The present study examined the roles of peroxisome proliferator-activated receptors (PPAR) in activation of hepatic stellate cells (HSC), a pivotal event in liver fibrogenesis. RNase protection assay detected mRNA for PPAR␥1 but not that for the adipocyte-specific ␥2 isoform in HSC isolated from sham-operated rats, whereas the transcripts for neither isoforms were detectable in HSC from cholestatic liver fibrosis induced by bile duct ligation (BDL). Semi-quantitative reverse transcriptasepolymerase chain reaction confirmed a 70% reduction in PPAR␥ mRNA level in HSC from BDL. Nuclear extracts from BDL cells showed an expected diminution of binding to PPAR-responsive element, whereas NF-B and AP-1 binding were increased. Treatment of culturedactivated HSC with ligands for PPAR␥ (10 M 15-deoxy-⌬ 12,14 -PGJ 2 (15dPGJ 2 ); 0.1ϳ10 M BRL49653) inhibited DNA and collagen synthesis without affecting the cell viability. Suppression of HSC collagen by 15dPGJ 2 was abrogated 70% by the concomitant treatment with a PPAR␥ antagonist (GW9662). HSC DNA and collagen synthesis were inhibited by WY14643 at the concentrations known to activate both PPAR␣ and ␥ (>100 M) but not at those that only activate PPAR␣ (<10 M) or by a synthetic PPAR␣-selective agonist (GW9578). 15dPGJ 2 reduced ␣1(I) procollagen, smooth muscle ␣-actin, and monocyte chemotactic protein-1 mRNA levels while inducing matrix metalloproteinase-3 and CD36. 15dPGJ 2 and BRL49653 inhibited ␣1(I) procollagen promoter activity. Tumor necrosis factor ␣ (10 ng/ml) reduced PPAR␥ mRNA, and this effect was prevented by the treatment with 15dPGJ 2 . These results demonstrate that HSC activation is associated with the reductions in PPAR␥ expression and PPAR-responsive element binding in vivo and is reversed by the treatment with PPAR␥ ligands in vitro. These findings implicate diminished PPAR␥ signaling in molecular mechanisms underlying activation of HSC in liver fibrogenesis and the potential therapeutic value of PPAR␥ ligands for liver fibrosis.

The unfolded protein response (UPR) is an evolutionarily conserved mechanism that activates both proapoptotic and survival pathways to allow eukaryotic cells to adapt to endoplasmic reticulum (ER) stress. Although the UPR has been implicated in tumorigenesis, its precise role in endogenous cancer remains unclear. A major UPR protective response is the induction of the ER chaperone GRP78/BiP, which is expressed at high levels in a variety of tumors and confers drug resistance in both proliferating and dormant cancer cells. To determine the physiologic role of GRP78 in in situ-generated tumor and the consequence of its suppression on normal organs, we used a genetic model of breast cancer in the Grp78 heterozygous mice where GRP78 expression level was reduced by about half, mimicking anti-GRP78 agents that achieve partial suppression of GRP78 expression. Here, we report that Grp78 heterozygosity has no effect on organ development or antibody production but prolongs the latency period and significantly impedes tumor growth. Our results reveal three major mechanisms mediated by GRP78 for cancer progression: enhancement of tumor cell proliferation, protection against apoptosis, and promotion of tumor angiogenesis. Importantly, although partial reduction of GRP78 in the Grp78 heterozygous mice substantially reduces the tumor microvessel density, it has no effect on vasculature of normal organs. Our findings establish that a key UPR target GRP78 is preferably required for pathophysiologic conditions, such as tumor proliferation, survival, and angiogenesis, underscoring its potential value as a novel therapeutic target for dual antitumor and antiangiogenesis activity. [Cancer Res 2008; 68(2):498-505]

Hepatic stellate cells (HSC) undergo transdifferentiation (activation) from lipid-storing pericytes to myofibroblastic cells to participate in liver fibrogenesis. Our recent work demonstrates that depletion of peroxisome proliferator-activated receptor ␥ (PPAR␥) constitutes one of the key molecular events for HSC activation and that ectopic expression of this nuclear receptor achieves the phenotypic reversal of activated HSC to the quiescent cells. The present study extends these findings to test a novel hypothesis that adipogenic transcriptional regulation is required for the maintenance of HSC quiescence. Transdifferentiation of vitamin A-storing hepatic stellate cells (HSC)1 to vitamin A-depleted myofibroblastic cells represents a key cellular event in the genesis of cirrhosis, for which no effective medial treatments are currently available except for liver transplantation. Transdifferentiated (activated) HSC are proliferative, proinflammatory, and fibrogenic with induced ability to synthesize and deposit extracellular matrices (1). Thus, better understanding of the mechanism underlying HSC transdifferentiation is the pivotal step toward identification of molecular targets for new and effective treatments for the disease. The most fundamental prerequisite for the understanding of HSC transdifferentiation is defining the cell type of differentiated HSC. This question relates to the origin of HSC that continues to puzzle the field. HSC are believed to serve as pericytes for hepatic capillaries called sinusoids. They represent 5-8% of total liver cells and 15-23% of nonparenchymal cells in the normal liver (2). HSC are positive for a mesenchymal marker such as vimentin. Rodent HSC express desmin (3) and glial fibrillary acidic protein (4), suggesting smooth muscle cell and glial cell lineage, respectively. Upon activation, both rodent and human HSC lose vitamin A and begin to express ␣-smooth muscle actin (5, 6). Interestingly, undifferentiated HSC in fetal livers that do not yet exhibit vitamin A storage also express ␣-smooth muscle actin (7), supporting a smooth muscle cell lineage. Synaptophysin, which controls exocytosis and the release of neurotransmitters in neurons and neuroendocrine cells, is also expressed in both rodent and human HSC (8). Neurotrophins such as nerve growth factor, brain-derived neurotrophic factor (BDNF), neutrophin NT-3, and NT-4/5 are also expressed (9), and so are their receptors, Trk-A, B, and C (9, 10), further supporting the neural and glial lineage.Peroxisome proliferator-activated receptor ␥ (PPAR␥) has been proposed as a potential molecular target for inhibition of HSC transdifferentiation (11-13). PPAR␥ level and activity are reduced in activated HSC, and the treatment of HSC with synthetic ligands for PPAR␥ such as thiazolidinediones effectively suppresses fibrogenic activity of and in vivo in experimental animals (13). However, these ligands are known to have PPAR␥-independent effects (14), and it was yet to be tested whether PPAR␥ per se had a direct effect to suppress ...

Depletion of peroxisome proliferator-activated receptor ␥ (PPAR␥) accompanies myofibroblastic transdifferentiation of hepatic stellate cells (HSC), the primary cellular event underlying liver fibrogenesis. The treatment of activated HSC in vitro or in vivo with synthetic PPAR␥ ligands suppresses the fibrogenic activity of HSC. However, it is uncertain whether PPAR␥ is indeed a molecular target of this effect, because the ligands are also known to have receptor-independent actions. To test this question, the present study examined the effects of forced expression of PPAR␥ via an adenoviral vector on morphologic and biochemical features of culture-activated HSC. The vector-mediated expression of PPAR␥ itself is sufficient to reverse the morphology of activated HSC to the quiescent phenotype with retracted cytoplasm, prominent dendritic processes, reduced stress fibers, and accumulation of retinyl palmitate. These effects are abrogated by concomitant expression of a dominant negative mutant of PPAR␥ that prevents transactivation of but not binding to the PPAR response element. PPAR␥ expression also inhibits the activation markers such as the expression of ␣-smooth muscle actin, type I collagen, and transforming growth factor ␤1; DNA synthesis; and JunD binding to the activator protein-1 (AP-1) site and AP-1 promoter activity. Inhibited JunD activity by PPAR␥ is not due to reduced JunD expression or JNK activity or to a competition for p300. But it is due to a JunD-PPAR␥ interaction as demonstrated by co-immunoprecipitation and glutathione S-transferase pull-down analysis. Further, the use of deletion constructs reveals that the DNA binding region of PPAR␥ is the JunD interaction domain. In summary, our results demonstrate that the restoration of PPAR␥ reverses the activated HSC to the quiescent phenotype and suppresses AP-1 activity via a physical interaction between PPAR␥ and JunD.