Vishakha Bhave

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;)

Excessive hepatomegaly of mice with hepatocyte‐targeted elimination of integrin linked kinase following treatment with 1,4‐bis [2‐(3,5‐dichaloropyridyloxy)] benzene

TCBOPOP (1,4‐bis [2‐(3,5‐dichaloropyridyloxy)] benzene) an agonist of the constitutive androstane receptor (CAR), produces rapid hepatocyte hyperplasia and hepatomegaly in the absence of hepatic injury. In this study we demonstrate that integrin‐linked kinase (ILK), which is involved in transmission of the extracellular matrix (ECM) signaling by way of integrin receptors, plays an important role in regulating TCPOBOP‐induced proliferation of hepatocytes and hepatomegaly. Hepatocyte‐specific ILK knockout mice (ILK/liver−/− mice) and wildtype mice (WT) were given a single dose of TCPOBOP (3 mg/kg) by oral gavage. Mice were sacrificed at days 1, 2, 5, and 7 after TCPOBOP administration. WT mice showed maximum proliferation on days 1 and 2, which came back to baseline levels by days 5 and 7 after TCPOBOP administration. The ILK/liver−/− mice, on the other hand, showed a prolonged and a sustained proliferative response as evident by an increased number of proliferative cell nuclear antigen assay (PCNA)‐positive cells even at days 5 and 7 after TCPOBOP administration. At day 7 the WT mice showed close to a 2.5‐fold increase in liver weight, whereas the ILK/liver−/− mice showed a 3.7‐fold increase in liver weight. The prolonged proliferative response in the ILK/liver−/− mice seems to be due to sustained induction of CAR leading to sustained induction of c‐Myc, which is known to be a key mediator of TCPOPOP‐CAR induced direct liver hyperplasia. Conclusion: The data indicate that ECM‐mediated signaling by way of ILK is essential for adjustment of final liver size and proper termination of TCPOBOP‐induced proliferation of hepatocytes. (HEPATOLOGY 2011;53:587‐595)

Liver-Specific Ablation of Integrin-Linked Kinase in Mice Results in Enhanced and Prolonged Cell Proliferation and Hepatomegaly after Phenobarbital Administration

We have recently demonstrated that disruption of extracellular matrix (ECM)/integrin signaling via elimination of integrin-linked kinase (ILK) in hepatocytes interferes with signals leading to termination of liver regeneration. This study investigates the role of ILK in liver enlargement induced by phenobarbital (PB). Wild-type (WT) and ILK:liver-/- mice were given PB (0.1% in drinking water) for 10 days. Livers were harvested on 2, 5, and 10 days during PB administration. In the hepatocyte-specific ILK/liver-/- mice, the liver:body weight ratio was more than double as compared to 0 h at day 2 (2.5 times), while at days 5 and 10, it was enlarged three times. In the WT mice, the increase was as expected from previous literature (1.8 times) and seems to have leveled off after day 2. There were slightly increased proliferating cell nuclear antigen-positive cells in the ILK/liver-/- animals at day 2 as compared to WT after PB administration. In the WT animals, the proliferative response had come back to normal by days 5 and 10. Hepatocytes of the ILK/liver-/- mice continued to proliferate up until day 10. ILK/liver-/- mice also showed increased expression of key genes involved in hepatocyte proliferation at different time points during PB administration. In summary, ECM proteins communicate with the signaling machinery of dividing cells via ILK to regulate hepatocyte proliferation and termination of the proliferative response. Lack of ILK in the hepatocytes imparts prolonged proliferative response not only to stimuli related to liver regeneration but also to xenobiotic chemical mitogens, such as PB.

Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Previous studies from our laboratory have found glypican 3 (GPC3) as a negative regulator of growth. CD81 was found to be a binding partner for GPC3, and its expression and co-localization with GPC3 increased at the end of hepatocyte proliferation. However, the mechanisms through which these two molecules might regulate liver regeneration are not known. We tested the hypothesis that GPC3 down-regulates the hedgehog (HH) signaling pathway by competing with patched-1 for HH binding. We found decreased GPC3-Indian HH binding at peak proliferation in mice followed by increase in glioblastoma 1 protein (effector of HH signaling). We performed a yeast two-hybrid assay and identified hematopoietically expressed homeobox (Hhex, a known transcriptional repressor) as a binding partner for CD81. We tested the hypothesis that Hhex binding to CD81 keeps it outside the nucleus. However, when GPC3 binds to CD81, CD81-Hhex binding decreases, resulting in nuclear translocation of Hhex and transcriptional repression. In support of this, we found decreased GPC3-CD81 binding at hepatocyte proliferation peak, increased CD81-Hhex binding, and decreased nuclear Hhex. GPC3 transgenic mice were used as an additional tool to test our hypothesis. Overall, our data suggest that GPC3 down-regulates cell proliferation by binding to HH and down-regulating the HH signaling pathway and binding with CD81, thus making it unavailable to bind to Hhex and causing its nuclear translocation.

Role of PINCH and Its Partner Tumor Suppressor Rsu-1 in Regulating Liver Size and Tumorigenesis

Particularly interesting new cysteine-histidine-rich protein (PINCH) protein is part of the ternary complex known as the IPP (integrin linked kinase (ILK)-PINCH-Parvin-α) complex. PINCH itself binds to ILK and to another protein known as Rsu-1 (Ras suppressor 1). We generated PINCH 1 and PINCH 2 Double knockout mice (referred as PINCH DKO mice). PINCH2 elimination was systemic whereas PINCH1 elimination was targeted to hepatocytes. The genetically modified mice were born normal. The mice were sacrificed at different ages after birth. Soon after birth, they developed abnormal hepatic histology characterized by disorderly hepatic plates, increased proliferation of hepatocytes and biliary cells and increased deposition of extracellular matrix. After a sustained and prolonged proliferation of all epithelial components, proliferation subsided and final liver weight by the end of 30 weeks in livers with PINCH DKO deficient hepatocytes was 40% larger than the control mice. The livers of the PINCH DKO mice were also very stiff due to increased ECM deposition throughout the liver, with no observed nodularity. Mice developed liver cancer by one year. These mice regenerated normally when subjected to 70% partial hepatectomy and did not show any termination defect. Ras suppressor 1 (Rsu-1) protein, the binding partner of PINCH is frequently deleted in human liver cancers. Rsu-1 expression is dramatically decreased in PINCH DKO mouse livers. Increased expression of Rsu-1 suppressed cell proliferation and migration in HCC cell lines. These changes were brought about not by affecting activation of Ras (as its name suggests) but by suppression of Ras downstream signaling via RhoGTPase proteins. In conclusion, our studies suggest that removal of PINCH results in enlargement of liver and tumorigenesis. Decreased levels of Rsu-1, a partner for PINCH and a protein often deleted in human liver cancer, may play an important role in the development of the observed phenotype.

Heat Shock Proteins and Phytochemicals: Role in Human Health and Disease

The use of plants for therapeutic purpose has a long history in many continents. However, the potential of phytochemicals, the bioactive components in the plant, in disease prevention and treatment is recognized and supported only recently by the scientific community. A lot of human diseases and health-related issues are related to the disruption of redox homeostasis by various stressors encountered constantly throughout human life. As a consequence, it leads to accumulation of abnormal proteins, inflammatory response, and oxidative stress, which are common denominators shared by many degenerative disorders. Heat shock proteins are stress proteins known to provide cytoprotection and play important roles in protein folding/unfolding. This chapter briefly describes recent findings in the effects of phytochemicals on oxidative stress-involved ischemia/reperfusion injury, obesity, and liver diseases through regulation of heat shock proteins. These phytochemicals include caffeic acid phenethyl ester from bee glue, synthetic oleanane triterpenoid CDDO-Im, curcumin from Indian spice turmeric, resveratrol from red grapes, naringin found in grapefruit, epigallocatechin-3-gallate from green tea, anthocyanins from pomegranate, and flavonoids.

Hepatocyte to Biliary Transdifferentiation: To Be(come) or Not to Be(come)?

Abstract Transdifferentiation or metaplasia exhibited by major cell types is yet another characteristic that adds to the uniqueness of liver as an organ. A discussion of the various types of transdifferentiation is presented, with special emphasis on hepatocyte to biliary transdifferentiation (HBT). Aspects such as conditions under which transdifferentiation occurs, the extent of its contribution in repair and recovery of the organ, the various animal models available to study this phenomenon, and the mechanisms and drivers behind the process are discussed. Finally, the impact of HBT in human liver disease and its potential usefulness in therapy are discussed.Transdifferentiation or metaplasia exhibited by major cell types is yet another characteristic that adds to the uniqueness of liver as an organ. A discussion of the various types of transdifferentiation is presented, with special emphasis on hepatocyte to biliary transdifferentiation (HBT). Aspects such as conditions under which transdifferentiation occurs, the extent of its contribution in repair and recovery of the organ, the various animal models available to study this phenomenon, and the mechanisms and drivers behind the process are discussed. Finally, the impact of HBT in human liver disease and its potential usefulness in therapy are discussed.