The role of parathyroid hormone-related protein in regulating neonatal lung development

PDF

Download

Open document

Flip pages

Copy asset link

Request this asset

Request accessible transcript

Transcript (if available)

Content

THE ROLE OF PARATHYROID HORMONE-RELATED PROTEIN IN
REGULATING NEONATAL LUNG DEVELOPMENT

by
Wei Deng

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
(CRANIOFACIAL BIOLOGY)

August 2009

Copyright 2009 Wei Deng

DEDICATION
This dissertation is dedicated to my loving parents for raising me to who I am today, for
believing and having deep faith in everything I do, for always supporting me and
boosting my confidence.

ii

ACKNOWLEDGEMENTS
From the starting stages to the final draft, I owe an immense debt of gratitude to my
advisor Dr. Michael Paine. This thesis would have never finished without his continuous
support. I am thankful for his continuous patience in teaching me and guiding me. He is
always there for all the help. I respect him as a great mentor and whom I can always look
up to.
I also want to thank Dr. Wei Shi and Dr. David Warburton for teaching the basics of
many techniques and for guiding me through my MS study. I am grateful to them for
accepting me in their lab. I want to thank our research collaborators Dr. John Torday and
Dr. Virender Rehan for their continuous advices on my research work and for helping me
prepare the publication. I want to specially thank Dr. Malcolm Snead for all the mistakes
he had to correct over and over in editing my thesis, for being such a great professor and
role model to me and for his positive impact on being a professional scientist. I want to
thank Marcelo Freire and Nikhil Chopra for providing the tremendous help on
constructing the thesis, for being such great friends and for supporting me during the
hardest time in my life. Without them, there will never be this thesis. I want to thank my
lab-members for being such a fun lab to work with and from whom I learned a lot of
technical stuffs. I also want to thank other administrative staffs and colleagues, for all
their time and effort in reviewing my progress.
Last but not the least, I want to thank my family for all the love and support they have
given me throughout my life, without them nothing is possible.
iii

TABLE OF CONTENTS
Dedication ii
Acknowledgements iii
List of Figures vi
Abstract vii
CHAPTER I: INTRODUCTION
Mammal lung development 1
Lung myofibroblasts and bronchopulmonary dysplasia disease 4
Parathyroid hormone-related protein 5
PTHrP signal transduction pathways 6

CHAPTER II: MATERIALS AND METHODS
Mouse strains and breeding 11
Histology and morphometric analysis 13
Tissue total RNA isolation and reverse transcription 14
Real-time PCR analysis 14
Immunohistochemistry 15
Cell proliferation 15
Tissue lysis and subcellular fractionation 16
Western blot 16
Data presentation and statistical analysis 17

CHAPTER III: RESULTS AND DISCUSSION
Conditional abrogation of PTHrP in mouse lung mesenchyme 18
The growth retardation and gross abnormalities in mesenchymal-specific 20
PTHrP knock-out mice
Conditional abrogation of PTHrP in lung epithelia does not affect lung 23
development
Failure of alveogenesis and severe bronchopulmonary dysplasia-like 26
changes in mesenchymal-specific PTHrP knock-out mice
Significant reduction of cell proliferation with dysregulation of cell 31
differentiation in mesenchymal-specific PTHrP knock-out mouse lungs
Loss of lung myofibroblasts in mesenchymal-specific PTHrP knock-out 33
mouse lungs

iv

Excessive and disordered elastin accumulated in mesenchymal-specific 38
PTHrP knock-out mouse lungs
Intracrine PTHrP regulates cell proliferation 40
Discussion 41
Conclusion 46

BIBLIOGRAPHY 47

v

LIST OF FIGURES

Figure 1: Overview of the lung development 3
Figure 2: The gene structure of mouse PTHrP 7
Figure 3: The phenotype of PTHrP

null mice 10
Figure 4: The breeding scheme 12
Figure 5: Lung mesenchymal-specific expression of Dermo1-Cre 19
Figure 6: The lethality rate and genotyping of PTHrP
Mesen-CKO
mice 21
Figure 7: The growth retardation and gross organ abnormalities of PTHrP
Mesen-CKO
22
mice at P6 or P3
Figure 8: The efficiency of the mesenchymal-specific PTHrP conditional knock-out 24
Figure 9: Lung epithelial-specific PTHrP mouse conditional knock-out 25
Figure 10: Phenotypes of mouse lung mesenchymal-specific PTHrP conditional 27
knock-out at P6
Figure 11: Changes in morphology and morphometric measurement of lung 30
mesenchymal-specific PTHrP conditional knock-out mouse lung
Figure 12: Abrogation of PTHrP in lung mesenchymal cells resulted in decreased 32
cell proliferation
Figure 13: Gene expression level of selected molecular markers of differentiated 34
lung cells in PTHrP
Mesen-CKO
mice
Figure 14: Protein expression level of selected molecular markers of differentiated 37
lung cells in wild-type littermates and PTHrP
Mesen-CKO
mice at
postnatal day6
Figure 15: Elastin staining of lung tissue at postnatal 6 days 39
Figure 16: The nuclear expression of PTHrP protein 42

vi

ABSTRACT
Parathyroid hormone-related protein (PTHrP) plays important roles in regulating a
variety of developmental processes in many organs, through paracrine, autocrine and
intracrine pathways. Conventional knockout of PTHrP in mice results in neonatal lethal
with multiple defects including asphyxia, skeletal deformities and osteochondrodysplasia.
In the present study, PTHrP conditional knockout in mouse lung epithelia versus
mesenchyme has been generated. Abrogation of PTHrP specifically in lung mesenchyme
results in a neonatal lethal due to lung alveolarization arrest and respiratory failure
accompanied with diminished cell proliferation, loss of myofibroblasts and increased
elastin accumulation. These manifestations are similar to those observed in
bronchopulmonary dysplasia. No abnormality has been found in PTHrP epithelial-
specific knock-out mice. Therefore, we conclude that PTHrP expression in lung
mesenchyme, but not in epithelia, is essential for postnatal lung development, possibly
through regulating cell proliferation, especially lung myofibroblasts, by an intracrine
pathway. The fundamental knowledge obtained from this study will help understanding
pathogenic mechanisms of neonatal lung disease such as bronchopulmonary dysplasia,
and may also provide clues for designing novel therapeutic strategies.

vii

CHAPTER 1: INTRODUCTION
1. Mammal lung development
Mammalian lung development is initiated by the formation of a pair of primary epithelial
buds that evaginate from the laryngo-tracheal groove in the ventral surface of the
primitive foregut endoderm into the surrounding splanchnic mesenchyme
(1, 2)
. The
respiratory tree then develops by branching morphogenesis, in which reiterated
outgrowth, elongation and subdivision of epithelial buds occurs, followed later by
alveolarization to form a large gas-exchange surface
(3, 4)
. Disruption of normal lung
developmental process can result in neonatal respiratory failure or distress if lung
formation is severely affected, or susceptibility to lung disease during later life if milder
changes occur in the developing lung
(9)
.
Since the lung developmental process is quite well conserved among species, mouse lung
development is an ideal model for studying the mechanism of lung organogenesis and
congenital respiratory diseases in humans. In mouse, lung development begins at
embryonic day (E) 9.5, and is divided histologically into 4 stages (Fig 1): 1)
Pseudoglandular stage (5-17 weeks of human pregnancy), E9.5-16.6 in mouse embryo:
the earliest stage. The embryonic lung undergoes rapid dichotomous branching,
developing epithelial tubular structures with lining cuboidal epithelial cells that resemble
an exocrine gland. However, this fluid containing respiratory tree structure is too
immature to perform gas exchange. 2) Canalicular stage (16-25 weeks of human
pregnancy), E16.6-17.4 in mouse embryo: The respiratory tree is further expanded in
1

diameter and length, accompanied by vascularization and angiogenenesis along the
airway. A massive increase in the number of capillaries occurs. The terminal bronchioles
are then divided into respiratory bronchioles and alveolar ducts, and the airway epithelial
cells are differentiated into peripheral squamous cells and proximal cuboidal cells. 3)
Terminal sac stage (24 weeks to late fetal period in human), E17.4-P5 in mouse: There is
the substantial thinning of the interstitium in this stage. The alveolar epithelial cells are
more clearly differentiated into mature squamous type I cells and type II epithelial cells.
The capillaries also grow rapidly in the mesenchyme surrounding the alveoli to form a
complex double network. Towards the end of this stage, the fetal lung supports relatively
inefficient air-exchange, but sufficient to maintain the life of prematurely born neonates.
Although human premature infants can breathe with the lung that has developed to the
end of terminal sac stage, the immature lung is nevertheless vulnerable to hyperoxic
injury and barotraumas, resulting in the alveolar hypoplasia phenotypes termed
bronchopulmonary dysplasia. Maturation of surfactant synthesis and secretion is a key
factor in determining whether the newborn lung can sustain gas exchange without
collapsing. Another key factor is the rapid switch from chloride ion driven fluid secretion
into the airway to sodium driven uptake of fluid out of the airway. This switch is driven
by the response of the adrenergic system to the umbilical cutting at birth. 4) Alveolar
stage (late fetal period to childhood in human), P5-P30 in mouse: Alveolarization is the
last step of lung development. Forming new septa within terminal sacs is the key step for
differentiation of the saccules into alveoli. This involves a complex interaction between
myofibroblasts that produce elastin matrix within the mesenchyme, adjacent airway
2

Fig 1. Overview of the lung development. Murine lung development begins at embryonic
day (E) 9.5, and is divided histologically into 4 stages: 1) Pseudoglandular stage (5-17
weeks of human pregnancy), E9.5-16.6 in mouse embryo; 2) Canalicular stage (16-25
weeks of human pregnancy), E16.6-17.4 in mouse embryo; 3) Terminal sac stage (24
weeks to late fetal period in human), E17.4-P5 in mouse; and 4) Alveolar stage (late fetal
period to childhood in human), P5-P30 in mouse. (Adapted from Human Embryology.
www.embryology.ch)

3
Mouse
gestation
days
Birth

epithelial cells and vascular endothelial cells. Controlled multiplication and
differentiation as well as migration of the myofibroblast progenitor cells within terminal
sac walls are therefore important for formation of new alveolar septa
(10)
.
2. Lung myofibroblasts and bronchopulmonary dysplasia disease.
Lung myofibroblasts are interstitial contractile cells that express α-smooth muscle actin
(α-SMA) and are found in the alveolar interstitium during lung development. Lung
myofibroblasts have morphologic and biochemical features intermediate between
fibroblasts and smooth muscle cells
(11)
. Although smooth muscle cells that both encircle
the airways and vasculature and lung myofibroblasts arise from lung mesenchyme and
express α-SMA, it is postulated that they are not from the same origin, for the depletion
of platelet-derived growth factor A (PDGF-A
-/-
) affects only lung myofibroblasts but not
smooth muscle cells
(30, 31)
.
Lung myofibroblasts are essential for secondary septation and elastin deposition in
alveogenesis. During this process, lung myofibroblasts were directed to specific sites in
the existing primary septa (the walls of the terminal air sac), followed by the subsequent
protrusion and extension of secondary septa that form alveoli
(29)
. Mice deficient in
platelet-derived growth factor A (PDGF-A
-/-
) lack lung myofibroblasts and septal elastin
deposit and developed an emphysema-like morphology as alveolar septa failing to form
(30, 31)
. Rishikof et al., using an elastase model of emphysema in mice, speculated that the
damaged areas of the lung represent a reactivation of the myofibroblast proliferation and
increased α-SMA content normally associated with postnatal alveolar septation
(12)
.
4

Torday and Rehan
(13)
suggested that the fibroblast-to-myofibroblast differentiation
associated with bronchopulmonary dysplasia pathogenesis in newborns, and with chronic
lung disease in adults, represents an ontogenetic recapitulation of myofibroblast
development.
Bronchopulmonary dysplasia (BPD) is a form of chronic lung disease that occurs in
infants, usually in preterm infants (<28 weeks pregnancy) with very low birth weight (<
1000 g). In recent years, the radiographic and pathological features of BPD have changed
considerably due to the major advances in perinatal care, including widespread use of
antenatal glucocorticoid therapy, postnatal surfactant replacement and improved
respiratory and nutritional support. The new BPD is characterized by the simplified distal
lung acinus, fewer and larger alveoli, arrested secondary septa formation, variable airway
smooth muscle hyperplasia, progressive interstitial fibrosis, especially excessive and
disordered elastin accumulation, and fewer and dysmorphic capillaries
(14)
. The
molecular mechanism involved in the pathogenesis of BPD remains unclear. However,
the PTHrP level in the tracheal aspiration of BPD infants is reported significantly lower
than those normal infants. The author stated that a PTHrP level < 1.32 pg/mg protein
could predict the later development of BPD maximally [86.4% correct classification (true
positives + true negatives)], with a sensitivity = 76.9% and specificity = 88.5%
(15)
,
suggesting the strong correlation between PTHrP level and BPD disease.
3. Parathyroid hormone-related protein (PTHrP)
PTHrP was discovered as a result of a search for the circulating factor secreted by
5

cancers which causes the common paraneoplasitc syndrome humoral hypercalcemia of
malignancy
(16)
. Since the identification of the peptide in 1982 and the cloning of the
cDNA in 1987, it has become clear that PTHrP is a prohormone that is post-
translationally cleaved by prohormone convertases to yield a complex family of peptides.
It is also clear that the PTHrP gene is expressed not only in cancers but also in vast
majority of normal tissues during adult and/or fetal life. The tremendous conservation of
the PTHrP nucleotide and amino acid sequences among species (human, rat, mouse,
chicken and etc.) suggested that PTHrP must play an important role in cellular and
developmental biology
(17)
.
In human, the PTHrP gene is located on the short arm of chromosome 12, compromises 8
exons, uses at least three promoters and, by alternate splicing, gives rise to three major
isoforms of the mature peptide (139, 141, and 173 amino acids)
(17)
. In mouse, PTHrP is
located on the short arm of chromosome 6 and consists of 5 exons (Fig 2). Its exon4
encodes the start codon and most of the protein. Only one form of mature PTHrP protein
(139 amino acids) exists in mouse, rat or rabbit
(33)
.
4. PTHrP signal transduction pathways
PTHrP possesses distinct paracrine /autocrine and intracrine signaling pathways
(17)
: (1)
Paracrine/autocrine pathway. Similarity of the N-terminus (1-34) of PTHrP to that of
parathyroid hormone (PTH), the major hormone regulator of calcium and phosphorus
homeostasis, enables PTHrP to share the signaling properties of PTH by interacting with
the common PTH/PTHrP or PTH type 1 receptor, a member of the G-protein-coupled
6

Fig 2. The gene structure of mouse PTHrP. Mouse PTHrP gene comprises 5 exons.
Exon4 encodes the start codon and the most of the protein. Only one form of mature
PTHrP (139 amino acids) exists in mouse, rat or rabbit. (Coding exons are shown as solid
boxes. The hatched boxes denote the pre-pro peptide and open boxes, the 5’ and 3’ non-
coding regions)
(33)
.

7
1 2 3 4 5
-36 +1 139aa

receptor family B. Previous studies have shown that PTHrP 1-36 and other N terminal
PTHrP species bind to the classical PTH receptor 1 in osteoblasts, renal tubular cells and
smooth muscle cells to activate the adenylyl cyclase/protein kinase A pathway and / or
the cytosolic calcium/inositol phosphate/protein kinase C pathway
(5, 6, 7)
. (2) Intracrine
pathway. In vivo and in vitro studies have indicated that PTHrP displays other functions
largely relating to an intracrine signaling role in the nucleus/nucleolus in regulating
apoptosis and cell proliferation
(34, 35, 36, 26)
. This nuclear translocation requires the mid-
region (87-107) of the mature PTHrP protein
(34)
. Recent advances have shown that the
intracellularly expressed PTHrP is able to shuttle in cell-cycle- and signal-dependent
fashion between the nucleus and cytoplasm through the action of the distinct intracellular
transport receptors (importin beta 1, exportin 1), mediating nuclear import and export of
PTHrP respectively
(38)
. In addition, PTHrP can be targeted to the nucleus in vascular
smooth muscle cells. Nuclear targeting is associated with a striking increase in
proliferation, which is the diametric opposite effect of PTHrP resulting from the
interaction with cell surface receptors on vascular smooth muscle cells
(26)
. Furthermore,
studies have shown that the mice missing the PTHrP nuclear translocation region and the
C-terminal region can still signal through the PTHrP paracrine/autocrine pathway but
lose the PTHrP intracrine pathway; and the mutant showed retarded growth, early
senescence, postnatal demise, reduced cell proliferation and increased cell apoptosis with
decreased expression of Cyclin D, Cdk4/6, pRb and Bmi-1. It suggests that PTHrP
intracrine pathway may signal through pRb phosphorylation and G1/S checkpoint release
to promote cell cycle progression
(8)
.
8

PTHrP signaling plays a key role in various organs and tissues development. Null
mutation of PTHrP mice results in the neonatal lethal with multiple defects including
asphyxia, skeletal deformities (a domed skull, short snout and mandible, protruding
tongue, narrow throax and disproportionately short limbs) and osteochondrodysplasia
(Fig 3). The histological examination showed a diminution of chondrocyte proliferation,
associated with premature maturation of chondrocytes and accelerated bone formation
(18)
.
Furthermore, PTHrP null mice have reduced surfactant formation and arrested type II
epithelial cell formation
(37)
. However, the lung is a complex organ, so global alteration of
PTHrP level may affect PTHrP signaling activities differently in either lung epithelium or
mesenchyme, or indeed both, by changing autocrine and/ or paracrine signaling activities,
which may be difficult to distinguish.
Thus, in the present study, endogenous PTHrP was selectively abrogated in either
mesenchymal cells or lung epithelial cells of the developing mouse lung using Cre/ loxP
conditional knock-out approaches, and it was found that PTHrP signaling plays important
and distinct roles in mesenchymal cells versus lung epithelial cells to differentially
control neonatal mouse lung development.
This study will be the first one to investigate the role of PTHrP in different compartment
(mesenchyme versus epithelia) during lung development. The fundamental knowledge
obtained from this study will help understanding pathogenic mechanisms of neonatal
lung disease, and may also provide clue for designing novel therapeutic strategies.

9

A. B.

C.

Fig 3. The phenotype of PTHrP

null mice. A. The PTHrP
-/-
mice died within 2 hours
after birth due to the asphyxia, and exhibited the chondrodysplastic phenotype
characterized by a domed skull, short snout and mandible, protruding tongue, narrow
throax and disproportionately short limbs. B. hindlimbs, note the striking paucity of
cartilaginous (nonstaining) components in PTHrP
-/-
mice. C. Lateral view of the skull
showing the shortened and broadened mandible and doming of the mutant skull
(18)
. (fe,
femur; fi, fibula; ti, tibia; so, supraoccipital bone; eo, exoccipital bone; tb, tympanic bulla;
hc, hyoid bone)

10
WT KO WT KO
WT KO

CHAPTER 2: MATERIALS AND METHODS
1. Mouse strains and breeding
Floxed PTHrP (PTHrP
fx/fx
) mice were generated in the laboratory of A.C. Karaplis, as
previously described
(20)
. In PTHrP
fx/fx
, exon 4 of PTHrP gene, which encodes most of
the protein (Fig 2, Fig 4), was flanked with two loxP DNA elements. Deletion of exon 4
causes frameshift and eliminates functional PTHrP protein expression. Mesoderm-
specific Dermo1-Cre heterozygous knock-in mice (Dermo1-Cre
+/-)
were generated and
kindly provided by D.M. Ornitz
(22)
. Our lab has previously confirmed that Dermo1-
driven Cre expression is specifically localized in developing lung mesenchymal cells (Fig
4)
(19)
. Inducible lung epithelial-specific Cre transgenic mice (SPC-reverse tetracycline
transactivator (rtTA)/TetOCre) were generated and provided by J.A. Whitsett
(21)
.
Timed mating between PTHrP
fx/fx
and PTHrP
fx/+
/Dermo1-Cre
+
mice generated
mesoderm-specific PTHrP conditional knockout (Mesen-CKO) mice (PTHrP
fx/fx
/Dermo1-Cre
+
), heterozygous PTHrP knockout mice (PTHrP
fx/+
/Dermo1-Cre
+
) and
control mice (PTHrP
fx/+
or PTHrP
fx/fx
) (Fig 4). Lung development in the control mice
was the same as in wild-type mice (PTHrP
+/+
).
Timed mating between PTHrP
fx/fx
and PTHrP
-/+
/SPC-rtTA/TetO-Cre mice generated
lung epithelial-specific PTHrP conditional knock-out (Epi-CKO) mice (PTHrP
-/fx
/SPC-
rtTA/TetO-Cre), heterozygous PTHrP knock-out mice (PTHrP
fx/+
/SPC-rtTA/TetO-Cre)
and control mice (PTHrP
fx/+
, PTHrP
-/fx
, PTHrP
fx/+
/SPC-rtTA, PTHrP
fx/+
/TetO-Cre,
PTHrP
-/fx
/SPC-rtTA or PTHrP
-/fx
/TetO-Cre ) when the inducing agent doxycycline (Dox)
11

X
PTHrP
fx/+
/Dermo1-Cre
+
PTHrP
fx/fx

PTHrP
-/+
/SPCrtTA/TetOCre PTHrP
fx/fx

X

PTHrP
fx/+
/Dermo1-Cre
+
PTHrP
fx/fx
/Dermo1-Cre
+
PTHrP
fx/fx

A. Or PTHrP
fx/+

B.

C. PTHrP
-/fx
/SPCrtTA/TetOCre

Fig 4. (A) The breeding scheme for generating mesenchymal-specific PTHrP knock-out
mice. The odds of obtaining PTHrP
Mesen-CKO
mice are 25%. (B) Schematic diagram of
PTHrP genomic structure in the genetically manipulated mice. (C) Breeding scheme for
generating epithelial-specific PTHrP knock-out mice.
12

Dox from E6.5-P30

was present (Fig 4). Normal lung development in the control mice was the same as in
wild-type mice (PTHrP
+/+
). Administration of Dox started from early embryonic stage
(E6.5) to the end-point of the lung maturation (P30) by feeding the pregnant or young
mice with 625 gm/kg Dox in food (TestDiet, Richmond, IN, USA) and 0.5 mg/ml in
drinking water (Sigma-Aldrich Co., St Louis, MO, USA) (Fig 4).
Mice used in the study were housed in pathogen-free conditions according to the protocol
approved by the Institutional Animal Care and Use Committee at the Saban Research
Institute of Children’s Hospital (Los Angeles, CA, USA).
2. Histology and morphometric analysis
The mouse lungs were inflated with 4% buffered paraformaldehyde fixation solution by
cannulating the trachea under a fixed water pressure (25 cm for adult and 20 cm for
neonates), and the excised lungs with ligated trachea were submerged in fixative
overnight at 4 ˚C, followed by dehydration and paraffin embedding. Tissue sections with
5µm thickness were prepared and stained with haematoxylin and eosin (H&E) for
histological examination.
Elastin was stained using Hart’s resorcin-fuchsin solution, and counterstained with 0.5%
tartrazine.
For morphometric analysis, five sections from the same lobes of each sample were
randomly chosen at ~250- µm intervals and stained with H&E. The mean linear intercept
(MLI) was then measured according to established methods
(24, 25)
. Briefly, an image of
13

each section examined was digitally captured at 40X magnification. The horizontal
andvertical lines at ~0.9-mm intervals within a rectangular grid were then used to count
alveolar surface intersections using ImagePro software. The MLI was then calculated as
the sum of the length of all counting lines divided by the total number of counted
intercepts of alveolar septa. Results were analyzed with unpaired t-tests to compare the
differences between mean values, and considered significant if p<0.05. In order to avoid
the sex differences in fetal lung maturation, this quantitative comparison was performed
among fetuses with the same sex at each time-point
(27)
.
3. Tissue total RNA isolation and reverse transcription
The total RNA was isolated from snap-frozen lung tissues using a Qiagen RNeasy kit
(Qiagen, Sata Clarita, CA), followed by DNase treatment to remove any potentially
contaminated genomic DNA. The quality of isolated RNA was checked by formaldehyde
agrose gel electrophoresis before the reverse transcription (RT) reaction. One microgram
of total RNA was added to the RT reaction mixture (iScript; Bio-Rad Laboratories,
Hercules, CA) and incubated at 25˚C for 5 min, 42˚C for 45 min and 85˚C for 5 min. The
product of RT was diluted 5, 25 or 125-fold and applied to polymerase chain reaction
(PCR) analysis.
4. Real-time PCR analysis
Real-time quantitative PCR analysis was performed on iCycler-iQ system (Bio-Rad) by
SYBR Green I dye detection. The reactions were assembled following manufacturer’s
recommendation. Briefly, 25µl of reaction mixture contains 1X iQ SYBR Green
14

Supermix (Bio-Rad), 200nM forward and reverse primers, and the cDNA template from
the RT reaction sample. The PCR conditions are 3 min at 95˚C followed by 40 cycles of
30 s at 95˚C, 30 s at annealing temperature, and 30 s at 72˚C. The specificity of PCR was
verified by measuring the melting curve of the PCR product in the end of reaction.
Fluorescent data were specified for collection during the 72˚C step. The relative cDNA
ratio was calculated by using ∆∆Ct
(28)
. GAPDH was used as reference control to
normalize equal loading of template cDNA.
5. Immunohistochemistry
The following antibodies were used in the present study: CCSP (WRAB-CCSP) and SPC
(WRAB-SPC) antibodies from Seven Hills Bioreagents (Cincinnati, OH, USA);
Aquaporin (AQP) 5 goat polyclonal antibodies (sc-9890) from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA, USA); α-smooth muscle actin (SMA) (A2547) antibody from
Sigma-Aldrich Co.; and PECAM-1 (550274) antibody from BD Biosciences.
Immunohistochemical staining was performed using the HistoStain kit from Zymed
Laboratories, Inc. (South San Francisco, CA, USA), according to the manufacturer’s
instruction. Either 3-amino-9-ethylcarbazole or 3, 3’-diaminobenzidine was used as
chromogenic substrate.
6. Cell proliferation
Cell proliferation was analyzed by proliferating cell nuclear antigen (PCNA) staining
using a PCNA staining kit from Zymed Laboratories, Inc. (South San Francisco, CA).
15

Under microscope with magnification X40, five fields from the same section of each
sample will be randomly chosen. PCNA-positive cells and total cells in each field were
counted. The PCNA index was calculated as the percentage of the PCNA- positive cells
out of the total cells from the same field. Unpaired t-tests were used for comparison of
statistical difference and p-values<0.05 were considered to be significant.
7. Tissue lysis and subcellular fractionation
Fresh wild-type lung tissues at P6 were collected and processed using the Nuclear extract
kit from Active Motif (Carlsbad, CA, USA). Briefly, lung tissues were lyzed in ice-cold,
low detergent, Hypotonic buffer containing a mixture of protease inhibitors, including
phosphatase inhibitor, followed by the disruption with a Dounce homogenizer (tight
pestle). Lysates were centrifuged at 850 X g at 4 ˚C and supernatants constituting crude
cytoplasmic protein fractions (Cyt) were collected. Pellets, which are crude nuclei
preparations, were resuspended in Complete lysis buffer with protease inhibitors,
including phosphatase inhibitor. Nuclei suspensions then were subjected to centrifugation
at 14,000 X g at 4 ˚ C and nuclei protein fraction (Nu) were collected. As for whole-tissue
extract (W), they were prepared by lyzing and homogenizing fresh wild-type lung tissue
in Complete lysis buffer with protease inhibitors, including phosphatase inhibitor. All
protein concentrations were estimated using the Bio-Rad Laboratories, Inc. Bradford
Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA).
8. Western blot
Equal amounts (40 µg) of total tissue lysate proteins were separated in NuPAGE 4-12%
16

gradient SDS-PAGE gels using a MOPS or MES buffering system (Invitrogen, Carlsbad,
CA, USA). After protein was transferred onto polyvinylidene difluoride membrane
(Millipore), incubated with related primary antibody and detected by the enhanced
chemiluminescence method. Antibodies for PTHrP (YII-Y201-EX) were purchased from
Cosmo Bio Co. (Carlsbad, CA, USA), and anti-glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was obtained from Research Diagnostics, Inc. (Flanders, NJ,
USA).
9. Data presentation and statistical analysis
At least three pairs of PTHrP conditional knock-out (CKO) and wild-type or normal
control littermate mice from different dams were analyzed in each experimental subgroup.
All quantitative data were expressed as mean ± SD. Unpaired t-tests were used for
comparison of statistical difference and p-values<0.05 were considered to be significant.

17

CHAPTER 3: RESULTS AND DISCUSSION

1. Conditional abrogation of PTHrP in mouse lung mesenchyme
Mice with the conventional PTHrP null mutation (PTHrP
-/-
) were born alive but die soon
within 2 hours after birth due to the asphyxia and widespread skeletal deformities (Fig 3)
which was resulted by diminished proliferation and accelerated differentiation of
chondrocytes in the developing endochondral skeleton
(18)
. However, the perinatal
lethality of the conventional PTHrP knock-out mouse model precludes observation of
potential postnatal tissue-specific alterations arising in the complete absence of PTHrP.
Therefore, to circumvent these limitations, we generated the PTHrP conditional knockout
mice by using a Cre-loxP system for this in vivo study.
The floxed PTHrP mice were generated by two loxP sites flanking exon4 of PTHrP gene
(Fig 2), which encodes most of the protein
(20)
. The mesenchyme-specific Dermo1-Cre
knock-in mice were generated by introduction of Cre recombinase into Dermo1, a
transcription factor highly expressed in mesenchymal cells during embryogenesis
(21)
.
The expression pattern of Cre in Dermol-Cre mice has been verified by crossing
Dermo1-Cre mice with Rosa26R mice in which only cells with Cre-medicated loxP DNA
recombination expressed LacZ
19
. The whole-mount sagittal section from LacZ stained
embryonic day (E) 14.5 detected the Cre activity in multiple tissues, including the lung
mesenchyme, but not in the lung epithelia (Fig 5)
19
.

18

Fig 5. Lung mesenchymal-specific expression of Dermo1-Cre. Lung mesenchymal-
specific expression of Cre in Dermo-Cre mice was verified by crossing Dermol-Cre and
Rosa26R mice, followed by LacZ staining on E14.5. The whole-mount sagittal section
revealed the Cre activity in multiple tissues, including the lung mesenchyme, but not in
epithelial cells
19
.
19

PTHrP
fx/fx
mice were then crossed with Dermo1-Cre mice to generate PTHrP
mesenchymal-specific conditional knock-out (PTHrP
Mesen-CKO
) mice, as shown by their
genotypes (Fig 6). We found that heterozygous mice (PTHrP
fx/+
/Dermo1-Cre
+
) appears
normal, whereas homozygous conditional knock-out mice (PTHrP
fx/fx
/Dermo1-Cre
+
)
exhibited 75% lethality at P7-P9 due to the respiratory failure (Fig 6).
2. The growth retardation and gross abnormalities in mesenchymal-specific PTHrP
knock-out mice
We observed the phenotype of the postnatal PTHrP
Mesen-CKO
mice. At birth, PTHrP
Mesen-
CKO
mice were similar in weight and size to their wild-type littermates. There was no
distinguishable difference for milk-intake and feeding. However, by three days
postpartum, PTHrP
Mesen-CKO
mice failed to grow relative to their wild-type littermates
and died at P7-P9 due to the respiratory failure (Fig 7). From P3 to P6, the PTHrP
Mesen-
CKO
mice exhibited marked phenotypic changes consistent to the original PTHrP
-/-
mice,
including an unstable gait, sluggish movement and slow reaction, cachexia, skeletal
dwarfism, domed skull, shortened snout and mandible, osteopenia with kyphosis (Fig 4).
The autopsy of the P6 PTHrP
Mesen-CKO
mice showed thinner skin, a profound decrease in
fat deposition and severe hypoxemia (data not shown). These non-pulmonary
abnormalities might be due to the disrupted PTHrP signaling in other key mesoderm-
derived tissues.

20

PTHrP+/+
PTHrP fx
PTHrP del/+
PTHrP del/del
A.
Litter Litter size PTHrP
Mesen-CKO
Lethality
#1 8 pups 2 KO P7.
P90
#2 6 pups 1 KO P9
#3 8 pups 1 KO P7
#4 6 pups 1 KO P7
#5 9 pups 2 KO P7
P60
#6 8 pups 1 KO P9
N= 8 6 died @ P7-P9
B.

Fig 6. (A) The lethality rate of PTHrP
Mesen-CKO
mice. We observed 6 litters and obtained
eight PTHrP
Mesen-CKO
mice, in which six died between P7-P9 and other two survived till
the 3
rd
and 2
nd
month respectively. Thus the lethality rate of PTHrP
Mesen-CKO
at P7 to P9
is 75%. (B) PCR genotypes of tail genomic DNA.

21

Dermol-Cre
+

PTHrP
fx
PTHrP
+

P6WT P6CKO P3WT P3CKO

A. B.
C.
Fig 7. The growth retardation and gross organ abnormalities of PTHrP
Mesen-CKO
mice at
P6 (A) or P3 (B). At P3, PTHrP
Mesen-CKO
mice failed to grow relative to their littermate
and died by P7-P9 due to the respiratory failure. At P6, PTHrP
Mesen-CKO
mice exhibited
significant phenotype, including the unstable gait, sluggish movement and slow reaction,
cachexia, skeletal dwarfism, domed skull, shortened snout and mandible with kyphosis.
(C) The growth parameters of wild-type versus PTHrP
Mesen-CKO
on P6. All values are
recorded as Mean ± SD; n=4 for wild-type or n=3 for PTHrP
Mesen-CKO
. Statistical
comparisons between groups were performed using unpaired t-tests and indicated the
differences of wild-type versus PTHrP
Mesen-CKO
. wLW, wet lung weight. wHW, wet
heart weight. BW, body weight. NS, not significant. WT, wild-type mice. KO, PTHrP
Mesen-CKO
mice. 22
Parameter Wild type PTHrP
Mesen-CKO
Significance
Body weight (mg) 338.3±0.28 171.9±0.23 P<0.001
Wet lung weight (mg) 95.5±0.1 44.5±0.1 P<0.001
Wet heart weight (mg) 31.0±0.1 14.5±0.1 P<0.001
wLW/BW 0.3±0.1 0.28±0.1 P<0.001
wHW/BW 0.9±0.1 0.9±0.1 NS

We next assessed the growth parameters on P6 PTHrP
Mesen-CKO
mice. We found that
more than a 50% reduction in the body weight, lung weight and heart weight (p<0.001).
Interestingly, a significant reduction of lung weight/body weight (p<0.001) was also
found on PTHrP
Mesen-CKO
mice, but not that of heart weight/body weight, suggesting the
loss of the mass in lungs from PTHrP
Mesen-CKO
mice (Fig 7). The efficiency of Cre
recombination on PTHrP
Mesen-CKO
mice was confirmed by genomic DNA PCR and
Western blotting using P6 wild-type, PTHrP
fx/fx
or PTHrP
Mesen-CKO
mice whole lung
tissue (Fig 8). The PCR primers sequences are P1: 5’-AGT CCT GCC TCA GTC TTC
TTG CC-3’ and P2: 5’-GAA ATG CTT ATA ATC CCA GCA TCT GAG-3’.
3. Conditional abrogation of PTHrP in lung epithelia does not affect lung
development.
Lung epithelium-specific PTHrP conditional knock-out (PTHrP
Epi-CKO
) mice were
generated by crossing PTHrP
-/fx
mice with SPC-rtTA/ TetO-Cre transgenic mice, in
which Cre expression was induced in airway epithelial cells of the whole lung and distal
bronchus by a lung epithelium specific SPC promoter-driven rtTA transgene, in
combination with the inducing agent Dox given prior to lung formation (at E6.5) and
until the end of the lung maturation at P30
(21)
. As the result of Cre-mediated loxP DNA
recombination, floxed-PTHrP exon 4, which encodes most of the PTHrP protein, was
deleted in lung epithelia. The genotype has been verified by tail DNA PCR. PTHrP
Epi-
CKO
mice exhibited similar growth and appearance comparing to their littermates without
mortality or lethality (Fig 9), suggesting that PTHrP in mouse lung epithelia is not critical
during the murine lung development.
23

Fig 8. The efficiency of the mesenchymal-specific PTHrP conditional knock-out. (A) The
PCR of P6 wild-type, PTHrP
fx/fx
or PTHrP
Mesen-CKO
mice lung tissue DNA showed the
correct and significant reduction of PTHrP gene on PTHrP
Mesen-CKO
mice. The positions
of PCR primers (P1, P2) and the size of the corresponding PCR product were indicated.
(B) The western blotting of P6 wild-type and PTHrP
Mesen-CKO
lung tissues showed the
significant reduction of the protein expression level of PTHrP, suggesting the sufficient
mesenchymal-specific PTHrP ablation on mouse lung. WT, wild-type mice. KO, PTHrP
Mesen-CKO
mice.

24
fx

P30 Epi-CKO P30 WT

A.

Fig 9. Lung epithelial-specific PTHrP mouse conditional knock-out. (A) Doxycycline
induction from the early embryonic stage (E6.5) till P30 showed that PTHrP
Epi-CKO
mice
have no phenotypic difference comparing to their littermates with no mortality or
lethality. (B) The PCR genotypes of tail genomic DNA. WT, wild-type mice. Epi-CKO,
PTHrP
Epi-CKO
mice.

25
PTHrP-
PTHrPfx
PTHrP+

SPC-rtTA
TetOCre

PTHrP fx

PTHrP-/+

PTHrPdel/del
PTHrP-/+

4. Failure of alveogenesis and severe bronchopulmonary dysplasia-like changes in
mesenchymal-specific PTHrP knock-out mice.
Newborn PTHrP
Mesen-CKO
mice breathed normally without any signs of respiratory
distress. However, PTHrP
Mesen-CKO
mice exhibited lethality at P7~P9 due to the
respiratory failure. Thus, in the present investigation, we have focused our attention on
the phenotype of PTHrP
Mesen-CKO
mice at P6. The autopsy of PTHrP
Mesen-CKO
mice at P6
showed dysplastic lung with fewer and abnormally enlarged air-filled sacs throughout the
lung parenchyma with thinner tissue septa (Fig 10). Upon autopsy, the hyperinflated lung
tissue appeared very fragile, and, during dissections under phosphate-buffered saline, air
bubbles were frequently observed to escape from the mutant mice bronchus. However,
pneumothorax or bleeding in the lung has not been observed in any of the postnatal
PTHrP
Mesen-CKO
mice. The lung tissue sections with H&E staining showed markedly
arrested alveogenesis, failure of secondary septa formation, fewer but hyperinflated
terminal air sacs and the attenuated tissue septa, a condition reminiscent of
bronchopulmonary dysplasia (Fig 11). We also tracked back in developmental time the
initiation of this phenotype and we found that these morphological changes started after
birth and gradually were aggravated till P6, for no detectable changes were found in
E18.5 lung and PTHrP
Mesen-CKO
mice die at P7~P9 due to the respiratory distress.
During mouse postnatal lung alveogenesis, secondary crests develop and extend to make
new secondary septa that further subdivide terminal air sac structures, accompanied by
decreased mean alveolar size. Therefore, the larger the alveolar size, the fewer the alveoli;
thus, alveogenesis can be quantified by calculating MLI. At P6, MLI in PTHrP
Mesen-CKO

26

A.

B.

C.
Fig 10. Phenotypes of mouse lung mesenchymal-specific PTHrP conditional knock-out at
P6. There were fewer but abnormally enlarged air-filled sacs throughout the lung
parenchyma with the thinner tissue septa, a condition reminiscent of bronchopulmonary
dysplasia. Upon autopsy, the hyperinflated lung tissue appeared very fragile, and, during
dissections under phosphate-buffered saline, air bubbles were frequently observed to
escape from the mutant mice bronchus. However, no pneumothorax or bleeding in the
lung has been seen in all postnatal PTHrP
Mesen-CKO
mice. (A)X1.25; (B) X1.6; (C) X4.0.
WT, wild-type. KO, PTHrP
Mesen-CKO
mice. 27
P6WT P6KO

Fig 11.
WT KO
A B
P6X10 P6X10
C. D.
P6X40 P6X40
E. F.
P3X10 P3X10

28

Fig 11, Continued
WT KO
G. H.
P1X10 P1X10

I. E18.5X10 J. E18.5X10
K. 29
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
MLI um
P6 WT P6 KO
MLI P6
P6 WT
P6 KO
*

30
Fig 11. Changes in morphology and morphometric measurement of lung mesenchymal-
specific PTHrP conditional knock-out mouse lung. Haematoxylin and eosin-stained lung
tissue sections from different embryonic and postnatal developmental stages in wild-
type (A, C, E, G and I) and PTHrP
Mesen-CKO
mice (B, D, F, H and J). At P6, PTHrP
Mesen-CKO
mice lung exhibited strikingly delayed respiratory maturation and arrested
alveogenesis, accompanied by fewer but hyperinflated terminal air sacs, failure of
secondary septa formation and the attenuated tissue septa. These morphological changes
were initiated after birth and gradually aggravated till P6. PTHrP
Mesen-CKO
mice would
die at P7~P9 due to the respiratory failure. No detectable changes in E18.5 lung were
found in PTHrP
Mesen-CKO
mice compared with the wild-type. (K) Morphometric
quantification of alveolar sizes by mean linear intercept (MLI) at P6 showed significant
enlarged alveolar size in PTHrP
Mesen-CKO
mice comparing with the wild-type. However,
no atelektasis was found in lung tissue sections at all postnatal and embryonic stages.
(*: P<0.005). WT, wild-type mice. KO, PTHrP
Mesen-CKO
mice. P6, A and BX10, C and
DX40; P3, E and FX10; P1, G and HX10; E18.5, I and JX10.

lung was significantly elevated (P<0.005) suggesting that little or no sub-
compartmentalization of the mutant lung parenchyma takes place. Instead, the pre-
alveolar saccules dilate to form large air-filled sacs (Fig 11). However, no atelectasis was
found in lung tissue sections at all postnatal and embryonic stages.
5. Significant reduction of cell proliferation with dysregulation of cell differentiation
in mesenchymal-specific PTHrP knock-out mouse lungs.
Secondary septa formation during alveogenesis is a complicated process requiring fine
coordination of alveolar myofibroblasts position and correct deposition of the elastic
interstitial matrix, the extension and simplification of capillary networks and the
outgrowth of epithelial cells. This is regulated by many factors including PTHrP
signaling. Abrogation of PTHrP in lung mesenchymal cells resulted in strikingly reduced
cell proliferation during the alveogenesis, as indicated by markedly diminished PCNA-
positive cells in lung parenchyma of PTHrP
Mesen-CKO
mice at P6 (Fig 12). Furthermore,
the decreased cell proliferation was also verified by calculation the percentage of PCNA-
positive cells over total cells per field (PCNA index). The PCNA index was significantly
decreased to 30.3% ± 4.2% in PTHrP
Mesen-CKO
mice lung versus 46.4% ± 8.6% in their
wild-type littermates, a 35% reduction in PCNA index (P<0.005) (Fig 12). This suggests
an obligatory requirement of PTHrP in the lung mesenchymal cell proliferation; and the
loss of lung parenchymal cells may be the direct cause of the reduced wLW/BW (Fig 7),
the thinner tissue septa and the arrested septa formation in the PTHrP
Mesen-CKO
mice lung.
Cell differentiation was also evaluated by quantitative RT-PCR and immuno-
histochemistry analyses for the related cell markers. Surfactant protein C (SPC) and
31

WT KO
A. B.

C.
Fig 12. Abrogation of PTHrP in lung mesenchymal cells resulted in decreased cell
proliferation. Histological staining with PCNA (dark brown) at P6 in (A) wild-type and
(B) PTHrP
Mesen-CKO
mice showed remarkably diminished PCNA-positive cells in the
PTHrP
Mesen-CKO
mice lung. (C) The PCNA index was measured based on the above
PCNA immunostaining, as presented by the percentage of PCNA-positive cells over the
total cells per field. The PCNA index was significantly reduced to 30.3% ± 4.2% in
PTHrP
Mesen-CKO
mice lung versus 46.4% ± 8.6% in their wild-type littermates, a 35%
reduction in PCNA index. (*: P< 0.005) (A, BX40). WT, wild-type mice. KO, PTHrP
Mesen-CKO
mice.
32
0
10
20
30
40
50
60
PCNA Index (%)
P6WT P6KO
PCNA
P6WT
P6KO
*

surfactant protein A (SPA) are cell-specific markers for alveolar epithelial cells type II.
Aquaporin 5 (AQP5) is an alveolar epithelial cells type I marker. Ciliated cells in the
proximal bronchial

and bronchiolar epithelia express Forkhead box J1 (FoxJ1). Clara cell
specific protein (CCSP) is secreted by the non-ciliated bronchial

and bronchiolar Clara
cells. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) reflects the angiogenesis
level during the lung development and is expressed by basal endothelial cells in the blood
vessels and other circulating cells.
At the mRNA level, expression of the distal conducting airway epithelial, lung epithelial
cell differentiation markers and angiogenesis markers, FoxJ1, SPC, SPA, AQP5 and
PECAM-1, had no significant change; whereas expression of Clara cell marker CCSP
increased about 2 times in both P6 and P3 PTHrP
Mesen-CKO
mice comparing with their
wild-type littermates (Fig 13). Consistent with the quantitative RT-PCR, protein
immunostaining showed no significant change of epithelial and endothelial cell markers
Fox J1, SPC, SPA, AQP5 and PECAM-1 in P6 lung of PTHrP
Mesen-CKO
mice (Fig 14)
(Fox J1, SPC, SPA and PECAM-1 data not shown). Interestingly, although there was
about 2 times increase in CCSP mRNA expression, no distinguishable difference has
been found in P6 lung of PTHrP
Mesen-CKO
mice comparing to their wild-type littermates
(Fig 14).
6. Loss of lung myofibroblasts in mesenchymal-specific PTHrP knock-out mouse
lungs.
Smooth muscle cells exist at three locations in the lung, in vascular and bronchial walls

33

SPC SPA AQP5 CCSP FoxJ1 α-SMA PECAM-1
P6KO/WT 0.95 1.59 0.63 1.7 1.55 0.55 1.26
P3KO/WT 1.66 1.5 1.51 2.18 1.29 0.88 0.67

Fig 13. Gene expression level of selected molecular markers of differentiated lung cells
in PTHrP
Mesen-CKO
mice. Gene expression at mRNA level was quantified by real-time
RT-PCR. The expression ratios of paired PTHrP
Mesen-CKO
mice and wild-type littermates
were calculated and marked as P6KO/WT and P3KO/WT respectively. Thus, the
expression level of P6 WT and P3 WT should be 1.0 in each group. As the result,
expression of distal conducting airway, lung epithelial cell differentiation markers and
angiogenesis cell markers FoxJ1, SPC, SPA, AQP5 and PECAM-1 did not show
significant change, whereas expression of Clara cells marker CCSP increased about 2
times in both P6KO/WT and P3KO/WT. α-SMA mRNA level was consistently decreased
to 0.55 times in P6KO/WT group.

34
0
0.5
1
1.5
2
2.5
SPC SPA AQP5 CCSP FoxJ1 α-SMA PECAM
Gene expression ratio (KO/WT)
Gene Expression at P6 and P3
P3KO
P6KO
*
*
*

and in alveolar septae. Septal smooth muscle cells (contractile interstitial cells or lung
myofibroblasts)
(40)
have the morphology of fibroblasts, but can be shown by
ultrastructural analysis to contain contractile elements
(41, 42, 43)
. They also express α-
smooth muscle actin
(44)
. Thus, these septal smooth muscle cells are termed lung
myofibroblasts.
α-SMA-expressing myofibroblasts in lung parenchyma are essential for secondary
septation in the postnatal lung alveolarization. During this process, lung myofibroblasts
were directed to specific sites in the existing primary septa (the walls of the terminal air
sac), followed by the subsequent protrusion and extension of secondary septa that form
alveoli
(29)
. Mice deficient in platelet-derived growth factor A (PDGF-A
-/-
) lack α-SMA-
positive lung myofibroblasts and developed an emphysema-like morphology as alveolar
septa fail to form
(30, 31)
. Moreover, developmental dysregulation of lung myofibroblasts
contributes to bronchopulmonary dysplasia in newborns, a chronic lung disease
characterized by the simplified lung structure and progressive fibrosis
(32)
.
The quantitative RT-PCR revealed that α-SMA gene expression was consistently
decreased in both P3 and P6 PTHrP
Mesen-CKO
mice (0.88 and 0.55 respectively)
comparing to their wild-type littermates (Fig 13). The protein immunostaining of α-
smooth muscle actin in wild-type showed that, under normal condition, lung fibroblasts
localized both at the thick-walled primary septa and the septal tips that protruded slightly
into the airspaces, the presumptive sites of future secondary septa formation. In contrast,
a profound deficiency of lung myofibroblasts was revealed in P6 PTHrP
Mesen-CKO
mouse
35

Fig 14.
WT KO
A. B.
P6WTX20 P6KOX20

C. D.
P6WTX40 P6KOX40
E. F.
P6WT X10 P6KOX10

36

Fig 14. Continued

WT KO

G. H.
P6WT X40 P6KOX40
I. J.
P6WTX20 P6KOX20

Fig 14. Protein expression level of selected molecular markers of differentiated lung cells
in wild-type littermates (A, C, E, G and I) and PTHrP
Mesen-CKO
mice (B, D, F, H and J) at
postnatal day 6. (A-D) Immunostaining for α-SMA (brown staining, arrows) showed a
profound deficiency in lung myofibroblasts in P6 PTHrP
Mesen-CKO
mice. The loss of α-
smooth muscle actin-staining cells was limited to the lung parenchyma. Vascular and
bronchial smooth muscles were clearly seen in PTHrP
Mesen-CKO
mouse lungs. (E-H)
Although there was about 2 times increase in CCSP mRNA expression, no significant
difference (red staining) has been found in P6 lung of PTHrP
Mesen-CKO
mice comparing to
their wild-type littermates. (I, J) Immunostaining of AQP5 (red staining) was used to
detect the alveolar epithelial cells type I. No significant change of AQP5 has been found
in P6 lungs of PTHrP
Mesen-CKO
mice, as well as other epithelial or endothelial cell
markers such as FoxJ1, SPC, SPA and PECAM-1 (data not shown). (E-J) Haematoxylin
counter stain (blue stain).

37

lungs (Fig 14). The defect of lung myofibroblasts may be the direct cause of the failed
secondary septa formation and arrested alveogenesis in the PTHrP
Mesen-CKO
mice lungs.
Considering lung myofibroblast is one of the major mesenchymal-derived cell lineages,
thus, the loss of the mesenchymal-derived cells, especially lung myofibroblasts, due to
the markedly reduced cell proliferation appears to be the explanation of failure of
alveogenesis and thinner tissue septa in PTHrP
Mesen-CKO
mice lung.
Moreover, similar to PDGF-A
-/-
mice
(30, 31)
, the loss of α-smooth muscle actin-staining
cells was limited to the lung myofibroblasts. Vascular and bronchial smooth muscles
were clearly seen in PTHrP
Mesen-CKO
mouse lungs (Fig 14). This suggests that the origin
of the alveolar myofibroblast may not be identical to that of vascular or bronchial smooth
muscle cells. PTHrP therefore might be required in the ontogeny of alveolar
myofibroblasts, but not in the ontogeny of vascular or bronchial smooth muscle cells.
7. Excessive and disordered elastin accumulated in mesenchymal-specific PTHrP
knock-out mouse lungs.
In addition to the loss of lung parenchymal cells, there is an excessive and disorganized
elastin accumulation in the PTHrP
Mesen-CKO
mouse lungs (Fig 15). Elastin is normally
deposited at the tips of alveolar septa, which are, when visualized in three dimensions,
alveolar entrance rings where it bears significant mechanical pressure during the
breathing
(45)
. Elastin can also be found in the vascular and bronchial walls or alveolar
walls for the structure support. In P6 PTHrP
Mesen-CKO
mouse lungs, the thickened and
disordered elastin accumulation could due to the dysregulation of cell differentiation by
the deficiency of PTHrP or due to the prolonged and excessive lung stretch compensating
38

P6WT P6KO

A. B.

Fig 15. Elastin staining of lung tissue at postnatal 6 days. (A) In wild-type littermates,
elastin fibers (black stain) were detected at alveolar septal tips, alveolar walls or
surrounding the bronchial and vascular walls. (B) In PTHrP
Mesen-CKO
mice, there’s
massive accumulation of disorganized, bundle-like elastic fibers at alveolar secondary
crests and within alveolar walls, an elastosis changes. (Counter-stained by taratrazine:
yellow color), (A, B X40)

39

to the loss of lung volume. Collagen expression has not been tested in this study so far,
thus we cannot draw the conclusion whether this lung remodeling only involves elastosis
or generally fibrosis.
8. Intracrine PTHrP regulates cell proliferation.
From previous studies, PTHrP composes two signaling pathways: (1) Paracrine
/autocrine pathway, by PTHrP binding to the classical PTH receptor 1 (G-protein-coupled
receptor) and activating the downstream adenylyl cyclase/protein kinase A and/or the
cytosolic calcium/inositol phosphate/protein kinase C
(5, 6, 7)
; or (2) Intracrine pathway, by
PTHrP direct translocating to nucleus and upregulating the Cyclin D, Cdk 4, Cdk 6 and
pRb to promote the cell proliferation
(8)
. Other studies have also shown that PTHrP
nuclear translocation in vascular smooth muscle cells (A-10 cells) produced a striking
increase in cell proliferation, whereas the paracrine PTHrP resulted in the opposite effect,
cell apoptosis
(26)
.
In our study, PTHrP deletion in lung mesenchyme caused a strikingly diminished cell
proliferation, resulted in the massive loss of lung mesenchymal cells. Thus, we interested
in examine whether the nuclear translocation is one of the methods that PTHrP regulates
cell proliferation in vivo. We at first prepared three different protein fractions
(cytoplasmic, nuclear and whole) from P6 wild-type mice lung tissues. To further
ascertain that fractions prepared from isolated nuclei were free of cytoplasmic
contamination, we analyzed nuclear protein extract by using GAPDH antibody. A band
corresponding to this GAPDH protein was detected in both whole-tissue and cytoplasmic
40

preparation, but negligible in nuclear extracts (Fig 16). This confirmed that the
purification procedure precluded cytoplasmic protein from nuclear extracts. Next, the
immunoblotting analysis using the PTHrP antibody revealed the abundant PTHrP protein
in the nucleus with two different forms presented, indicating there is the nuclear
translocation of PTHrP protein during the normal lung development. The larger form in
size could be the phosphorylated PTHrP. Corresponding forms were also present in
whole-tissue preparations, but the signal intensity was weaker because PTHrP is more
likely to be enriched in nuclear extracts with respect to its relative content in whole-tissue
lysates.
Given that there is the nuclear translocation of PTHrP protein in vivo, we postulated that
PTHrP might regulate cell proliferation through the intracrine pathway during the
postnatal lung development.
9. Discussion
PTHrP is a factor that regulates the growth and development in virtually every tissue in
which it has been examined
(16)
. For example, studies in transgenic mice have
demonstrated that PTHrP is required for normal chondrocyte maturation and
differentiation in the epiphyseal growth plate
(18)
, for normal epidermal and hair follicle
development
(39)
, for normal mammary development
(23)
. In the pancreatic islet, targeted
overexpression of PTHrP leads to a dramatic increase in pancreatic β-cell mass
(46)
.
Conventional deletion of PTHrP resulted in reduced surfactant formation and arrested
type II epithelial cell formation
(37)
. In present study, we have approached the biological
41

Cyt Nu W
anti-PTHrP
anti-GAPDH
Fig 16. The nuclear expression of PTHrP protein. Immunoblotting of cytoplasmic (Cyt),
nuclear (Nu) and whole (W) lung tissue protein fractions from P6 wild-type mice have
been examined by using a GAPDH antibody (anti-GAPDH) or a PTHrP antibody (anti-
PTHrP). GAPDH band was present in both (W) and (Cyt) but negligible in the (Nu)
extract, indicating that nuclear preparation was free of cytoplasmic protein
contaminations. PTHrP protein has been found abundantly in the nuclear extract and was
presented in two different forms, indicating there is the nuclear translocation of PTHrP
protein during the normal lung development and the larger form in size could be the
phosphorylated PTHrP. Corresponding forms were also present in (W), but the signal
intensity was weaker because PTHrP is more likely to be enriched in nuclear extracts
with respect to its relative content in whole-tissue lysates.

42

function of PTHrP signaling in lung epithelial versus mesenchymal cells during lung
development by selectively abrogated PTHrP intracellular signaling activity using Cre/
loxP methods, by deleting PTHrP protein specifically in either lung epithelium or
mesenchyme with SPC-rtTA/ TetO-Cre and Dermo1-Cre driver lines, respectively.
Interestingly, blockage of endogenous PTHrP function in airway epithelial cells alone
(Epi-CKO) failed to elicit any detectable alterations in vivo. The PTHrP
Epi-CKO
mice
breathed normally and displayed similar appearance without any mortality and lethality.
However, there is a failure of secondary septa formation, significantly arrested
alveogenesis, loss of lung mesenchyme-derived cells (especially lung myofibroblasts)
and accumulation of elastin accompanied by a 75% neonatal lethality, growth retardation,
skeletal deformity in PTHrP
Mesen-CKO
mice. Therefore, PTHrP expression in lung
mesenchyme, but not in epithelia, must be essential for postnatal lung development.
Previous in vivo studies have reported that PTHrP null mice
(37)
, as well as PTHrP (1-84)
KI mice
(8)
(lack of nuclear localization region and C-terminal region), demonstrated
dysplastic bone formation and postnatal lethality (within 2 hours or 2-3 weeks after birth
respectively). Our PTHrP mesenchymal-specific knockout mice model confirmed with
this skeleton phenotype. Similar but severer than PTHrP (1-84) KI mice, PTHrP
Mesen-CKO

also exhibited the postnatal growth retardation, thinner skin, decrease in fat deposition
and other non-pulmonary abnormalities. This could due to the partially reserved
paracrine/ autocrine pathway in PTHrP (1-84) KI mice.
Alveogenesis is known to be a complicated process, with coordinated growth of alveolar
epithelial cells (both type I and type II epithelial cells), capillary endothelial cells and
43

myofibroblasts and deposition of extracellular matrix, particularly elastin. Cell
proliferation and differentiation in each of these cell lineages contribute to the secondary
septa and, thus, to the related alveoli formation, generating sufficient gas-blood exchange
surface. By measuring the cell cycle-specific protein markers PCNA, it was found that
cell proliferation in the PTHrP
Mesen-CKO
lung was diminished, suggesting that PTHrP
signaling in lung mesenchymal cells is essential for directly regulating mesenchymal cell
growth and the loss of lung mesenchymal cells may be the direct cause of the failure of
secondary septa formation and arrested alveogenesis.
To further narrow the affected cell lineages in PTHrP
Mesen-CKO
lungs, expression patterns
of cell-specific markers were examined in the different types of cells. No significant
changes in epithelial cells and endothelial cells were observed in PTHrP
Mesen-CKO
lungs,
as assessed by SPC, SPA, AQP5, Fox J1, CCSP and PECAM-1. However, the dramatic
loss of myofibroblasts, a mesenchymal cell lineage, was confirmed by both quantitative
RT-PCR and immunohistochemistry. Similarly, conventional deletion of PDGF-A caused
the specific absence of lung myofibroblasts and, subsequently, the failure of secondary
septa formation and alveogenesis, which is consistent with our current observation. These
data suggest that alveogenesis, controlled by the PTHrP pathway, which promotes
mesenchymal cell growth, specially myofibroblast growth, is directly affected by
abrogating mesenchymal PTHrP signaling activity in PTHrP
Mesen-CKO
mouse model.
Interestingly, the loss of mesenchymal cell types was specific to myofibroblasts but not
bronchial or vascular smooth muscle cells in both PTHrP
Mesen-CKO
and PDGF-A
-/-
models,
suggesting that these cell types have separate ontogeny, and PTHrP and PDGF-A are
44

both required in myofibroblasts genesis.
However, different from PDGF-A
-/-
mouse lung, we did not observe the decrease of
elastin fiber in PTHrP
Mesen-CKO
lung. On the contrary, elastin fiber increased significantly
in P6 PTHrP
Mesen-CKO
lung with disordered arrangement. This is consistent with the
observation in neonatal BPD disease which is known to occur in respiratory failure
managed with the surfactant replacement and ventilation assistant after premature birth,
characterized by the progressive fibrosis, especially the excessive and disordered elastin
accumulation, failure of secondary septa formation and alveogenesis. The reason of
elastin accumulation is still unclear, but the prolonged and excessive lung stretch for
compensating the loss of lung volume could be one of the reasons for PTHrP
Mesen-CKO

mice.
To further understand the PTHrP signaling pathway in regulating cell proliferation, in
vivo study has shown that PTHrP (1-84) KI (lack of nuclear localization region and C-
terminal region) mice exhibited systematic reduction of cell proliferation and increased
cell apoptosis in bone, central nerve system, bone marrow, thymus, spleen and other
tissues
(8)
. The author believed that the decreased cell proliferation and increased cell
apoptosis were due to the loss of PTHrP intracrine signaling pathway whereas intact
paracrine/ autocrine pathway. Other studies also confirmed with this observation, for
example, PTHrP was found in the nucleolus of mouse chondrocytes, osteoblasts,
embryonic fibroblasts, untransfected vascular smooth muscle cells as well as in PTHrP-
overexpressing COS cells by immunocytochemistry and immunoelectron microscopy
(26,
48)
. Deletion of the entire nuclear translocation region (87-107) prevented the nuclear
45

targeting in embryonic fibroblasts, vascular smooth muscle cells and COS cells.
Moreover, this nuclear translocation of PTHrP appears to trigger pRb phosphorylation
and release of G1/S arrest and thereby cell cycle progression
(49)
. In our study, deletion of
PTHrP in mesenchyme resulted in a significant decrease in total cell number and cell
proliferation. The existence of nuclear PTHrP suggests that the regulatory function of
PTHrP in cell proliferation could be mediated by the intracrine pathway.

10. Conclusion
In conclusion, the deletion of PTHrP in mesenchyme resulted in the massive loss of lung
mesenchymal cells, especially lung myofibroblasts, the direct cause of alveogenesis
failure, attenuated tissue septa and BPD-like morphological changes. Other phenotypes
include growth retardation, skeleton deformity, thinner skin, loss of fat tissues and
hypoxemia could result from the deletion of PTHrP in other key mesenchymal tissues.
No phenotype has been found in PTHrP lung epithelial-specific knock-out mice.
Therefore, PTHrP expression in lung mesenchyme, but not in epithelia, is essential for
postnatal lung development, possibly through regulating cell proliferation, especially
lung myofibroblasts, by an intracrine pathway. This study will be the first of its kind to
investigate the PTHrP’s function in lung mesenchymal versus epithelial development. It
will not only elucidate the novel mechanisms of PTHrP in regulating lung development,
but also help to design novel therapeutic strategies for neonatal lung diseases such as
bronchopulmonary dysplasia.

46

BIBLIOGRAPHY
7. Abou-Samra AB, Jüppner H, Force T, Freeman MW, Kong XF, Schipani E, Urena P,
Richards J, Bonventre JV, Potts JT Jr, et al. Expression cloning of a common receptor for
parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like
cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol
trisphosphates and increases intracellular free calcium. Proc Natl Acad Sci U S A. 1992
Apr 1; 89(7):2732-6
41. Adler KB, Low RB, Leslie KO, Mitchell J, Evans JN. Contractile cells of the normal
and fibrotic lung. Lab Invest 1989; 60:473-485.
30. Bostrom H, Willetts K, Pekny M, Leveen P, Lindahl P, Hedstrand H, Pekna M,
Hellstrom M, Gebre-Medhin S, Schalling M, Nilsson M, Kurland S, Tornell J, Heath JK,
Betsholtz C. PDGF-A signaling is a critical event in lung alveolar myofibroblast
development and alveogenesis. Cell 1996; 85:863-873.
32. Bourbon J,Boucherat O,Chailley-Heu B,Delacourt C. 2005. Control mechanisms of
lung alveolar development and their disorders in bronchopulmonary dysplasia. Pediatr
Res 57: 38R-46R
19. Chen H, Zhuang F, Liu YH, Xu B, Moral PD, Deng W, Chai Y, Kolb M, Gauldie J,
Warburton D, Moses HL, Shi W. TGF-β receptor II in epithelia versus mesenchyme
plays distinct roles in the developing lung. Eur Respir J 2008; 32:285-295.
14. Coalson JJ. Pathology of new bronchopulmonary dysplasia. Semin Neonatol 2003;
8:73-81.
25. Dunnill MS. Quantitative methods in the study of pulmonary pathology. Thorax 1962;
17:320-328.
49. Fiaschi-Taesch N, Takane KK, Masters S, Lopez-Talavera JC, Stewart AF.
Parathyroid-hormone-related protein as a regulator of pRb and the cell cycle in arterial
smooth muscle. Circulation 2004; 110:177-185.
11. Gabbiani G. 1992. The biology of the myofibroblast. Kidney Int 41: 30-532
17. Gensure RC, Gardella TJ, Jüppner H. Parathyroid hormone and parathyroid hormone-
related peptide, and their receptors. Biochem Biophys Res Commun. 2005 Mar
18;328(3):666-78
20. He B, Deckelbaum RA, Miao D, Lipman ML, Pollak M, Goltzman D, Karaplis AC.
Tissue-specific targeting of the pthrp gene: the generation of mice with floxed alleles.
Endocrinology. 2001 May; 142(5):2070-7
48. Henderson JE, Amizuka N, Warshawsky H, Biasotto D, Lanske BM, Goltzman D,
Karaplis AC. Nucleolar localization of parathyroid hormone-related peptide enhances
survival of chondrocytes under conditions that promote apoptotic cell death. Mol Cell
Biol. 1995 Aug;15(8):4064-75. 47

34. Henderson JE, Amizuka N, Warshawsky H, Biasotto D, Lanske BMK, Goltzman D,
Karaplis AC. Nucleolar localization of parathyroid hormonerelated peptide enhances
survival of chondrocytes under conditions that promote apoptotic cell death. Mol Cell
Biol 1995 15:4064–4075
35. Henderson JE, He B, Goltzman D, Karaplis AC. Constitutive expression of
parathyroid hormone-related peptide (PTHrP) stimulates growth and inhibits
differentiation of CFK2 chondrocytes. J Cell Physiol 1996 169:33–41
2. Hilfer SR. Morphogenesis of the lung: control of embryonic and fetal branching. Annu
Rev Physiol 1996; 58:93-113.
4. Hogan BL, Grindley J, Bellusci S, Dunn NR, Emoto H, Itoh N. Branching
morphogenesis of the lung: new models for a classical problem. Cold Spring Harb Symp
Quant Biol 1997; 62:249-256.
3. Hogan BL. Morphogenesis. Cell 1999; 96:225-233.
6. Jüppner H, Abou-Samra AB, Freeman M, Kong XF, Schipani E, Richards J,
Kolakowski LF Jr, Hock J, Potts JT Jr, Kronenberg HM, et al. A G protein-linked
receptor for parathyroid hormone and parathyroid hormone-related peptide. Science.
1991 Nov 15; 254(5034):1024-6.
5. Jüppner H, Abou-Samra AB, Uneno S, Gu WX, Potts JT Jr, Segre GV. The
parathyroid hormone-like peptide associated with humoral hypercalcemia of malignancy
and parathyroid hormone bind to the same receptor on the plasma membrane of ROS
17/2.8 cells. J Biol Chem. 1988 Jun 25; 263(18):8557-60
40. Kapanchi Y, Assimacopoulos A, Irle C, Zwahlen A, Gabbiani G. Contractile
interstitial cells in pulmonary alveolar septa: a possible regulator of ventilation/perfusion
ratio? J Cell Biol 1974; 60:375-392.
18. Karaplis AC, Luz A, Glowacki J, Bronson RT, Tybulewicz VL, Kronenberg HM,
Mulligan RC. Lethal skeletal dysplasia from targeted disruption of the parathyroid
hormone-related peptide gene. Genes Dev. 1994 Feb 1; 8(3):277-89
29. Kim N, Vu TH. Parabronchial smooth muscle cells and alveolar myofibroblasts in
lung development. Birth Defects Res C Embryo Today. 2006 Mar; 78(1):80-9.
45. Kojic M, Butler J, Vlastelica I, Stojanovic B, Rankovic V, Tsuda A. Geometric
hysteresis of alveolated duct architecture. FASEB 2006; 20:A1258-A1259.
38. Lam MHC, Briggs LJ, Hu W, Martin TJ, Gillespie MT, JansDA. Importin beta
recognizes parathyroid hormone-related protein with high affinity and mediates its
nuclear import in the absence of importin alpha. J Biol Chem 1999 274:7391–7398
36. Lam MHC, Olsen SL, Rankin WA, Ho PWM, Martin TJ, Gillespie MT,Moseley JM.
PTHrP and cell division: expression and localization of PTHrP in a keratinocyte cell line
(HaCaT) during the cell cycle. J Cell Physiol 1997 48

44. Leslie K, Mitchell JJ, Woodcock-Mitchell JL, Low RB. α-Smooth muscle actin
expression in developing and adult lung. Differentiation 1990; 44:143-149.
31. Lindahl P, Karlsson L, Hellstrom M, Gebre-Medhin S, Willetts K, Heath JK,
Betsholtz C. Alveogenesis failure in PDGF-A-deficient mice is coupled to lack of distal
spreading of alveolar smooth muscle cell progenitors during lung development.
Development 1997; 124:3943-3953.
26. Massfelder T, Dann P, Wu TL, Vasavada R, Helwig JJ, Stewart AF. Opposing
mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular
smooth muscle cells: a critical role for nuclear targeting. Proc Natl Acad Sci U S A. 1997
Dec 9; 94(25):13630-5.
8. Miao DS, Su H, He B, Gao JJ, Xia QW, Zhu M, Gu Z, Goltzman D, Karaplis AC.
Severe growth retardation and early lethality in mice lacking the nuclear localization
sequence and C-terminus of PTH-related protein. Proc Natl Acad Sci 2008;
105(51):20309-20314.
42. Noguchi A, Reddy R, Kursar JD, Parks WC, Mecham RP. Smooth muscle isoactin
and elastin in fetal bovine lung. Exp Lung Res 1989; 4:537-552.
21. Perl AK, Wert SE, Nagy A, Lobe CG, Whitsett JA. Early restriction of peripheral and
promixal cell lineages during formation of the lung. Proc Natl Acad Sci 2002; 99:10482-
10487.
28. Pfaffl MW. A new mathematical model for relative quantification in real-time PCR.
Nucleic Acids Res 2001; 17:3023-3028.
16. Philbrick WM, Wysolmerski JJ, Galbraith S, Holt E, Orloff JJ, Yang KH, Vasavada
RC, Weir EC, Broadus AE, Stewart AF. Defining the roles of parathyroid hormone-
related protein in normal physiology. Physiol Rev. 1996 Jan;76(1):127-73
33. Power DM, Ingleton PM, Flanagan J, Canario AV, Danks J, Elgar G, Clark MS.
Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP)
in a teleost, Fugu rubripes. Gene. 2000 May 30;250(1-2):67-76
15. Rehan VK, Torday JS. Lower parathyroid hormone-related protein content of tracheal
aspirates in very low birth weight infants who develop bronchopulmonary dysplasia.
Pediatr Res 2006; 60:216-220.
12. Rishikof DC,Lucey EC,Kuang PP,Snider GL,Goldstein RH. 2006. Induction of the
myofibroblast phenotype following elastolytic injury to mouse lung. Histochem Cell Biol
125: 527-534.
37. Rubin LP, Kovacs CS, De Paepe ME, Tsai SW, Torday JS, Kronenberg HM.
Arrested pulmonary alveolar cytodifferentiation and defective surfactant synthesis in
mice missing the gene for parathyroid hormone-related protein. Dev Dyn. 2004
Jun;230(2):278-89 49

43. Schürch W, Seemayer TA, Gabbiani G. Myofibroblasts. In Histology for Pathologists,
S.S. Sternberg, ed. (New York: Raven Press), pp. 109-144.
10. Shi W, Bellusci S, Warburton D. Lung development and adult lung diseases. Chest
2007 Aug; 132(2):651-6
1. Ten Have-Opbroek AA. Lung development in the mouse embryo. Exp Lung Res 1991;
17:111-130.
24. Thurlbeck WM. Measurement of pulmonary emphysema. Am Rev Respir Dis 1967;
95:752-764.
13. Torday J,Rehan VK. 2007. The evolutionary continuum from lung development to
homeostasis and repair. Am J Physiol Lung Cell Mol Physiol 292: L608-L611.
27. Torday JS, Nielsen HC, Fencl Mde M, Avery ME. Sex differences in fetal lung
maturation. Am Rev Respir Dis 1981; 123:205-208.
46. Vasavada RC, Cavaliere C, D'Ercole AJ, Dann P, Burtis WJ, Madlener AL, Zawalich
K, Zawalich W, Philbrick W, Stewart AF. Overexpression of parathyroid hormone-
related protein in the pancreatic islets of transgenic mice causes islet hyperplasia,
hyperinsulinemia, and hypoglycemia. J Biol Chem. 1996 Jan 12;271(2):1200-8
9. Warburton D, Gauldie J, Bellusci S, Shi W. Lung development and susceptibility to
chronic obstructive pulmonary disease. Proc Am Thorac Soc 2006; 3:668-672.
47. Warburton D, Gauldie J, Bellusci S, Shi W. Lung development and susceptibility to
chronic obstructive pulmonary disease. Proc Am Thorac Soc 2006; 3: 668-672.
23. Wysolmerski JJ, Broadus AE, Zhou J, Fuchs E, Milstone LM, Philbrick WM.
Overexpression of parathyroid hormone-related protein in the skin of transgenic mice
interferes with hair follicle development. Proc Natl Acad Sci U S A. 1994 Feb
1;91(3):1133-7
39. Wysolmerski JJ, McCaughern-Carucci JF, Daifotis AG, Broadus AE, Philbrick WM.
Overexpression of parathyroid hormone-related protein or parathyroid hormone in
transgenic mice impairs branching morphogenesis during mammary gland development.
Development 1995 Nov;121(11):3539-47.
22. Yu K, Xu J, Liu Z, Sosic D, Shao J, Olson EN, Towler DA, Ornitz DM. Conditional
inactivation of FGF receptor 2 reveals an essential role for FGF signaling in the
regulation of osteoblast function and bone growth. Development. 2003 Jul;
130(13):3063-74

50

Asset Metadata

Creator Deng, Wei (author)

Core Title The role of parathyroid hormone-related protein in regulating neonatal lung development

Contributor Electronically uploaded by the author (provenance)

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 07/20/2009

Defense Date 05/27/2009

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

Tag BPD,chronic lung disease development,neonatal lung,OAI-PMH Harvest,parathyroid hormone-related protein

Language English

Advisor Paine, Michael L. (committee chair), Snead, Malcolm L. (committee member), Warburton, David (committee member)

Creator Email dengwei.dengwei@yahoo.com,dengwei_07@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2379

Unique identifier UC1202466

Identifier etd-Deng-3034 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-405853 (legacy record id),usctheses-m2379 (legacy record id)

Legacy Identifier etd-Deng-3034.pdf

Dmrecord 405853

Document Type Thesis

Rights Deng, Wei

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email uscdl@usc.edu

Abstract (if available)

Abstract Parathyroid hormone-related protein (PTHrP) plays important roles in regulating a variety of developmental processes in many organs, through paracrine, autocrine and intracrine pathways. Conventional knockout of PTHrP in mice results in neonatal lethal with multiple defects including asphyxia, skeletal deformities and osteochondrodysplasia. In the present study, PTHrP conditional knockout in mouse lung epithelia versus mesenchyme has been generated. Abrogation of PTHrP specifically in lung mesenchyme results in a neonatal lethal due to lung alveolarization arrest and respiratory failure accompanied with diminished cell proliferation, loss of myofibroblasts and increased elastin accumulation. These manifestations are similar to those observed in bronchopulmonary dysplasia. No abnormality has been found in PTHrP epithelial-specific knock-out mice. Therefore, we conclude that PTHrP expression in lung mesenchyme, but not in epithelia, is essential for postnatal lung development, possibly through regulating cell proliferation, especially lung myofibroblasts, by an intracrine pathway. The fundamental knowledge obtained from this study will help understanding pathogenic mechanisms of neonatal lung disease such as bronchopulmonary dysplasia, and may also provide clues for designing novel therapeutic strategies.