Shirish Paranjpe

Cell Cycle Effects Resulting from Inhibition of Hepatocyte Growth Factor and Its Receptor c-Met in Regenerating Rat Livers by RNA Interference

Hepatocyte growth factor (HGF) and its receptor c‐Met are involved in liver regeneration. The role of HGF and c‐Met in liver regeneration in rat following two‐thirds partial hepatectomy (PHx) was investigated using RNA interference to silence HGF and c‐Met in separate experiments. A mixture of 2 c‐Met‐specific short hairpin RNA (ShRNA) sequences, ShM1 and ShM2, and 3 HGF‐specific ShRNA, ShH1, ShH3, and ShH4, were complexed with linear polyethylenimine. Rats were injected with the ShRNA/PEI complex 24 hours before and at the time of PHx. A mismatch and a scrambled ShRNA served as negative controls. ShRNA treatment resulted in suppression of c‐Met and HGF mRNA and protein compared with that in controls. The regenerative response was assessed by PCNA, mitotic index, and BrdU labeling. Treatment with the ShHGF mixture resulted in moderate suppression of hepatocyte proliferation. Immunohistochemical analysis revealed severe suppression of incorporation of BrdU and complete absence of mitosis in rats treated with ShMet 24 hours after PHx compared with that in controls. Gene array analyses indicated abnormal expression patterns in many cell‐cycle‐ and apoptosis‐related genes. The active form of caspase 3 was seen to increase in ShMet‐treated rats. The TUNEL assay indicated a slight increase in apoptosis in ShMet‐treated rats compared with that in controls. Conclusion: The data indicated that in vivo silencing of c‐Met and HGF mRNA by RNA interference in normal rats results in suppression of mRNA and protein, which had a measurable effect on proliferation kinetics associated with liver regeneration. (HEPATOLOGY 2007.)

RNA Interference Against Hepatic Epidermal Growth Factor Receptor Has Suppressive Effects on Liver Regeneration in Rats

Liver regeneration after a two-thirds partial hepatectomy (PHx) is a complex process requiring interaction and cooperation of many growth factors and cytokines and cross talk between multiple pathways. Along with hepatocyte growth factor and its receptor MET (HGF-MET), the epidermal growth factor receptor (EGFR) signaling pathway is activated within 60 minutes after PHx. To investigate the role of EGFR in liver regeneration, we used two EGFR-specific short hairpin silencing RNAs to inhibit EGFR expression in regenerating normal rat liver. Suppression of EGFR mRNA and protein was evident in treated rats. There was also a demonstrable decrease but not complete elimination of bromo-deoxyuridine incorporation and mitoses at 24 hours after PHx. In addition, we observed up-regulation of MET and Src as well as activation of the ErbB-3-ErbB-2-PI3K-Akt pathway and down-regulation of STAT 3, cyclin D1, cyclin E1, p21, and C/EBP beta. The decrease in the ratio of C/EBP alpha to C/EBP beta known to occur after PHx was offset in shEGFR-treated rats. Despite suppression of hepatocyte proliferation lasting into day 3 after PHx, liver weight restoration occurred. Interestingly, hepatocytes in shEGFR-treated rats were considerably larger when compared with ScrRNA-treated controls. The data indicate that although the MET and EGFR pathways are similar, the contributions made by MET and EGFR are unique and are not compensated by each other or other cytokines.

Suppression of liver regeneration and hepatocyte proliferation in hepatocyte‐targeted glypican 3 transgenic mice

Glypican 3 (GPC3) belongs to a family of glycosylphosphatidylinositol‐anchored, cell‐surface heparan sulfate proteoglycans. GPC3 is overexpressed in hepatocellular carcinoma. Loss‐of‐function mutations of GPC3 result in Simpson‐Golabi‐Behmel syndrome, an X‐linked disorder characterized by overgrowth of multiple organs, including the liver. Our previous study showed that GPC3 plays a negative regulatory role in hepatocyte proliferation, and this effect may involve CD81, a cell membrane tetraspanin. To further investigate GPC3 in vivo, we engineered transgenic (TG) mice overexpressing GPC3 in the liver under the control of the albumin promoter. GPC3 TG mice with hepatocyte‐targeted, overexpressed GPC3 developed normally in comparison with their nontransgenic littermates but had a suppressed rate of hepatocyte proliferation and liver regeneration after partial hepatectomy. Moreover, gene array analysis revealed a series of changes in the gene expression profiles in TG mice (both in normal mice and during liver regeneration). In unoperated GPC3 TG mice, there was overexpression of runt related transcription factor 3 (7.6‐fold), CCAAT/enhancer binding protein alpha (2.5‐fold), GABA A receptor (2.9‐fold), and wingless‐related MMTV integration site 7B (2.8‐fold). There was down‐regulation of insulin‐like growth factor binding protein 1 (8.4‐fold), Rab2 (5.6‐fold), beta‐catenin (1.7‐fold), transforming growth factor beta type I (3.1‐fold), nodal (1.8‐fold), and yes‐associated protein (1.4‐fold). Changes after hepatectomy included decreased expression in several cell cycle–related genes. Conclusion: Our results indicate that in GPC3 TG mice, hepatocyte overexpression of GPC3 suppresses hepatocyte proliferation and liver regeneration and alters gene expression profiles, and potential cell cycle–related proteins and multiple other pathways are involved and affected. (HEPATOLOGY 2010;52:1060–1067)

Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Glypicans are heparan sulfate proteoglycans that are bound to the cell surface by glycosylphosphatidylinositol. While six members of the glypican family are known in mammals, our study focused on glypican 3 (GPC3). Loss-of-function mutations of GPC3 result in the Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by pre- and postnatal liver and other organ overgrowth. GPC3 is overexpressed in human hepatocellular carcinoma; however, its role in normal liver regeneration and hepatocyte proliferation is unknown. Here we investigated the role of GPC3 in hepatocyte proliferation. GPC3 mRNA and protein levels begin to increase 2 days after hepatectomy with peak expression levels by day 5. In hepatocyte cultures, GPC3 reaches a plateau when hepatocyte proliferation decreases. In vitro studies using Morpholino oligonucleotides showed that blocking GPC3 expression promoted hepatocyte growth. Yeast two-hybrid assays revealed that GPC3 interacts with CD81, a member of the tetraspanin family that is reported to be involved in hepatitis C virus infection and cell proliferation. We found that CD81 levels also increased 2 days after partial hepatectomy and toward the end of regeneration. Immunofluorescence showed that CD81 and GPC3 colocalize by 2 and 6 days after hepatectomy. Co-immunoprecipitation validated the interaction of GPC3 and CD81. Our results indicate that GPC3 may be a negative regulator of liver regeneration and hepatocyte proliferation, and that this regulation may involve CD81.

Synthesis of IL-6 by hepatocytes is a normal response to common hepatic stimuli

Exogenous interleukin 6 (IL-6), synthesized at the initiation of the acute phase response, is considered responsible for signaling hepatocytes to produce acute phase proteins. It is widely posited that IL-6 is either delivered to the liver in an endocrine fashion from immune cells at the site of injury, or alternatively, in a paracrine manner by hepatic immune cells within the liver. A recent publication showed there was a muted IL-6 response in lipopolysaccharide (LPS)-injured mice when nuclear NFκB was specifically inactivated in the hepatocytes. This indicates hepatocellular signaling is also involved in regulating the acute phase production of IL-6. Herein, we present extensive in vitro and in vivo evidence that normal hepatocytes are directly induced to synthesize IL-6 mRNAs and protein by challenge with LPS, a bacterial hepatotoxin, and by HGF, an important regulator of hepatic homeostasis. As the IL-6 receptor is found on the hepatocyte, these results reveal that induction of the acute phase response can be regulated in an autocrine as well as endocrine/paracrine fashion. Further, herein we provide data indicating that following partial hepatectomy (PHx), HGF differentially regulates IL-6 production in hepatocytes (induces) versus immune cells (suppresses), signifying disparate regulation of the cell sources involved in IL-6 production is a biologically relevant mechanism that has previously been overlooked. These findings have wide ranging ramifications regarding how we currently interpret a variety of in vivo and in vitro biological models involving elements of IL-6 signaling and the hepatic acute phase response.

Genes inducing iPS phenotype play a role in hepatocyte survival and proliferation in vitro and liver regeneration in vivo

Reprogramming factors have been used to induce pluripotent stem cells as an alternative to somatic cell nuclear transfer technology in studies targeting disease models and regenerative medicine. The neuronal repressor RE‐1 silencing transcription factor (REST) maintains self‐renewal and pluripotency in mouse embryonic stem cells by maintaining the expression of Oct3/4, Nanog, and cMyc. We report that primary hepatocytes express REST and most of the reprogramming factors in culture. Their expression is up‐regulated by hepatocyte growth factor (HGF) and epidermal growth factor (EGF). REST inhibition results in down‐regulation of reprogramming factor expression, increased apoptosis, decreased proliferation, and cell death. The reprogramming factors are also up‐regulated after 70% partial hepatectomy in vivo. Conclusion: These findings show that genes inducing the iPS phenotype, even though expressed at lower levels than embryonic stem cells, nonetheless are associated with control of apoptosis and cell proliferation in hepatocytes in culture and may play a role in such processes during liver regeneration. (HEPATOLOGY 2011)

Hepatocyte proliferation and hepatomegaly induced by phenobarbital and 1,4‐bis [2‐(3,5‐dichloropyridyloxy)] benzene is suppressed in hepatocyte‐targeted glypican 3 transgenic mice

Glypican 3 (GPC3) is a family of glycosylphosphatidylinositol‐anchored, cell‐surface heparan sulfate proteoglycans. Loss‐of‐function mutations of GPC3 cause Simpson‐Golabi‐Behmel syndrome characterized by overgrowth of multiple organs, including liver. Our previous study showed that in GPC3 transgenic (TG) mice, hepatocyte‐targeted overexpression of GPC3 suppresses hepatocyte proliferation and liver regeneration after partial hepatectomy and alters gene expression profiles and potential cell cycle‐related proteins. This study investigates the role of GPC3 in hepatocyte proliferation and hepatomegaly induced by the xenobiotic mitogens phenobarbital (PB) and TCPOBOP (1, 4‐bis [2‐(3, 5‐dichloropyridyloxy)] benzene). Wildtype (WT) and GPC3 TG mice were given 0.1% PB in drinking water for 10 days or a single dose of TCPOBOP (3 mg/kg) by oral gavage. At day 5 the WT mice showed a 2.2‐ and 3.0‐fold increase in liver weight, whereas the GPC3 TG mice showed a 1.3‐ and 1.6‐fold increase in liver weight after PB and TCPOBOP administration, respectively. There was a significant suppression of proliferative response in the GPC3 TG mice, as assessed by percent of Ki67‐positive hepatocyte nuclei. Moreover, gene array analysis showed a panel of changes in the gene expression profile of TG mice, both before and after administration of the xenobiotic mitogens. Expression of cell cycle‐related genes in the TG mice was also decreased compared to the WT mice. Conclusion: Our results indicate that in GPC3 TG mice, hepatocyte‐targeted overexpression of GPC3 plays an important role for regulation of liver size and termination of hepatocyte proliferation induced by the xenobiotic mitogens PB and TCPOBOP, comparable to the effects seen in the GPC3 TG mice during liver regeneration after partial hepatectomy. (HEPATOLOGY 2011;)

Combined systemic elimination of MET and epidermal growth factor receptor signaling completely abolishes liver regeneration and leads to liver decompensation

Receptor tyrosine kinases MET and epidermal growth factor receptor (EGFR) are critically involved in initiation of liver regeneration. Other cytokines and signaling molecules also participate in the early part of the process. Regeneration employs effective redundancy schemes to compensate for the missing signals. Elimination of any single extracellular signaling pathway only delays but does not abolish the process. Our present study, however, shows that combined systemic elimination of MET and EGFR signaling (MET knockout + EGFR‐inhibited mice) abolishes liver regeneration, prevents restoration of liver mass, and leads to liver decompensation. MET knockout or simply EGFR‐inhibited mice had distinct and signaling‐specific alterations in Ser/Thr phosphorylation of mammalian target of rapamycin, AKT, extracellular signal–regulated kinases 1/2, phosphatase and tensin homolog, adenosine monophosphate–activated protein kinase α, etc. In the combined MET and EGFR signaling elimination of MET knockout + EGFR‐inhibited mice, however, alterations dependent on either MET or EGFR combined to create shutdown of many programs vital to hepatocytes. These included decrease in expression of enzymes related to fatty acid metabolism, urea cycle, cell replication, and mitochondrial functions and increase in expression of glycolysis enzymes. There was, however, increased expression of genes of plasma proteins. Hepatocyte average volume decreased to 35% of control, with a proportional decrease in the dimensions of the hepatic lobules. Mice died at 15‐18 days after hepatectomy with ascites, increased plasma ammonia, and very small livers. Conclusion: MET and EGFR separately control many nonoverlapping signaling endpoints, allowing for compensation when only one of the signals is blocked, though the combined elimination of the signals is not tolerated; the results provide critical new information on interactive MET and EGFR signaling and the contribution of their combined absence to regeneration arrest and liver decompensation. (Hepatology 2016;64:1711‐1724)

Leukocyte‐Specific Protein 1: A Novel Regulator of Hepatocellular Proliferation and Migration Deleted in Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most commonly diagnosed form of liver cancer with high morbidity and mortality. Copy number variation (CNV) analysis of human HCC revealed that leukocyte‐specific protein 1 (LSP1) had the highest number of cases with CNV. LSP1, a F‐actin‐binding protein, is expressed in hematopoietic cells and interacts with kinase suppressor of Ras (KSR), a scaffold for the extracellular signal‐related kinase/mitogen‐activated protein kinase pathway. Expression of LSP1 in liver, and its role in normal hepatocellular function and carcinogenesis, remains unknown. Therefore, LSP1 messenger RNA and protein levels were analyzed in normal hepatocytes in culture, rat liver following partial hepatectomy (PHx), and hepatoma cell lines. In culture and after PHx, LSP1 increased after the termination of hepatocyte proliferation. To investigate LSP1 function in HCC, short hairpin RNA was utilized to stably knock down LSP1 expression in the JM1 rat hepatoma cell line. Loss of LSP1 in JM1 cells resulted in dramatic up‐regulation of cyclin D1 and phosphorylated ERK2, increased cell proliferation, and migration. Coimmunoprecipitation and immunofluoresence analysis displayed an interaction and colocalization between LSP1, KSR, and F‐actin in JM1 cells and liver during regeneration. Conversely, expression of LSP1 in the JM2 rat hepatoma cell line led to decreased proliferation. Enhanced expression of LSP1 in mouse hepatocytes during liver regeneration after injection of an LSP1 expression plasmid also led to decreased hepatocyte proliferation. Conclusion: LSP1 is expressed in normal hepatocytes and liver after PHx after termination of proliferation. In rat hepatoma cell lines and mouse liver in vivo, LSP1 functions as a negative regulator of proliferation and migration. Given the high frequency of LSP1 CNV in human HCC, LSP1 may be a novel target for diagnosis and treatment of HCC. (Hepatology 2015;61:537‐547)

TCPOBOP-induced hepatomegaly & hepatocyte proliferation is attenuated by combined disruption of MET & EGFR signaling.

TCPOBOP (1,4‐Bis [2‐(3,5‐Dichloropyridyloxy)] benzene) is a constitutive androstane receptor (CAR) agonist that induces robust hepatocyte proliferation and hepatomegaly without any liver injury or tissue loss. TCPOBOP‐induced direct hyperplasia has been considered to be CAR‐dependent with no evidence of involvement of cytokines or growth factor signaling. Receptor tyrosine kinases (RTKs), MET and epidermal growth factor receptor (EGFR), are known to play a critical role in liver regeneration after partial hepatectomy, but their role in TCPOBOP‐induced direct hyperplasia, not yet explored, is investigated in the current study. Disruption of the RTK‐mediated signaling was achieved using MET knockout (KO) mice along with Canertinib treatment for EGFR inhibition. Combined elimination of MET and EGFR signaling [MET KO + EGFR inhibitor (EGFRi)], but not individual disruption, dramatically reduced TCPOBOP‐induced hepatomegaly and hepatocyte proliferation. TCPOBOP‐driven CAR activation was not altered in [MET KO + EGFRi] mice, as measured by nuclear CAR translocation and analysis of typical CAR target genes. However, TCPOBOP‐induced cell cycle activation was impaired in [MET KO + EGFRi] mice due to defective induction of cyclins, which regulate cell cycle initiation and progression. TCPOBOP‐driven induction of FOXM1, a key transcriptional regulator of cell cycle progression during TCPOBOP‐mediated hepatocyte proliferation, was greatly attenuated in [MET KO + EGFRi] mice. Interestingly, TCPOBOP treatment caused transient decline in hepatocyte nuclear factor 4 alpha expression concomitant to proliferative response; this was not seen in [MET KO + EGFRi] mice. Transcriptomic profiling revealed the vast majority (~40%) of TCPOBOP‐dependent genes primarily related to proliferative response, but not to drug metabolism, were differentially expressed in [MET KO + EGFRi] mice. Conclusion: Taken together, combined disruption of EGFR and MET signaling lead to dramatic impairment of TCPOBOP‐induced proliferative response without altering CAR activation.