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Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through JNK dependent and independent pathways

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Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through JNK dependent and independent pathways

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i

PROTEIN KINASE C (PKC) PARTICIPATES IN ACETAMINOPHEN
HEPATOTOXICITY THROUGH JNK DEPENDENT AND INDEPENDENT
PATHWAYS

by
Maria Cecilia D. Ybanez

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
May 2013

Copyright 2013 Maria Cecilia D. Ybanez

ii

ACKNOWLEDGEMENTS
I would like to thank Dr. Neil Kaplowitz for giving me the opportunity to work in his lab
and in this project. His vast intellectual knowledge and passion for science has been such
an inspiration to me throughout these years. The guidance he has provided during my
presentations in the lab meetings has been invaluable in enabling me to plan well-
designed experiments. He has always been supportive of my decisions and has kindly
provided fantastic letter of recommendations when I needed one. I also would like to
thank Dr. Derick Han for his direction throughout these years. His instructive and
detailed requirements towards scientific research pushed me to be a better researcher. I
have shared a lot of hard work and delight in working with them. I have gained much
knowledge from them and will keep it with me as I grow in my scientific career.
I would also like to express my deepest gratitude to Dr. Zoltan Tokes my program
advisor and chair of the committee. His kindness, encouragement and support throughout
these years has given me so much and enabled me to succeed in my pursuit of academic
advancement. I am infinitely grateful for all he has done for me.
Special thanks to the Kalra lab and my lab members in Kaplowitz lab. Research won’t be
as enjoyable without them.
I would like to thank Behnam Saberi, MD for his contribution to this project, his hard
work and innovativeness has led to some great experiments. We devised a cell culture
model where we eliminated the DMSO effect on APAP and we called it Post-treatment.
Lastly, I am eternally grateful to my family especially my mom for instilling the
confidence in me, supported my decisions, and unconditional support in my education
and research. I am the person I am today because of them.

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

LIST OF FIGURES iv

ABREVIATIONS viii
ABSTRACT x
CHAPTER 1: INTRODUCTION 1

1.1 Specific aims 13

CHAPTER 2: MATERIALS AND METHODS 15
2.1 Materials 15
2.2 Animals 15
2.3 Isolation of Liver Mitochondria and Cytoplasm 16
2.4 Measurements of respiration in isolated mitochondria 16
2.5 Cell culture 17
2.6 Western blot samples 17
2.7 Compound C experiment 17
2.8 AMPK activator III, DHPO, experiment 18
2.9 Autophagic flux experiment 18
2.10 Determination of apoptosis and necrosis 18
2.11 Protein Measurement 19
2.12 Western Blot Analysis 19
2.13 Histological Analysis 20
2.14 HPLC measurements for GSH and GSSG 20
2.15 Statistical Analysis 20

CHAPTER 3: RESULTS 21

3.1 PKC plays an important role in APAP-induced necrosis in primary cultured
hepatocytes. 21
3.2 PKC inhibitors protect against APAP hepatotoxicity through JNK
dependent and independent pathways. 24
3.3 Broad-spectrum PKC inhibitors modulate p-AMPK to protect hepatocytes
from APAP hepatotoxicity. 26
3.4 PKC inhibitor Ro-31-8425 protects against APAP induced liver injury
in vivo. 32
3.5 Silencing PKC-α using antisense protects against APAP-induced liver
injury through a JNK-dependent pathway in vivo. 36

iv

3.6 PKC-α translocates to mitochondria and inhibits mitochondrial respiration
during APAP-induced liver injury. 40

CHAPTER 4: DISCUSSION 46

4.1 PKC plays an important role in APAP hepatotoxicity. 46
4.2 Broad-spectrum PKC inhibitors protect against APAP hepatotoxicity
through an AMPK-dependent, JNK-independent pathway. 46
4.3 Inhibition of PKC-α protects against APAP hepatotoxicity through a JNK
dependent pathway. 48

CHAPTER 5: FUTURE DIRECTIONS 50

CHAPTER 6: REFERENCES 52

v

LIST OF FIGURES
Figure 1. Two hit hypothesis to mitochondria 3
Figure 2. Domain structure of protein kinase C (PKC) isoforms 5
Figure 3. Domain structure of AMPK subunit isoforms and splice variants 8
Figure 4. AMPK as a master regulator of metabolism 10
Figure 5. A model of the p70S6K pathway 12
Figure 6. Pre-treatment of PKC inhibitors protects primary cultured hepatocytes
against APAP hepatotoxicity 22

Figure 7. Post-treatment of PKC inhibitors protect primary cultured hepatocytes
against APAP hepatotoxicity 22

Figure 8. Effect of PKC inhibitors on covalent binding 23

Figure 9. APAP treatment to hepatocytes increases PKC activity (proteins
phosphorylated by PKC 23

Figure 10. Time course of JNK phosphorylation subsequent to APAP treatment
in hepatocytes 25

Figure 11. Effect of broad-spectrum PKC inhibitors or classical PKC inhibitor
on JNK phosphorylation in hepatocytes after treatment of APAP 25mM 25

Figure 12. Densitometry of p-JNK in primary cultured hepatocytes 26

Figure 13. Time course of p-AMPK following APAP treatments in hepatocytes 27

Figure 14. Effect of broad-spectrum PKC inhibitors or classical PKC inhibitor
on p-AMPK levels in hepatocytes after treatment with APAP 25mM 27

Figure 15. Densitometry of p-AMPK in primary cultured hepatocytes 28

Figure 16. p-AMPK plays a key role in APAP hepatotoxicity in primary mouse
hepatocytes 29

Figure 17. Effect of compound C on p-AMPK levels in cultured hepatocytes
after treatment with APAP 20mM 29

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Figure 18. AMPK activator treatment protects hepatocytes from APAP
hepatotoxicity 30

Figure 19. Effect of lysosomal protease inhibitor, NH
4
Cl/Leupeptin on LC3-II
levels after APAP treatment 31

Figure 20. Effect of PKC inhibitors on LC3-II levels after APAP treatment
with or without lysosomal protease inhibitor 32

Figure 21. Effect of PKC inhibitors on effectors of protein translation (p70S6K)
and autophagy (p62, LC3-II) following APAP treatments 32

Figure 22. Broad-spectrum PKC inhibitor protects against APAP-induced liver
injury in vivo through a JNK-independent pathway 34

Figure 23. Broad-spectrum PKC inhibitor markedly reduced centrilobular
necrosis caused by APAP in vivo 34

Figure 24. Effect of Ro-31-8425 treatment on GSH levels in the liver and
isolated mitochondria subsequent to APAP treatment (2 hours) 35

Figure 25. Effect of Ro-31-8425 treatment on JNK activation and translocation to
mitochondria following APAP treatment in vivo 35

Figure 26. Effect of Ro-31-8425 on p-AMPK, p70S6K, and S6 activation levels
in the liver following APAP treatment in vivo 36

Figure 27. Silencing PKC-α protects against APAP-induced liver injury through
a JNK dependent pathway in vivo 37

Figure 28. Silencing PKC-α markedly reduced centrilobular necrosis caused
by APAP in vivo 38

Figure 29. JNK activation and translocation to mitochondria in vivo 38

Figure 30. Densitometry of p-JNK in PKC-α silenced mice in vivo 39

Figure 31. PKC-ε knock out mice are not protected from APAP-induced
liver injury 40

Figure 32. PKC-α translocates to the mitochondria and phosphorylates
mitochondrial proteins during APAP hepatotoxicity 42

Figure 33. PKC-α translocation to the mitochondria parallels a decline in
mitochondria respiration 43

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Figure 34. Silencing JNK decreases PKC-α translocation to mitochondria and
phosphorylation of mitochondrial proteins 44

Figure 35. Role of PKC, AMPK, and JNK in APAP hepatotoxicity 45

viii

ABREVIATIONS
ACC- Acetyl-coa carboxylase
AKT – Protein kinase B
ALT- Alanine aminotransferase
AMPK – AMP- activated kinase
APAP – Acetaminophen
ASK-1 – Apoptosis signal-regulating kinase 1
ASO- Antisense
ATG7 – Autophagy related protein 7
CYP2e1 – Cytochrome P450 2E1
DAG – Diacylglycerol
eF2 – Eukaryotic elongation factor 2
4EBP1 – 4E binding protein 1
ERK – Extracellular signal regulated kinase
GSK-3β – Glycogen synthase kinase 3β
GLUT4 – Glucose transporter type 4
GS – Glycogen synthase
HMGR- HMG-CoA reductase
H
2
O
2
– Hydrogen peroxide
HSL- Hormone-sensitive lipase
JNK – C-Jun N-Terminal Kinase
LK β1- Liver kinase β1
MAPK – Mitogen-activated protein kinase

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MLK-3 – Mixed lineage kinase – 3
MPT – Mitochondrial permeability transition
mTORC1- Mammalian target of rapamycin
NAC – N-acetyl cysteine
NAPQI – N-acetyl-p-benzoquinone imine
PAR6 – Prostate apoptosis response – 6
P62 – Nucleoporin 62
PDK1 – Phosphoinositide dependent kinase -1
PFK2 – Phosphofructokinase
PGC – α – PPARγ coactivator-1α
PMA – Phorbol ester phorbol-12-myristate-13-acetate
P70S6K – p70 ribosomal S6 kinase
PKC – Protein kinase C
PIP3 – Phosphatidylinositol (3,4,5) – triphosphate
ROS – Reactive oxygen species
S6 – S6 ribosomal protein
Sirt1 – Mammalian ortholog of silent information regulator
ULK- UNC-51-like kinase 1

x

ABSTRACT
Our previous studies have shown that acetaminophen (APAP)-induced hepatocyte
necrosis is mediated by JNK. In the present study we show that protein kinase C (PKC)
plays an important role in APAP-induced liver injury through JNK-dependent and
independent pathways. Treatment of primary mouse hepatocytes with two different
broad-spectrum PKC inhibitors (Ro-31-8245, Go6983), protected against APAP
hepatotoxicity without inhibiting JNK activation. Ro-31-8245 treatment to mice also
resulted in upregulation of p-AMPK in the liver and protection against APAP-induced
liver injury in vivo, despite sustained JNK activation. APAP treatment caused a
decreased p-AMPK, which was prevented by broad-spectrum PKC inhibitors. AMPK
inhibition by compound C or activation using AMPK activator oppositely modulated
APAP hepatotoxicity. This suggests PKC-dependent downregulation of AMPK-regulated
survival pathways is an important component of APAP hepatotoxicity. In contrast to
broad-spectrum inhibitors, treatment of hepatocytes with a more specific classical PKC
inhibitor (Go6976) that inhibits mainly PKC-α and PKC-βI protected against APAP by
inhibiting JNK activation. Knockdown of PKC-α using antisense (ASO) in mice
protected against APAP-induced liver injury by inhibiting JNK activation. APAP
treatment resulted in PKC-α translocation to mitochondria, phosphorylation of
mitochondrial proteins, and decline in mitochondria respiration in the liver. JNK 1 and 2
silencing using ASO in mice decreased APAP-induced PKC-α translocation to
mitochondria, suggesting PKC-α and JNK act together through a feed forward
mechanism to mediate APAP-induced liver injury. Conclusion: PKC-α and other PKC(s)
regulate death (JNK) and survival (AMPK), to modulate APAP-induced liver injury.

1

CHAPTER 1
INTRODUCTION
Acetaminophen (APAP) is the most common cause of acute liver failure in the
United States, accounting for 46% of all cases (Lee et al., 2008). Patterns of presentation
differ between countries with more accidental cases reported in United States. Annually,
400-500 deaths occur in United States as a result of acetaminophen related liver failure
(Lee et al., 2008). Currently the standard treatment for acetaminophen induced liver
injury is N-acetyl cysteine, NAC, which is a specific antidote that supplies glutathione.
Administration of NAC within 24 hours of overdose with acetaminophen significantly
decreases the risk of liver injury and due to the minimal side effects NAC is given up to
72 hours after overdose (Lee et al., 2008). In the case of chronic alcoholics, they develop
APAP hepatotoxicity even at moderate doses. Predisposition to APAP hepatotoxicity is
presumably caused by induction of cytochrome P-4502EI by ethanol and by depletion of
glutathione (GSH) because of the effects of alcohol, the malnutrition often associated
with alcoholism, and the depletion associated with chronic use of APAP and impaired
glucuronidation caused by fasting perhaps as well (Zimmerman et al., 1995). APAP
hepatotoxicity involves the active participation of signal transduction pathways that
activate JNK (Gunawan et al., 2006). Inhibition of JNK prevents APAP-induced liver
injury even in the presence of extensive GSH depletion and covalent binding (Hanawa et
al., 2008). A two hit hypothesis to mitochondria was proposed as the central mechanism
mediating APAP-induced liver injury. APAP is metabolized to NAPQI by CYP2e1,
which depletes GSH and causes covalent binding in cytoplasm and mitochondria (first
hit) (Figure 1). Mitochondrial GSH depletion and covalent binding causes increase in

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mitochondrial reactive oxygen species (ROS) generation that activates JNK, through
GSK-3b-MLK-3 (early phase) and ASK-1 (late phase) dependent MAP kinase pathways
(Han et al., 2010). Activated JNK translocates to mitochondria to bind to Sab (second
hit), an outer membrane protein that is phosphorylated by JNK and is required for
toxicity (Figure 1). JNK binding to Sab on mitochondria leads to further enhancement of
ROS generation by a mechanism not yet understood; the enhanced ROS is important in
sustaining JNK activation and inducing the mitochondrial permeability transition (MPT)
to mediate hepatocyte necrosis (Win et al., 2011). APAP-induced liver injury is primarily
associated with necrosis, although a small amount of apoptosis has been suggested in
both mice and humans (Han et al., 2010). APAP injury therefore represents a type of
“programmed necrosis”, with apoptosis minimized by caspase inhibition due to extensive
redox perturbation and ATP depletion. JNK signaling is essential for APAP-induced
programmed necrosis, and other signaling proteins such as GSK-3b, MLK-3, and ASK-1
that mediate APAP-induced liver injury act upstream to modulate JNK signaling (Han et
al., 2010). JNK signaling pathway appears to be the primary prodeath pathway mediating
hepatocyte death following hepatotoxic doses of Acetaminophen (Han et al., 2010;
Nakagawa et al., 2008; Sharma et al., 2012; Shinohara et al., 2010).

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Figure
1.
Two
hit
hypothesis
to
mitochondria.
Aside from MAPK, other signaling pathways may be activated by ROS.
Previously we have shown that hydrogen peroxide induced necrosis of primary mouse
hepatocytes is modulated by activation of Protein Kinase C (PKC) and subsequent
inactivation of AMP-activated kinase (AMPK). Treatment of broad-spectrum PKC
inhibitors alone induced greater AMPK phosphorylation suggesting that there is some
basal activity inhibiting AMPK. Broad-spectrum PKC inhibitors, Ro-31-8425 and
bisindolymaleimide I) also induced activation of AMPK in primary mouse hepatocytes
treated with hydrogen peroxide and even when PKC-α was knocked down (Saberi et al.,
2008). Classical PKC inhibitor, Go 6976, which inhibits PKC-α and β1 did not
upregulate AMPK and partially prevented H
2
O
2
- induced necrosis. Knockdown of PKC-α
also partially protected against H
2
O
2
, however the protection was smaller ~22% than
when treated with Go 6976 (~45%), suggesting that Go 6976 may work through other
pathways to protect hepatocytes from H
2
O
2
- induced necrosis (Saberi et al., 2008).

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Overall, broad-spectrum PKC inhibitors significantly protect against H
2
O
2
induced
hepatocyte necrosis through activation of an AMPK kinase survival pathway (Saberi et
al., 2008). However the role of PKC and AMPK in APAP hepatotoxicity is not known.
PKC is part of Serine/Threonine Kinase family that regulates various cellular
functions including cell proliferation, differentiation and apoptosis (Gutcher et al., 2003;
Rosse et al., 2010; Spitaler et al., 2004). These kinases contain a highly conserved
catalytic domain, which includes motifs (hydrophobic motif, turn motif) required for
ATP/substrate – binding and catalysis and a regulatory domain, which maintains the
enzyme in an inactive conformation (Figure 2). PKC regulatory domains reside in the
NH
2
terminus of the protein and contain an autoinhibitory pseudosubstrate domain and
two discrete membrane targeting modules, C1 and C2 (Figure 2)(Steinberg, 2008). PKC
isoforms are subdivided into three subfamilies based on differences in their NH
2
terminal
regulatory domain structure: classical PKCs, novel PKCs and atypical PKCs (Figure 2).
The regulatory domains of cPKC isoforms (α, βI, βII, and γ) contain a C1 domain
(consisting of tandem ~ 50 residue long sequences, C1A and C1B, each with cysteines
and 2 histidines that coordinate the 2 Zn
2+
) that serves the role of diacylglycerol binding
motif. Classical PKC (cPKC) also has a C2 domain that binds anionic phospholipids in a
calcium dependent manner (Steinberg, 2008). Novel PKCs (δ, ε, η, and θ) also have a
C1A and C1B domain. However, the C2 domains lack the critical calcium coordinating
acidic residues, which are determinants for calcium binding. Novel PKCs are primarily
activated by agonists that promote DAG accumulation or by PMA without a calcium
requirement (Steinberg, 2008). Atypical PKCs (ζ and λ/ι) do not have a calcium –
sensitive C2 domain. They have a C1 domain that binds PIP
3
or ceramide but not DAG or

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PMA and a protein – protein interaction PB1 domain that mediates interactions with
other PB1 – containing scaffolding proteins (i.e. p62, PAR6) (Steinberg, 2008). Atypical
PKC activity is regulated mainly by protein – protein interactions and phosphorylation by
phosphoinositide dependent kinase-1 (Steinberg, 2008). They are insensitive to Ca
2+
,
diacylglycerol, and phorbol esters (Rosse et al., 2010).

Figure 2. Domain structure of protein kinase C (PKC) isoforms. Top: PKCs has a
conserved kinase domain (teal) and more variable regulatory domains. All PKC
substrates have a pseudosubstrate motif (shown in green) NH
2
terminal to the C1 domain
(shown in pink). Molecular sensors of the phorbol 12-myristate 13-acetate
(PMA)/diacylglycerol (DAG) in cPKC and nPKCs are the tandem C1 domains whereas
the single aPKC C1 domain does not bind to DAG/PMA. nPKCs C2 domain do not bind
calcium; the PKCδ-C2-domain is a phosphotyrosine interaction module. PKC variable
regions are shown in gray. Bottom: ribbon diagrams of PKC C1B domain, C2 domain,
and kinase domain structure. Adapted from Steinberg. (88) 1341-1378. 2008.

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Distribution of PKC isoforms in different organs and tissues is variable (Bareggi
et al., 1995; Wetsel et al., 1992). Five isoforms (α, βII, δ, ε, ζ) have been shown to be
present in the liver (Croquet et al., 1996). Whether PKC activation or inhibition protects
or promotes cell death may depend on the model of injury, cell type and different
isoforms that are involved (Domenicotti et al., 2000; Maher, 2001). In hepatocytes PKC
activation or inhibition have opposite effects against oxidative stress (Jimenez-Lopez et
al., 2005; Saberi et al., 2008). Inhibition of PKC in hepatocyte cell line RALA cells
increases their susceptibility against oxidative stress through sustained JNK activation
(Y. Wang et al., 2004). On the contrary, PKC inhibition ameliorates the ischemia and
reperfusion injury in rat orthotopic liver transplantation model (Jimenez-Lopez et al.,
2005). One of the other known roles of PKC is in energy homeostasis, insulin signaling
and glucose metabolism. Inhibition of atypical PKC has shown to cause activation and
regulation of energy sensor AMP-activated kinase (AMPK) (Xie et al., 2006).
AMPK is another serine-threonine kinase with a heterotrimeric complex
consisting of catalytic α subunit and two regulatory subunits (β and ϒ) and serves as an
important energy sensor in cells responding to AMP: ATP ratio (Towler et al., 2007;
Yang et al., 2010). The α subunit contains the kinase domain in the N-terminal half with
the C-terminal regions being required to form a complex with the β and γ subunits
(Figure 3) (Towler et al., 2007). The β subunit contains 2 conserved regions located in
the central and C-terminal region (Figure 3) (Towler et al., 2007). The C-terminal domain
is sufficient to form a functional αβγ complex that is regulated by AMP whereas the
central region is known to be a glycogen - binding domain. The γ subunit (γ
1
, γ
2
, γ
3
)
contains variable N – terminal regions followed by 4 tandem repeats of 60 amino acid

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sequence called Bateman domains (Figure 3) (Towler et al., 2007). The 2 CBS motifs
also known currently as Bateman domain antagonistically bind ATP with a lower affinity
than AMP. The tandem domains bind AMP with a high cooperativity suggesting that the
second site is inaccessible until AMP was bound to the first (Figure 3) (Towler et al.,
2007). Therefore, this suggests the increase sensitivity of AMPK system to small
changes in AMP. The activity of AMPK can be regulated by upstream kinases, which
include liver kinase β1 (LK β1) and Ca
2+
/calmodulin-dependent protein kinase kinase
where both directly phosphorylate threonine – 172 in the α subunit (Yang et al., 2010).
Phosphorylation at Thr 172 site in the α subunit is essential for AMPK activation (Yang
et al., 2010). This phosphorylation at Thr 172 produces a 100-fold activation (Towler et
al., 2007).

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Figure 3. Domain structure of AMPK subunit isoforms and splice variants. Regions that
are shown in the same color are related and the functions are indicated in the figure.
Adapted from Towler and Hardie. (100) 328-34. 2007.

In hepatocytes in low energy state, intracellular AMP increases and this leads to AMPK
phosphorylation and activation. AMPK activation causes ATP production by switching
off the anabolic processes and turning on the catabolic pathways (Yang et al., 2010).
AMPK marks as one of the master sensors and regulators of nutrient stress in maintaining
intracellular energy homeostasis, which is essential for normal cell function and survival
under physiological and pathological conditions (Figure 4) (S. Wang et al., 2012). AMPK

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not only regulates the energy homeostasis but it also have been shown to have
cytoprotective effects in hepatocytes by inhibition of apoptosis and activation of
autophagy, mitochondrial biogenesis and protection against mitochondrial injury (Choi et
al., 2010; Hoyer-Hansen et al., 2007; Nakada et al., 2010; Peralta et al., 2001; Samari et
al., 1998; Shin et al., 2009). Figure 3 shows AMPK regulation of energy balance at
multiple organs by targeting various downstream substrates. AMPK has been shown to
inactivate ACC (acetyl-CoA carboxylase) resulting in the inhibition of fatty acid
synthesis and promotion of mitochondrial β-oxidation. AMPK can inhibit HMG-CoA
reductase to reduce cholesterol synthesis (Figure 4) (S. Wang et al., 2012). Cellular
NAD
+
levels and Sirt1 activity is increased by AMPK, causing the deacetylation and
activation of PGC1α thus resulting in an increase in mitochondrial gene expression
leading to mitochondrial biogenesis. At the same time, activation of AMPK leads to
increased food intake and increase in glucose transporter 4 expression and glucose
transport in skeletal muscle (Figure 4) (S. Wang et al., 2012). AMPK activation inhibits
mTORC1 by direct phosphorylation of tuberous sclerosis complex 2 thus inhibiting
protein synthesis (Figure 4) (S. Wang et al., 2012).

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Figure 4. AMPK as a master regulator of metabolism. AMPK. AMPK regulates lipid,
protein and glucose metabolism by modulating various downstream targets. Activation of
AMPK promotes fatty acid oxidation and glucose utilization but inhibits protein, fatty
acid, and glycogen synthesis. GS, glycogen synthase; PFK2, phosphofructokinase 2,
HMGR, HMG-Coa reductase; HSL, hormone - sensitive lipase. Adapted from Wang et.
al. (122) 555-573. 2012.

As mentioned previously AMPK activation has been shown to inhibit mTORC1
(Ni et al., 2012). This regulates two major pathways, autophagy and protein synthesis.
Autophagy is the cellular process of ‘self engulfment’ in which the cell breaks down its
own organelles (macroautophagy) and cytosolic components (microautophagy) to ensure
sufficient metabolites when nutrients run low (Mihaylova et al., 2011). The most
upstream components of the autophagy pathway include the serine/threonine kinase Atg1

11

and its associated regulatory subunits Atg13 and Atg17 (Mihaylova et al., 2011). In
mammals, orthologs of the yeast Atg1 (autophagy-related protein 1) have been identified,
ULK1 and ULK2 (Egan et al., 2011). Phosphorylation of ULK1 by AMPK is required
for the ULK1 function in response to nutrient deprivation (Egan et al., 2011). It has been
shown that APAP inhibits mTORC1 and this leads to activation of autophagy. Induction
of autophagy protects against APAP-induced hepatotoxicity by removal of injured
mitochondria (Ni et al., 2012). Similarly, autophagy- related protein 7 (Atg 7) knockout
mice are more susceptible to APAP induced liver injury (Igusa et al., 2012). Conversely,
activation of mTORC1 activates serine threonine kinase, p70S6K that is shown to
activate S6K on 40S subunit of ribosome and increase mRNA translation and protein
synthesis (Berven et al., 2000). Two isoforms of the p70S6K have been identified: a 70
kDa cytoplasmic form and an 85 kDa nuclear form. P70S6K has been shown to
phosphorylate ribosomal S6 protein in vitro (Kawasome et al., 1998). The mechanism of
activation of p70S6K involves a complex series of phosphorylation events on eight serine
and threonine residues (Krause et al., 2002).

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Figure 5. A model of the p70S6K pathway. Adapted from Berven and Crouch (78), 447-
451, 2000.

These phosphorylation sites have been identified as S404, S411, S418, S424 and T421 on
the C-terminal autoinhibitory domain and T229, S371 and T389, which are crucial for
catalytic activity (Berven et al., 2000). Studies have shown that rapamycin and
wortmannin inhibit the phosphorylation of sites T229, T389, S404 and S411 (Figure 5).
The autoinhibitory sites are thought to be phosphorylated by members of the mitogen
activated protein kinase (MAPK) family, p38 and extracellular signal-regulated kinases
(ERK) (Berven et al., 2000). Phosphorylation at the autoinhibitory sites induces a
conformational change that allows phosphorylation at the catalytic sites (Berven et al.,

13

2000). Other signaling proteins such as phosphoinositide dependent kinase -1 (PDK1),
AKT, atypical PKC, cyclin dependent kinase and mammalian target of rapamycin have
been linked to phosphorylation of these sites, although the mechanism is not well
understood (Figure 5) (Berven et al., 2000). Cellular functions of p70S6K are: protein
synthesis/cell growth, cell cycle control and cell migration. During cell growth, the
physiological target of activated p70S6K is the 40S subunit of the ribosomal S6 protein.
Phosphorylation of S6 stimulates translation of 5’ TOP mRNA, a subset of mRNA which
encodes components of the translational machinery such as elongation factors, ribosomal
proteins, poly (A) – binding protein thus modulation efficiency of translation (Berven et
al., 2000). Rapamycin, which inhibits mTOR activity, blocks translation of the 5’ TOP
mRNA as well as the phosphorylation of the 4EBP-1, a protein that binds to the
translational initiation factor, eIF4E (Berven et al., 2000). Studies have demonstrated the
involvement of p70S6K in cell cycle control. Rapamycin inhibits cell proliferation in T
lymphocytes and delays G
1
- S phase transition in fibroblasts. Rapamycin-induced effects
on cell cycle are due to inactivation of p70S6K and were demonstrated by the
microinjection of anti-p70S6K antibodies, which blocked progression of fibroblast S-
phase (Berven et al., 2000). It may be that p70S6K regulates induction of cyclin and cdk
because studies have shown the presence of 5’ TOP sequence in cyclin D1 mRNA
(Berven et al., 2000). Lastly, p70S6K also functions in cell migration. First, p70S6K
colocalizes with actin stress fibers suggesting its role in actin polymerization (Berven et
al., 2000). Second, stimulation of thrombin results in a shape change that is described by
elongation and organization of stress fibers and this effect was blocked by rapamycin
(Berven et al., 2000).

14

1.1 Specific Aims
We hypothesize that PKC and AMPK play a role in Acetaminophen toxicity. In the
present study, the specific aim is to examine the possible role of PKC in modulating
acetaminophen-induced programmed necrosis. We focused on the effects of broad-
spectrum PKC inhibitors and inhibition of PKC-α on AMPK, master regulator of energy
in hepatocytes, and JNK signaling in APAP-induced liver injury.

15

CHAPTER 2
MATERIALS AND METHODS
2.1 Materials—All inhibitors (Ro-31-8425, Go 6983, Go 6976, Compound C,
Leupeptin) and the activator (AMPK activator III, DHPO) used in this study were
purchased from Calbiochem (San Diego, CA). Ammonium chloride was purchased from
Sigma Aldrich (St. Louis, MO). Antisense oligonucleotide (ASO) targeting mouse PKC-
α (Isis pharmaceuticals, Carlsbad, CA) and a chemical control oligonucleotide were
synthesized as 20-nt uniform phosphorothioate chimeric oligonucleotide and purified.
Oligonucleotide was chimeric oligonucleotides containing five nuclease resistant 2´-O-
methoxyethylribose-modified phosphorothioate residues on the 5´ and 3´-ends, flanking a
2´-deoxyribonucleotide/phosphorothioate region that supports RNase H-based cleavage
of the targeted mRNA.
2.2 Animals—Male mice (C57 BL/6; 6-8 weeks of age) were obtained from Harlan
Bioproducts for Science Inc. (Indianapolis, IN). The animals were fasted overnight (no
food, but water was available) prior to experiments. All the treatments were administered
intraperitoneally; APAP (Sigma) was dissolved in warm PBS (55°C) and cooled to 37°C
before injection into mice. For antisense, ASO experiments, the animals were given
50mg/kg in PBS (intraperitoneally, 6 injections total, one every other day, ~0.3ml volume
Cont ASO or PKCα ASO) prior to APAP administration. The last dose of antisense was
given 1 day prior to APAP administration. In experiments with PKC inhibitor (Ro-31-
8425), PKC inhibitor was dissolved in DMSO (8.3%, v/v) in PBS (1mg in 125 µl of
DMSO diluted with 1375 µl of PBS). PKC inhibitor (10 mg/kg) was injected
(intraperitoneally, 0.3 ml volume injected) into mice 1 h prior to APAP injection.

16

Controls received the same dose of DMSO. Blood was obtained after mice were
anesthetized at the indicated time periods and serum alanine aminotransferase (ALT) was
measured at the University of Southern California Pathology Reference Laboratory. All
animals received care under the institutional guidelines for the care and use of laboratory
animals in research. These studies were approved by the Institutional Animal Care and
Utilization Committee.
2.3 Isolation of Liver Mitochondria and Cytoplasm—Mitochondria were isolated from
liver of mice by differential centrifugation. Livers were excised, washed with 0.25 M
sucrose, and homogenized in mitochondria isolation buffer, H-medium (210 mM
mannitol, 70 mM sucrose, 2 mM HEPES, 0.05% bovine serum albumin (w/v), plus
protease and phosphatase inhibitors). The homogenate was centrifuged at 850 x g for 10
min, the pellet removed, and the centrifugation process repeated. The resulting
supernatant was centrifuged at 8,500 x g for 15 min. The supernatant (“cytoplasmic
fraction,” post mitochondrial S9 fraction) was collected and saved at − 80 °C for future
analysis. The pellet, which represents the mitochondria fraction, was washed with
mitochondria isolation buffer, H-medium and the centrifugation repeated at 8,500 x g.
The mitochondria were resuspended in H-medium before oxygen electrode and Western
blot analysis.
2.4 Measurements of respiration in isolated mitochondria— Respiration was
measured in freshly isolated mitochondria by monitoring oxygen consumption with a
Clark-type electrode (Hanstech, UK) in mitochondria respiration buffer containing 230
mM mannitol, 70 mM sucrose, 30 mM Tris-HCl, 5 mM KH
2
PO
4
, 1 ml of respiration
buffer and oxygen consumption was monitored in the presence of mitochondrial

17

substrates (glutamate/malate 7.5 mM – complex I substrates; succinate 7.5 mM –
complex II substrates) with or without ADP (250 mM) (Shinohara et al., 2010).

2.5 Cell culture—Primary mouse hepatocytes were isolated as previously described from
C57BL/6 mice (Saberi et al., 2008).
Briefly, the liver was perfused with collagenase, and
isolated hepatocytes (1.2 x 10
6
, viability >90%) were plated in individual 60-mm-
diameter LUX culture dishes coated with 0.03% rat-tail collagen (Millipore). After 3 h,
the culture medium was changed to serum-free medium containing 100 U/ml penicillin
and 0.1 mg/ml streptomycin. Cell culture experiments were performed in two different
ways with inhibitors being added before or after APAP treatment: Pre-treatment or post-
treatment. In pre-treatment, first hepatocytes were treated with various inhibitors. 1h later
hepatocytes were treated with various doses of acetaminophen in medium in the presence
of inhibitors. After 2 h exposure to APAP, the culture media was changed. In post-
treatment, hepatocytes were first exposed to different doses of APAP in media. After 2h
exposure to APAP, the culture media was changed; then hepatocytes were treated with
various inhibitors.
2.6 Western blot samples—hepatocytes were exposed to various doses of
acetaminophen in medium and were incubated for 2 h. After 2 h exposure to APAP, the
culture media was changed; then hepatocytes were treated with various PKC inhibitors
with or without compound C, an AMPK inhibitor, or AMPK activator III then incubated
for 2h. After 2h incubation with the inhibitors and activator, the cells were scraped using
200 µl per plate of H-Medium with protease and phosphatase inhibitors (lysis buffer).
The cell lysates were saved at − 80 °C for future analysis.
2.7 Compound C experiment—hepatocytes were exposed to various doses of

18

acetaminophen in medium and were incubated for 2h. After 2 h exposure to APAP, the
culture media was changed; then the hepatocytes were treated with PKC inhibitors: Ro-
31-8425 (5 µM) Go 6983 (10 µM) Go 6976 (10 µM) and compound C (40 µM)
(Calbiochem). The hepatocytes were incubated for another 2 h before scraping of the
cells for western blot or incubated overnight, 16 h for determination of apoptosis and
necrosis.
2.8 AMPK activator III, DHPO, experiment—hepatocytes were exposed to various
doses of acetaminophen in medium and were incubated for 2h. After 2 h exposure to
APAP, the culture media was changed; then the hepatocytes were treated with PKC
inhibitors: Ro- 31-8425 (5 µM), Go 6983 (10 µM), Go 6976 (10 µM) and AMPK
activator III, DHPO (100 µM) (Calbiochem). The hepatocytes were incubated for another
2 h before scraping of the cells for western blot or incubated overnight, 16 h for
determination of apoptosis and necrosis.
2.9 Autophagic flux experiment—hepatocytes were exposed to various doses of
acetaminophen in medium and were incubated for 2h. After 2 h exposure to APAP, the
culture media was changed; then the hepatocytes were treated with PKC inhibitors: Ro-
31-8425 (5 µM), Go 6983 (10 µM), Go 6976 (10 µM) and ammonium chloride (20 mM)
and Leupeptin (100 µM). After 2h incubation with the inhibitors, the cells were washed
with PBS and scraped using 200 µl per plate of lysis buffer. The cell lysates were saved
at − 80 °C for future analysis.
2.10 Determination of apoptosis and necrosis—After 16 h of various treatments, cells
were double stained with 8 µg/ml Hoechst 33258 and 1 µM Sytox green. Hepatocytes
were incubated with Hoechst 33258 for 15 min. Sytox green was added just before

19

analysis. After cells have been stained, culture dishes were observed under an
OLYMPUS fluorescent microscope. Quantitation of total and necrotic cells (Sytox green
positive) was performed as previously described by counting > 1,000 cells in 10 different
fields (Saberi et al., 2008).
2.11 Protein Measurement—Dye reagent from Bio-rad was prepared by diluting 1 part
dye reagent concentrate with 4 parts of distilled deionized water. For the protein
standards, quickstart bovine serum albumin standard (Bio-rad) was used and diluted in H-
medium with 0% BSA. The standard concentrations were 0.125mg/ml, 0.25mg/ml,
0.5mg/ml, 0.75mg/ml, 1mg/ml, and 1.5mg/ml. The 2mg/ml bio-rad quickstart BSA was
diluted to the various concentrations mentioned in H-medium. Precisely 800 µl of diluted
dye reagent, 10 µl of protein standards and 10 µl of sample was pipetted in the
microcentrifuge tubes. The protein standards and samples mixed with diluted dye
reagent was pipetted into a 96 well plate. Protein solutions were assayed triplicate in the
96 well plate. Absorbance was measured at 595 nm.
2.12 Western Blot Analysis—Aliquots of cytoplasmic, mitochondria extracts or whole
cell lysates were fractionated by electrophoresis on 7.5, 10 or ANY KD% SDS-
polyacrylamide gel (Bio-Rad). Subsequently, the proteins were transferred to PVDF
membrane, and blots were blocked with 5% (w/v) non-fat milk or 5% (w/v) BSA
dissolved in TBS-T (TBS with 0.1% Tween 20). 5% (w/v) BSA dissolved in TBST was
used to block membranes that were going to be incubated with phosphorylated antibodies
because the casein in milk may interfere with binding or increase background. The blots
were then incubated with the desired primary and secondary antibodies. Finally, the
proteins were detected by luminol ECL reagent (Thermo Scientific, Hudson, NH).

20

AMPK-α, p-AMPK-α (thr172), JNK, p-JNK (p46 and p54), p-PKC substrate, GSK-3β, p-
GSK-3β, p-p70S6K (ser371), phospho-p70S6K (thr389), phospho-S6 ribosomal protein,
p70S6K, LC3A/B, LC3B, SQSTMI/P62, prohibitin, and β-actin were obtained from Cell
Signaling Technologies (Danvers, MD). PKC-α and PKC-ε antibodies were obtained
Santa Cruz (Santa Cruz, CA). Antiserum to NAPQI protein adducts provided by Dr. Jack
A. Hinson of University of Arkansas. All gels shown were representative samples from
three experiments. Densitometry was performed using the Image J program from the
NIH.
2.13 Histological Analysis—Livers were removed, fixed with 10% buffered formalin,
embedded in paraffin, and cut into 5-µm thick sections. All specimens were stained with
hematoxylin/eosin and evaluated under a light microscope.
2.14 HPLC measurements for GSH and GSSG—GSH and GSSG were detected using
reverse-phase HPLC and a Coulochem II electrochemical detector (ESA Laboratories,
Chelmsford, MA) as previously described. At collection time points, hepatocytes were
washed with cold PBS and then treated with 5% metaphosphoric acid to prevent GSH
autoxidation. Samples were centrifuged (12,000 g for 5 min), and the supernatant was
injected into the HPLC.
2.15 Statistical Analysis—Statistical analyses were performed using Paired Student’s t
test p < 0.05 was defined as statistically significant.

21

CHAPTER 3
RESULTS
3.1 PKC plays an important role in APAP-induced necrosis in primary cultured
hepatocytes. Pre-treatment of primary cultured hepatocytes with two different broad-
spectrum PKC inhibitors (Ro-31-8425, Go6983) and a classical PKC inhibitor (Go6976)
significantly protected against APAP toxicity (Figure 6). To further insure that PKC
inhibitors protect against APAP hepatotoxicity without affecting APAP metabolism, PKC
inhibitors were added 2 hours after APAP treatment at the time of removal of APAP
(post-treatment). Similar to pre-treatment experiments, post-treatment of PKC inhibitors
significantly protected against APAP induced hepatocyte necrosis (Figure 7). As
expected, post-treatment with PKC inhibitors did not affect covalent binding of NAPQI
(Figure 8). PKC activation (PKC substrate protein phosphorylation) was observed
following APAP treatment, which increased in a dose dependent manner and was
inhibited by PKC inhibitor treatment (post-treatment; Figure 9). These findings suggest
that PKC(s) is activated by APAP treatment in hepatocytes and plays an important role in
mediating necrotic cell death induced by APAP.

22

Figure 6. Pre-treatment of PKC inhibitors protects primary cultured hepatocytes against
APAP hepatotoxicity. Hepatocytes were pre-treated 1 hour with PKC inhibitors - Ro-31-
8425 (5µM; ▲), Go6983 (10µM; ●) or classical PKC inhibitor - Go6976 (10µM; ■), or
DMSO (control, ◇) prior to addition of different doses of APAP. Necrotic cells were
determined using Sytox green 16-24 h after APAP treatment. Results are mean ± S.D. *
p value ≤ 0.05 versus APAP treatment alone.

Figure 7. Post-treatment of PKC inhibitors protect primary cultured hepatocytes against
APAP hepatotoxicity. Hepatocytes were treated with APAP for 2 hours. After 2hours,
APAP was removed, media was changed and hepatocytes were treated with different
PKC inhibitors - Ro-31-8425 (5µM; ▲), Go6983 (10µM; ●) or classical PKC inhibitor -
Go6976 (10µM; ■) or DMSO (control, ◇). Necrotic cells were determined using Sytox
green 16-24 h after APAP treatment. Results are mean ± S.D. * p value ≤ 0.05 versus
APAP treatment alone.

23

Figure 8. Effect of PKC inhibitors on covalent binding. Primary mouse hepatocytes were
treated with APAP 25mM. After 2 hours, APAP was removed and cells were treated
with different PKC inhibitors [Ro-31-8425 (5 µM), Go 6983 (10 µM), Go 6976 (10 µM)].
2 hours post PKC inhibitor treatment; hepatocytes were washed and scraped with lysis
buffer. Western blot analysis was performed with antisera against NAPQI protein adducts
in whole cell lysates to measure covalent binding to liver proteins.

Figure 9. APAP treatment to hepatocytes increases PKC activity (proteins

24

phosphorylated by PKC). Hepatocytes were treated with various doses of APAP with or
without Ro-31-8425, post-treatment. PKC activity was activity was assessed in whole
cell lysates by western blotting using an antibody that recognizes proteins phosphorylated
by PKC (PKC recognition motif: serine residues surrounded by arginine or lysine at the
−2 and +2 positions of hydrophobic residue at +1 position). (n = three experiments)

3.2 PKC inhibitors protect against APAP hepatotoxicity through JNK dependent
and independent pathways. We next examined if the protection of PKC inhibitors
against APAP in cultured hepatocytes was mediated through an effect on JNK. As
expected, APAP treatment in cultured hepatocytes activated JNK, starting around 2 hours
(Figure 10). Treatment of hepatocytes with broad-spectrum PKC inhibitors, Go6983 and
Ro-31-8425 (post-treatment), did not alter the enhanced p-JNK levels induced by APAP
treatment (Figure 11). In contrast, the classical PKC inhibitor, Go 6976, decreased p-JNK
levels under basal conditions and in the presence of APAP. This suggests broad-spectrum
PKC inhibitors protected against APAP through a JNK-independent pathway, while
classical PKC inhibitor protected through a JNK-dependent pathway. Densitometry using
image J from NIH showed enhanced p-JNK levels with broad-spectrum PKC inhibitors
(Ro-31-8425 and Go 6983) and decreased p-JNK levels with classical PKC inhibitor, Go
6976 which was consistent with the western blots (Figure 12).

25

Figure 10. Time course of JNK phosphorylation subsequent to APAP treatment in
hepatocytes. Western blot analysis was performed using antisera against p-JNK, JNK and
β-actin.

Figure 11. Effect of broad-spectrum PKC inhibitors – Ro-31-8425 (5 µM) and Go 6983
(10 µM) or classical PKC inhibitor – Go 6976 (10 µM) on JNK phosphorylation in
hepatocytes after treatment of APAP 25mM. The hepatocytes were incubated with APAP
in media for 2 hours and then another 2 h after change of media and addition of inhibitors
prior to scraping of the cells. Western blot analysis was performed using antisera against
p-JNK, JNK and β-actin.

26

Figure 12. Densitometry of p-JNK in primary cultured hepatocytes. Densitometry was
determined using NIH Image J. Normalized to actin. (n = three experiments)

3.3 Broad-spectrum PKC inhibitors modulate p-AMPK to protect hepatocytes from
APAP hepatotoxicity. APAP treatment caused a decline in p-AMPK levels, which was
due in part to an overall decline in AMPK levels in cultured hepatocytes (Figure 13).
Broad-spectrum PKC inhibitors increased the basal levels of p-AMPK and increased p-
AMPK levels in the presence of APAP, even though total AMPK levels still remained
suppressed (Figure 14). In contrast, classical PKC inhibitor Go6976 decreased the basal
levels of p-AMPK compared to control and did not affect the decline in p-AMPK and
AMPK caused by APAP treatment. This difference between the effect of broad-spectrum
and classical PKC inhibitors suggests that APAP treatment activates novel and/or
atypical PKC(s) that have an inhibitory effect on p-AMPK, which is blocked by broad-
spectrum PKC inhibitors, but not classical PKC inhibitor. Densitometry using image J

27

from NIH showed enhanced p-AMPK levels with broad-spectrum PKC inhibitors (Ro-
31-8425 and Go 6983) and decreased p-AMPK levels with classical PKC inhibitor, Go
6976 which was consistent with the western blots (Figure 15).

Figure 13. Time course of p-AMPK following APAP treatments in hepatocytes. Western
blot analysis was performed using antisera against p-AMPK, AMPK (thr 172) and β-
actin.

Figure 14. Effect of broad-spectrum PKC inhibitors – Ro-31-8425 (5 µM), Go 6983 (10
µM) or classical PKC inhibitor – Go 6976 (10 µM) on p-AMPK levels in hepatocytes
after treatment with APAP 25mM. Hepatocytes were incubated with APAP in media for
2 hours and then another 2 h after change of media and addition of inhibitors prior to
scraping of the cells. Western blot analysis was used using antisera against p-AMPK (Thr
172), AMPK and β-actin.

28

Figure 15. Densitometry of p-AMPK in primary cultured hepatocytes. Densitometry was
done using NIH Image J. Normalized to actin. (n = three experiments)

To confirm the importance of p-AMPK in APAP hepatotoxicity, we modulated
AMPK activity using inhibitors and activators. The AMPK inhibitor, compound C (Cpd
C), prevented p-AMPK upregulation induced by the broad-spectrum PKC inhibitor
Go6983 and reversed its protective effect against APAP hepatotoxicity (Figure 16 and
17). Cpd C, however, did not reverse the protective effect of PKC inhibitor Go6976
against APAP hepatotoxicity. This data supports the interpretation that broad-spectrum
PKC inhibitors protect through an AMPK-dependent but JNK-independent pathway,
while the classical PKC inhibitor Go6976 protects through an AMPK-independent but
JNK-dependent pathway. To further confirm that activation of AMPK is important in
APAP hepatotoxicity, hepatocytes were treated with an AMPK activator (AMPK
activator III, Calbiochem). AMPK activator significantly protected against APAP toxicity

29

in primary cultured hepatocytes in the post-treatment protocol (Figure 18).

Figure 16. p-AMPK plays a key role in APAP hepatotoxicity in primary mouse
hepatocytes. Compound C; an AMPK inhibitor, abrogates the protective effects of broad-
spectrum PKC inhibitor Go 6983 but not the classical PKC inhibitor Go 6976 against
APAP hepatotoxicity. Hepatocytes were treated with PKC inhibitor Go 6983 or Go 6976
(2 hour post APAP treatment) in the presence or absence of compound C (Cpd C; 10
µM).
#P ≤ 0.05 compared to control (PKC inhibitors with no APAP). *P ≤ 0.05 compared
to Go 6983 with APAP (without compound C). (n = three experiments)

Figure 17. Effect of compound C on p-AMPK levels in cultured hepatocytes after
treatment with APAP 20mM. Western blot analysis was used using antisera against p-
AMPK (Thr 172), AMPK and β-actin.

30

Figure 18. AMPK activator treatment protects hepatocytes from APAP hepatotoxicity.
After 2 hours of APAP treatments, APAP was removed and hepatocytes were treated
with AMPK activator III, DHPO, (50 µM) or DMSO for controls. Western blot (insets)
were performed on hepatocytes treated with DMSO or AMPK activator III for 2 hours to
confirm activation of AMPK by the small molecule inhibitor. DMSO alone had no effect
on APAP toxicity when given 2 hours post APAP (data not shown). *P ≤ 0.05 compared
to APAP. (n = three experiments)

We next examined possible downstream targets of p-AMPK that may be involved
in protecting hepatocytes against APAP. p-AMPK is known to activate autophagy
through inhibition of mTORC1, which has been suggested to be protective against APAP.
mTORC1 also increases mRNA translation and protein synthesis by activation of
p70S6K, a serine threonine kinase that is shown to activate S6K on the 40S subunit of
ribosome. Although APAP treatment appears to trigger autophagy as shown by the
dramatic decline in p62 levels, there was no increase in LC3II levels even when
lysosomal function was inhibited thus no increase in autophagic flux (Figure 19 and 20).

31

Our findings regarding the effect of APAP on p62 are similar to a previous study but our
LC3II results are different. However our experiments involved short-term culture after
plating for 3 hours while the previous study used hepatocytes rested overnight. PKC
inhibitors did not seem to affect autophagy markers p62 and LC3II, suggesting autophagy
was not affected by PKC inhibitor treatment (Figure 21). However, autophagy is difficult
to accurately measure and more experiments are needed to definitively assert the effects
of broad-spectrum PKC inhibitors and classical PKC inhibitor on autophagy.
Surprisingly, broad-spectrum and classical PKC inhibitors prevented a decline in p-
p70S6K levels (active form) caused by APAP treatment. This could be a contributing
additional factor in protection. Total p70S6K levels declined with APAP treatment with
or without PKC inhibitor treatment. Therefore one possible mechanism by which the
PKC inhibitors may protect against APAP toxicity is by increasing p70S6K activity,
which increase protein synthesis that is normally downregulated by APAP treatment.

Figure 19. Effect of lysosomal protease inhibitor, NH
4
Cl/Leupeptin on LC3-II levels after
APAP treatment. Western blot analysis was performed using antisera against LC3B, p62,
β-actin.

32

Figure 20. Effect of PKC inhibitors on LC3-II levels after APAP treatment with or
without lysosomal protease inhibitor. Western blot analysis was performed using antisera
against LC3B, p62, β-actin.

Figure 21. Effect of PKC inhibitors on effectors of protein translation (p70S6K) and
autophagy (p62, LC3-II) following APAP treatments. Hepatocytes were treated with
APAP 25mM for 2 hours, then media was changed and PKC inhibitors were added and
the hepatocytes were incubated for another 2 hours subsequent to scraping of the cells.
Western blot analysis was performed using antisera against LC3B, p62, p-p70S6K,
p70S6K and β-actin.

3.4 PKC inhibitor Ro-31-8425 protects against APAP induced liver injury in vivo.
We next determined if PKC inhibitors could protect against APAP in vivo. Mice were

33

pretreated with PKC inhibitor Ro-31-8425 (10mg/kg dissolved in DMSO and PBS) 1 h
prior to treatment with various doses of APAP. Ro-31-8425 treatment significantly
protected against an APAP dose of 500mg/kg, but not at higher doses of APAP, as
observed with JNK inhibitor treatment (Figure 22). Centrilobular necrosis caused by
APAP was markedly reduced with Ro-31-8425 pre-treatment (Figure 23). Ro-31-8425
pre-treatment protected despite extensive GSH depletion and JNK phosphorylation and
translocation to mitochondria remained elevated, even possibly enhanced (Figure 24, 25).
Also, as seen in vitro, Ro-31-8425 treatment enhanced p-AMPK levels, despite some
decline in total AMPK levels (Figure 26). P-p70S6K and its downstream target p-S6
ribosomal protein (active form), which are associated with enhanced translation of
mRNA transcripts and protein synthesis, were decreased with APAP treatment, which
was prevented by Ro-31-8425 treatment. This suggests that APAP treatment activates a
PKC isoform(s) that downregulates protein translation. Other PKC inhibitors, Go6983
and Go6976, could not be dissolved without increasing levels of DMSO, which affects
APAP hepatotoxicity and thus were not tested in vivo. AMPK activators also suffered
from solubility issues (AMPK activator III) or did not maintain p-AMPK levels in the
presence of APAP (metformin, AICAR; data not shown). Thus although enhanced
AMPK phosphorylation correlates with protective effects of Ro-31-8425 in vivo, we
could not fully explore p-AMPK effects on APAP hepatotoxicity in vivo as we were able
to do in cultured hepatocytes due to technical issues.

34

Figure 22. Broad-spectrum PKC inhibitor protects against APAP-induced liver injury in
vivo through a JNK-independent pathway. A. Serum ALT (control = no fill, Ro-31-8425
= black, JNK inhibitor = gray). Mice were pre-treated with Ro-31-8425 (10mg/kg in
DMSO (8.3%) and PBS), or JNK inhibitor (SP600125; 10mg/kg in DMSO (8.3%)) or
PBS with equivalent amounts of DMSO for control, 1 h prior to APAP treatment. Results
are mean S.D. *p value ≤ 0.05 versus APAP treatment alone. N=4-10 mice per group. (n
= three experiments)

Figure 23. Broad-spectrum PKC inhibitor markedly reduced centrilobular necrosis caused
by APAP in vivo. H&E histology of mice treated with APAP 500 mg/kg with or without
Ro-31-8425.

35

Figure 24. Effect of Ro-31-8425 treatment on GSH levels in the liver and isolated
mitochondria subsequent to APAP treatment (2 hours).

Figure 25. Effect of Ro-31-8425 treatment on JNK activation and translocation to
mitochondria following APAP treatment in vivo. Western blot analysis was performed
using antisera p-JNK, JNK, β-actin and prohibitin (PHB, mitochondria loading control).

36

Figure 26. Effect of Ro-31-8425 on p-AMPK, p70S6K, and S6 activation levels in the
liver following APAP treatment in vivo. Western blot analysis was performed using
antisera p-p70S6K, p-AMPK, p-S6, AMPK, β-actin and prohibitin (PHB, mitochondria
loading control).

3.5 Silencing PKC-α using antisense protects against APAP-induced liver injury
through a JNK-dependent pathway in vivo. We next silenced PKC-α expression using
antisense (ASO) to determine if PKC-α was responsible for the JNK-dependent
protection observed with classical PKC inhibitor Go6976 treatment in cultured
hepatocytes. Silencing PKC-α using ASO protected against APAP-induced liver injury in
mice, as seen by significant decline in ALT levels and decreased centrilobular necrosis
(Figure 27 and 28). Silencing PKC-α did not affect the rate of GSH depletion caused by
APAP (data not shown). As observed with Go 6976 treatment in vitro, silencing PKC-α
significantly inhibited JNK phosphorylation and translocation to mitochondria (Figure
29). Densitometry using image J from NIH showed decreased p-JNK levels after
silencing PKC-α, which is consistent with the western blots (Figure 30). This suggests
that PKC-α plays a key role in activating and/or sustaining JNK during APAP
hepatotoxicity. This presents an apparent contradiction with the observation that Ro-31-

37

8425, a strong PKC-α inhibitor, did not inhibit JNK activation caused by APAP (Figure
25). However the broad-spectrum PKC inhibitors mediated by atypical or unconventional
PKCs may be inhibiting pathways while at the same time activating AMPK survival
mechanisms either downstream of JNK or completely separate that downregulate p-JNK,
resulting in enhanced p-JNK levels.

Figure 27. Silencing PKC-α protects against APAP-induced liver injury through a JNK
dependent pathway in vivo. Mice were treated with PKC-α or control ASO for 2 weeks,
and then treated with APAP (300 mg/kg). Serum ALT levels. Increase in ALT represents
liver damage. Western blot analysis (insets) was performed on liver samples from mice
treated to confirm the silencing of PKC-α. N=5 mice per group. * p value ≤ 0.05 versus
control ASO. (n = three experiments)

38

Figure 28. Silencing PKC-α markedly reduced centrilobular necrosis caused by APAP in
vivo. H&E histology of mice treated with cont ASO and PKC-α ASO.

Figure 29. JNK activation and translocation to mitochondria in vivo. Western blot
analysis was performed using antisera p-JNK, JNK, β-actin and prohibitin (PHB,
mitochondria loading control).

39

Figure 30. Densitometry of p-JNK in PKC-α silenced mice in vivo. Densitometry was
determined using NIH Image J. Normalized to actin. (n = three experiments)

We previously observed that silencing PKC-α also affects PKC-ε levels, possible
due to regulation of PKC-ε by PKC-α. To confirm that the protective effects of PKC-α
ASO against APAP was due to PKC-α and not PKC-ε, APAP hepatotoxicity was tested
in PKC-ε knock out mice. PKC-ε knock out mice were not protected against APAP-
induced liver injury (Figure 31), suggesting PKC-ε is not involved in APAP
hepatotoxicity.

40

Figure 31. PKC-ε knock out mice are not protected from APAP-induced liver injury.
Mice were treated with acetaminophen (300 mg/kg; ip). Mice were sacrificed at 24 hours,
and ALT levels were measured.

3.6 PKC-α translocates to mitochondria and inhibits mitochondrial respiration
during APAP-induced liver injury. PKC-α, like JNK, has been reported to translocate
to mitochondria to decrease respiration and enhance ROS generation. We observed that
APAP treatment causes PKC-α translocation to mitochondria, which was associated with
increased levels of mitochondrial proteins phosphorylated by PKC (Figure 32). The
silencing of PKC-α with ASO decreased the level of mitochondrial proteins

41

phosphorylated by PKC. APAP-induced mitochondrial dysfunction was also reduced in
PKC-α silenced mice (Figure 33), suggesting that PKC-α translocation and
phosphorylation may play a role in mitochondrial dysfunction during APAP
hepatotoxicity. The fact that silencing PKC-α reduced JNK activation and translocation
to mitochondria suggested that JNK might be downstream of PKC-α. To confirm the
sequence of signaling events, we silenced JNK using ASO and examined its effect on
PKC-α translocation to mitochondria. Surprisingly, silencing JNK 1 and 2 reduced PKC-
α translocation to mitochondria and reduced the level of mitochondrial proteins
phosphorylated by PKC (Figure 34). This suggests that PKC-α and JNK participate
jointly through a feed-forward mechanism to mediate APAP-induced liver injury.

42

Figure 32. PKC-α translocates to the mitochondria and phosphorylates mitochondrial
proteins during APAP hepatotoxicity. PKC-α ASO or control ASO mice were treated
with APAP (300 mg/kg). At various times liver mitochondria were isolated using
differential centrifugation.

43

Figure 33. PKC-α translocation to the mitochondria parallels a decline in mitochondria
respiration (Empty bar = control ASO; black bars = PKC-α ASO). Mitochondrial
respiration was measured in the presence of succinate and ADP (State III respiration).

44

Figure 34. Silencing JNK decreases PKC-α translocation to mitochondria and
phosphorylation of mitochondrial proteins. Mitochondria were isolated from JNK ASO
or control ASO treated mice at various times following APAP treatment. * p value ≤ 0.05
versus control ASO. N = 4.

45

Figure 35. Role of PKC, AMPK, and JNK in APAP hepatotoxicity.

46

CHAPTER 4
DISCUSSION
4.1 PKC plays an important role in APAP hepatotoxicity. APAP hepatotoxicity is
mediated by a signaling transduction pathway involving JNK activation and translocation
to mitochondria. In this study, by using wide variety of PKC inhibitors and by silencing
PKC-α, we observed that PKC family members also play an important role in APAP
hepatotoxicity. The mechanism of protection by PKC inhibitors differed markedly, with
broad-spectrum PKC inhibitors (Go 6983 or Ro-31-8425) protecting through JNK-
independent, AMPK-dependent pathway, and classical PKC inhibitor (Go 6976) or
silencing PKC-α protected through a JNK-dependent, AMPK-independent pathway
(Figure 35).
4.2 Broad-spectrum PKC inhibitors protect against APAP hepatotoxicity through
an AMPK-dependent, JNK-independent pathway. Our findings with broad-spectrum
PKC inhibitors suggest that APAP activates various PKC isoforms that modulate p-
AMPK. However like all inhibitors, broad-spectrum PKC inhibitors could have off-target
effects and other proteins besides PKC might be inhibited. Regardless of whether it is a
PKC isoform or a protein similar to PKC, our work with PKC inhibitors uncovered the
importance of p-AMPK in modulating APAP hepatotoxicity. The protective of effects of
broad-spectrum PKC inhibitors against APAP hepatotoxicity, at least in cultured
hepatocytes was due to sustained p-AMPK levels in hepatocytes. p-AMPK has been
shown to regulate energy homeostasis and many survival pathways including autophagy,
mitochondrial biogenesis, and inhibition of apoptosis (Hoyer-Hansen et al., 2007; Nakada
et al., 2010; Peralta et al., 2001; Shin et al., 2009). APAP treatment decreased the level of

47

p-AMPK in hepatocytes and in the liver. The loss of p-AMPK may therefore be an
important contributing mechanism in mediating APAP hepatotoxicity. In APAP, despite
inhibition of AMPK, mTOR was inhibited (decreased p-p70S6K). However the broad-
spectrum PKC inhibitors increased AMPK but restored p-p70S6K suggesting mTOR
independent activation of this kinase. We also show for the first time that protection
against APAP hepatotoxicity can occur despite sustained JNK activation (Figure 35).
Many signaling pathways have been shown to modulate APAP hepatotoxcity, but they do
so by modulating JNK signaling. Thus it was surprising that we observed Ro-31-8425
protected against APAP hepatotoxicity both in cultured hepatocytes and in vivo despite
enhanced JNK phosphorylation and translocation to mitochondria. Thus although, APAP
hepatotoxicity requires activation of JNK-mediated cell death pathways, AMPK may
concomitantly contribute by suppressing or counteracting. Thus, broad-spectrum PKC
inhibitors upregulate AMPK, which promotes survival in parallel with JNK death
pathways, resetting the threshold for cell death. At present we do not know precisely
which of the many AMPK targets are critical in counteracting APAP toxicity.
Based on work with the broad-spectrum PKC inhibitor, our data suggest that APAP
treatment activates a PKC isoform that inhibits p-AMPK, p-p70S6K, and S6. Since
classical PKC inhibitor did not affect AMPK and PKCε knockout mice were not
protected, PKCδ and other PKCs are the leading candidate. One study has shown that
PKC-ζ regulates AMPK phosphorylation through an LKβ1 dependent pathway in
endothelial cells. PKC-ζ has also been shown to directly regulate p70S6K activity in
cells. Whether PKC-ζ is involved in AMPK and p70Sk6 regulation, as well as APAP
hepatotoxicity will require further investigation. It is also possible that APAP activates a

48

PKC isoform that activates proteosome or protease pathways that degrade p-AMPK,
AMPK and other signaling proteins or activates a phosphatase that dephosphorylates p-
AMPK and other proteins. As previously mentioned PKC inhibitors may have non-
specific targets and the pathways outlined above may not definitively involve PKC.
Further studies will be needed to determine which PKC isoforms or other proteins
inhibited by PKC inhibitors regulate p-AMPK and p-p70S6K levels during APAP
hepatotoxicity.
4.3 Inhibition of PKC-α protects against APAP hepatotoxicity through a JNK
dependent pathway. Classical PKC inhibitor Go6976 protected against APAP, through
an AMPK-independent (not inhibited by compound C) and JNK-dependent pathway. We
found that PKC-α plays an important role in activating and/or sustaining JNK to mediate
APAP hepatotoxicity. APAP treatment caused PKC-α translocation to mitochondria and
concomitantly increased PKC-dependent phosphorylation of mitochondrial proteins,
which was accompanied by mitochondrial dysfunction. Other previous studies have also
suggested that PKC-α translocates to mitochondria to inhibit mitochondrial respiration.
Silencing of PKC-α expression reduced mitochondrial dysfunction caused by APAP
suggesting that PKC-α was involved in disrupting mitochondrial respiration (Gutcher et
al., 2003). However, whether mitochondria dysfunction was due directly to PKC-α or due
to decreased JNK translocation to mitochondria, which we observed when PKC-α was
silenced, will require further investigation. It may be that both JNK and PKC-α
translocation to mitochondria is needed for maximal inhibition of mitochondrial
respiration and consequent increased ROS generation important in sustaining JNK
activity during APAP hepatotoxicity. A study in a lung cancer cell line has also shown

49

that PKC-α activates JNK, which was inhibited by Go 6976 treatment through receptor
for activated C kinase 1 (RACK1), which serves as an adaptor for PKC-JNK interaction
(Lang et al., 2004; Lopez-Bergami et al., 2005). Further studies in hepatocytes are needed
to determine if RACK1 or mitochondrial ROS are key factors in PKC-α regulation of
JNK in the liver.

50

CHAPTER 5
FUTURE DIRECTIONS
We now understand that inhibiting protein kinase C protects against Acetaminophen
hepatotoxicity. We determined the protection of the broad-spectrum inhibitors were
through an AMPK dependent, JNK independent pathway. Broad-spectrum inhibitors
protected against acetaminophen hepatotoxicity despite sustained JNK activation. As
mentioned above, at this present time, the specific targets of AMPK crucial for
counteracting APAP toxicity are not known. It may be beneficial in the future do
proteomics to determine protein-protein interactions so as to determine potential partners
in the cell signaling cascade. In addition, further studies are needed to determine which
of the five PKC isoforms found in the liver or other proteins inhibited by PKC inhibitors
regulate p-AMPK and p-p70S6K levels during APAP hepatotoxicity. Studies have shown
that PKC-α translocates to the mitochondria and inhibit mitochondria respiration.
Conversely, PKC-α knockdown mice prevented mitochondria dysfunction and JNK
translocation to mitochondria was decreased. Therefore, further investigation is required
to determine whether mitochondria dysfunction was due directly to PKC-α or due to
decreased JNK translocation to mitochondria. Lastly, a study from lung cancer cell line
showed that PKC-α activated JNK, which was inhibited by the classical PKC inhibitor,
Go 6976 through RACK1. In primary mouse hepatocytes treated with APAP, we
determined that PKC-α activates JNK and Go 6976 inhibited JNK as well as
mitochondrial ROS. Further investigation is needed in the future to determine whether
RACK1 or mitochondrial ROS are key factors in PKC-α regulation of JNK in the liver.
Lastly, knockdown of PKC ζ should be considered in the future to determine its role in

51

the regulation of signaling pathways involved in APAP-induced liver injury because
recent studies on have shown that PKC ζ is upstream of p70S6K.

52

CHAPTER 6
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Abstract (if available)

Abstract Our previous studies have shown that acetaminophen (APAP)-induced hepatocyte necrosis is mediated by JNK. In the present study we show that protein kinase C (PKC) plays an important role in APAP-induced liver injury through JNK-dependent and independent pathways. Treatment of primary mouse hepatocytes with two different broad-spectrum PKC inhibitors (Ro-31-8245, Go6983), protected against APAP hepatotoxicity without inhibiting JNK activation. Ro-31-8245 treatment to mice also resulted in upregulation of p-AMPK in the liver and protection against APAP-induced liver injury in vivo, despite sustained JNK activation. APAP treatment caused a decreased p-AMPK, which was prevented by broad-spectrum PKC inhibitors. AMPK inhibition by compound C or activation using AMPK activator oppositely modulated APAP hepatotoxicity. This suggests PKC-dependent downregulation of AMPK-regulated survival pathways is an important component of APAP hepatotoxicity. In contrast to broad-spectrum inhibitors, treatment of hepatocytes with a more specific classical PKC inhibitor (Go6976) that inhibits mainly PKC-α and PKC-βI protected against APAP by inhibiting JNK activation. Knockdown of PKC-α using antisense (ASO) in mice protected against APAP-induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC-α translocation to mitochondria, phosphorylation of mitochondrial proteins, and decline in mitochondria respiration in the liver. JNK 1 and 2 silencing using ASO in mice decreased APAP-induced PKC-α translocation to mitochondria, suggesting PKC-α and JNK act together through a feed forward mechanism to mediate APAP-induced liver injury. Conclusion: PKC-α and other PKC(s) regulate death (JNK) and survival (AMPK), to modulate APAP-induced liver injury.

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Asset Metadata

Creator Ybanez, Maria Cecilia D. (author)

Core Title Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through JNK dependent and independent pathways

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 10/30/2013

Defense Date 03/14/2013

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

Tag acetaminophen (APAP),AMPK,Necrosis,OAI-PMH Harvest,p70S6K

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Advisor Tokes, Zoltan A. (committee chair), Kalra, Vijay K. (committee member), Kaplowitz, Neil (committee member)

Creator Email ybanez@usc.edu,ybanez0103@gmail.com

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