged hypoxia. Based on the data presented, we envision
two potential signaling pathways by which Etk activation and downstream events
enhance and stabilize epithelial barrier function. As depicted in Fig. 6, we postulate
that Etk restores epithelial TER properties by preventing the disassembly of
intercellular tight junctions through a novel signaling pathway, even in hypoxic
insult. In the first pathway, tight junctional seal and TER are enhanced/maintained by
Etk activation via stabilization of the actin cytoskeleton. In the second pathway, in
60
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response to hypoxia, Etk acts directly on elements of TJs (eg. occludin) to enhance
TJ integrity. Specific mechanisms and target proteins involved in the possible
regulation of actin filaments and TJ by Etk activation remain to be future challenges.
Hypoxia
Etk
Actin
Latmnculin
Genistein
TER
Fig. 6 A putative model for protection against the injurious effect of prolonged
hypoxia on TER by Etk activation. A schematic presentation of possible roles of
Etk signaling in the enhancement of TER. As shown, at least two major pathways
are involved: Etk activation leads to actin polymerization and/or prevents the
disassembly of intercellular tight junctions under hypoxic conditions. The exact
molecular mechanism by which Etk enhances TER and the downstream effector(s)
of Etk remain to be established.
61
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Asset Metadata

Creator Chang, Hung-Kang (author)

Core Title Etk/Bmx activation modulates barrier function in epithelial cells

School Graduate School

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, cell,biology, molecular,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Ann, David (committee chair), Kim, Kwang-Jin (committee member), Shen, Wei-Chiang (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-290823

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Rights Chang, Hung-Kang

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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