Shigang Xiong

Peroxisome Proliferator-activated Receptors and Hepatic Stellate Cell Activation

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 PPARγ1 but not that for the adipocyte-specific γ2 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 transcriptase-polymerase chain reaction confirmed a 70% reduction in PPARγ mRNA level in HSC from BDL. Nuclear extracts from BDL cells showed an expected diminution of binding to PPAR-responsive element, whereas NF-κB and AP-1 binding were increased. Treatment of cultured-activated HSC with ligands for PPARγ (10 μm15-deoxy-Δ12,14-PGJ2 (15dPGJ2); 0.1∼10 μm BRL49653) inhibited DNA and collagen synthesis without affecting the cell viability. Suppression of HSC collagen by 15dPGJ2 was abrogated 70% by the concomitant treatment with a PPARγ antagonist (GW9662). HSC DNA and collagen synthesis were inhibited by WY14643 at the concentrations known to activate both PPARα and γ (>100 μm) but not at those that only activate PPARα (<10 μm) or by a synthetic PPARα-selective agonist (GW9578). 15dPGJ2 reduced α1(I) procollagen, smooth muscle α-actin, and monocyte chemotactic protein-1 mRNA levels while inducing matrix metalloproteinase-3 and CD36. 15dPGJ2 and BRL49653 inhibited α1(I) procollagen promoter activity. Tumor necrosis factor α (10 ng/ml) reduced PPARγ mRNA, and this effect was prevented by the treatment with 15dPGJ2. These results demonstrate that HSC activation is associated with the reductions in PPARγ expression and PPAR-responsive element binding in vivo and is reversed by the treatment with PPARγ ligands in vitro. These findings implicate diminished PPARγ signaling in molecular mechanisms underlying activation of HSC in liver fibrogenesis and the potential therapeutic value of PPARγ ligands for liver fibrosis.

Critical Role of the Stress Chaperone GRP78/BiP in Tumor Proliferation, Survival, and Tumor Angiogenesis in Transgene-Induced Mammary Tumor Development

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.

Steatohepatitis induced by intragastric overfeeding in mice

Nonalcoholic steatohepatitis is prevalent among obese individuals with excessive caloric intake, insulin resistance, and type II diabetes. However, no animal models exist that recapitulate this important association. This study produced and characterized steatohepatitis (SH) caused by intragastric overfeeding in mice. C57BL/6, tumor necrosis factor (TNF) type I receptor–deficient, and genetically matched wild type mice were fed via an implanted gastrostomy tube a high‐fat diet for 9 weeks in the increasing amount up to 85% in excess of the standard intake. Animals were examined for weight gain, insulin sensitivity, and histology and biochemistry of liver and white adipose tissue (WAT). Overfed C57BL/6 mice progressively became obese, with 71% larger final body weights. They had increased visceral WAT, hyperglycemia, hyperinsulinemia, hyperleptinemia, glucose intolerance, and insulin resistance. Of these mice, 46% developed SH with increased plasma alanine aminotransferase (121 ± 27 vs. 13 ± 1 U/L), neutrophilic infiltration, and sinusoidal and pericellular fibrosis. Obese WAT showed increased TNFα and leptin expression and reciprocally reduced adiponectin expression. The expression of lipogenic transcription factors (SREBP‐1c, PPARγ, LXRα) was increased, whereas that of a lipolytic nuclear factor PPARα was reduced in SH. SH was associated with reduced cytochrome P450 (Cyp)2e1 but increased Cyp4a. TNF type I receptor deficiency did not prevent obesity and SH. In conclusion, forced overfeeding with a high‐fat diet in mice induces obesity, insulin resistance, and SH in the absence of TNF signaling or Cyp2e1 induction. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;42:905–914.)

Signaling Role of Intracellular Iron in NF-κB Activation

Iron chelators inhibit endotoxin-induced NF-κB activation in hepatic macrophages (HMs), suggesting a role for the intracellular chelatable pool of iron in NF-κB activation. The present study tested this hypothesis. Analysis of Fe59-loaded HMs stimulated with lipopolysaccharide (LPS), revealed a previously unreported, transient rise in intracellular low molecular weight (LMW)·Fe59complex ([LMW·Fe] i ) at ≤2 min returning to the basal level within 15 min. The [LMW·Fe] i response preceded IκB kinase (IKK) (≥15 min) and NF-κB (≥30 min) activation. Iron chelators (1,2-dimethyl-3-hydroxypyridin-4-one andN,N′-bis-2-hydroxybenzylethylenediamine-N,N′-diacetic acid) abrogated the [LMW·Fe] i response and IKK and NF-κB activation. The [LMW·Fe] i response was also observed in tumor necrosis factor α (TNFα)-stimulated HMs and RAW264.7 cells treated with LPS and interferon-γ but not in primary rat hepatocytes or myofibroblastic cells exposed to LPS or TNFα. Both [LMW·Fe] i response and IKK activation in LPS-stimulated HMs were inhibited by diphenylene iodonium (nonspecific inhibitor for flavin-containing oxidases),l-N 6-(1-iminoethyl)lysine (selective iNOS inhibitor), and adenoviral-mediated expression of a dominant negative mutant of Rac1 or Cu,Zn-superoxide dismutase, suggesting the role of ⋅NO and O 2 ⨪ in mediating the iron signaling. In fact, this inhibition was recapitulated by a cell-permeable scavenger of ONOO−, 5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinato iron (III) chloride. Conversely, ONOO− alone induced both [LMW·Fe] i response and IKK activation. Finally, direct addition of ferrous iron to cultured HMs activated IKK and NF-κB. These results support a novel signaling role for [LMW·Fe] i in IKK activation, which appears to be induced by ONOO−and selectively operative in macrophages.

A Critical Role for GRP78/BiP in the Tumor Microenvironment for Neovascularization during Tumor Growth and Metastasis

Glucose-regulated protein 78 (GRP78)/BiP is a multifunctional protein which plays a major role in endoplasmic reticulum (ER) protein processing, protein quality control, maintaining ER homeostasis, and controlling cell signaling and viability. Previously, using a transgene-induced mammary tumor model, we showed that Grp78 heterozygosity impeded cancer growth through suppression of tumor cell proliferation and promotion of apoptosis and the Grp78(+/-) mice exhibited dramatic reduction (70%) in the microvessel density (MVD) of the endogenous mammary tumors, while having no effect on the MVD of normal organs. This observation suggests that GRP78 may critically regulate the function of the host vasculature within the tumor microenvironment. In this article, we interrogated the role of GRP78 in the tumor microenvironment. In mouse tumor models in which wild-type (WT), syngeneic mammary tumor cells were injected into the host, we showed that Grp78(+/-) mice suppressed tumor growth and angiogenesis during the early phase but not during the late phase of tumor growth. Growth of metastatic lesions of WT, syngeneic melanoma cells in the Grp78(+/-) mice was potently suppressed. We created conditional heterozygous knockout of GRP78 in the host endothelial cells and showed severe reduction of tumor angiogenesis and metastatic growth, with minimal effect on normal tissue MVD. Furthermore, knockdown of GRP78 expression in immortalized human endothelial cells showed that GRP78 is a critical mediator of angiogenesis by regulating cell proliferation, survival, and migration. Our findings suggest that concomitant use of current chemotherapeutic agents and novel therapies against GRP78 may offer a powerful dual approach to arrest cancer initiation, progression, and metastasis.

Iron-dependent activation of NF-κB in Kupffer cells: a priming mechanism for alcoholic liver disease

Alcoholic liver disease is associated with hepatic iron accumulation, and iron supplementation exacerbates alcoholic liver disease, suggesting the pathogenic role of iron in alcoholic liver disease. We have tested a hypothesis that iron plays a signaling role in activation of redox-sensitive nuclear factor-kappa B (NF-kappaB) and that increased iron content results in heightened expression of proinflammatory cytokines in Kupffer cells because of this signaling. In cultured Kupffer cells isolated from normal rats, treatment with a lipophilic iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one (L1), markedly reduced lipopolysaccharide (LPS)-induced NF-kappaB activation and expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6. Kupffer cells, isolated from rats with experimentally induced alcoholic liver disease, had significant increases in nonheme iron content, NF-kappaB binding, and mRNA expression for TNF-alpha and macrophage inflammatory protein-1. Ex vivo L1 treatment normalized all these parameters. Addition of ferrous iron to cultured normal rat Kupffer cells increased I-kappa B kinase (IKK) activity at 15 min and NF-kappaB binding at 30 min. L1 pretreatment completely abrogated both effects. Moreover, the iron treatment increased TNF-alpha release and TNF-alpha promoter activity in a NF-kappaB-dependent manner. Ferrous iron also transiently decreased cytoplasmic I-kappa B-alpha (IkappaB-alpha), with concomitant increases in nuclear p65 protein and DNA binding of p65/p50. Taken together, these results support the existence of iron-dependent signaling for activation of IKK/NF-kappaB in Kupffer cells, and this iron signaling serves as a target for a potential priming effect for the pathogenesis of experimental alcoholic liver disease.

Peroxisome Proliferator-activated Receptor γ Suppresses Proximal α1(I) Collagen Promoter via Inhibition of p300-facilitated NF-I Binding to DNA in Hepatic Stellate Cells

Depletion of peroxisome proliferator-activated receptor γ (PPARγ) represents one of the key molecular changes that underlie transdifferentiation (activation) of hepatic stellate cells in the genesis of liver fibrosis (Miyahara, T., Schrum, L., Rippe, R., Xiong, S., Yee, H. F., Jr., Motomura, K., Anania, F. A., Willson, T. M., and Tsukamoto, H. (2000) J. Biol. Chem. 275, 35715-35722; Hazra, S., Xiong, S., Wang, J., Rippe, R. A., Krishna, V., Chatterjee, K., and Tsukamoto, H. (2004) J. Biol. Chem. 279, 11392-11401). In support of this notion, ectopic expression of PPARγ suppresses hepatic stellate cells activation markers, most notably expression of α1(I) procollagen. However, the mechanisms underlying this antifibrotic effect are largely unknown. The present study utilized deletion-reporter gene constructs of proximal 2.2-kb α1(I) procollagen promoter to demonstrate that a region proximal to -133 bp is where PPARγ exerts its inhibitory effect. Within this region, two DNase footprints with Sp1 and reverse CCAAT box sites exist. NF-I, but not CCAAT DNA-binding factor/NF-Y, binds to the proximal CCAAT box in hepatic stellate cells. A mutation of this site almost completely abrogates the promoter activity. NF-I mildly but independently stimulates the promoter activity and synergistically promotes Sp1-induced activity. PPARγ inhibits NF-I binding to the most proximal footprint (-97/-85 bp) and inhibits its transactivity. The former effect is mediated by the ability of PPARγ to inhibit p300-facilitated NF-I binding to DNA as demonstrated by chromatin immunoprecipitation assay.

Iron Causes Interactions of TAK1, p21ras, and Phosphatidylinositol 3-Kinase in Caveolae to Activate IκB Kinase in Hepatic Macrophages

We recently discovered a novel signaling phenomenon involving a rapid and transient rise in intracellular low molecular weight iron complex(es) in activation of IκB kinase (IKK) in hepatic macrophages. We also showed direct treatment with ferrous iron substitutes for this event to activate IKK. The present study used this model to identify upstream kinases responsible for IKK activation. IKK activation induced by iron is abrogated by overexpression of a dominant negative mutant (DN) for transforming growth factor β-activated kinase-1 (TAK1), NF-κB-inducing kinase, or phosphatidylinositol 3-kinase (PI3K) and by treatment with the mitogen-activated protein kinase (MAPK) kinase-1 (MEK1) inhibitor. Iron increases AKT phosphorylation that is prevented by DNTAK1 or DNp21ras. Iron causes ERK1/2 phosphorylation that is attenuated by DN-PI3K, prevented by DNp21ras, but unaffected by DNTAK1. Iron-induced TAK1 activity is not affected by the PI3K or MEK1 inhibitor, suggesting TAK1 is upstream of PI3K and MEK1. Iron increases interactions of TAK1 and PI3K with p21ras as demonstrated by co-immunoprecipitation and co-localization of these proteins with caveolin-1 as shown by immunofluorescent microscopy. Finally, filipin III, a caveolae inhibitor, abrogates iron-induced TAK1 and IKK activation. In conclusion, MEK1, TAK1, NF-κ-inducing kinase, and PI3K are required for iron-induced IKK activation in hepatic macrophages and TAK1, PI3K, and p21ras physically interact in caveolae to initiate signal transduction.

Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis

One prime feature of alcoholic liver disease (ALD) is iron accumulation in hepatic macrophages/Kupffer cells (KC) associated with enhanced NF-kappaB activation. Our recent work demonstrates a peroxynitrite-mediated transient rise in intracellular labile iron (ILI) as novel signaling for endotoxin-induced IKK and NF-kappaB activation in rodent KC. The present study investigated the mechanism of KC iron accumulation and its effects on ILI response in experimental ALD. We also tested ILI response in human blood monocytes. Chronic alcohol feeding in rats results in increased expression of transferrin (Tf) receptor-1 and hemochromatosis gene (HFE), enhanced iron uptake, an increase in nonheme iron content, and accentuated ILI response for NF-kappaB activation in KC. Ex vivo treatment of these KC with an iron chelator abrogates the increment of iron content, ILI response, and NF-kappaB activation. The ILI response is evident in macrophages derived from human blood monocytes by PMA treatment but not in vehicle-treated monocytes, and this differentiation-associated phenomenon is essential for maximal TNF-alpha release. PMA-induced macrophages load iron dextran and enhance ILI response and TNF-alpha release. These effects are reproduced in KC selectively loaded in vivo with iron dextran in mice and more importantly aggravate experimental ALD. Our results suggest enhanced iron uptake as a mechanism of KC iron loading in ALD and demonstrate the ILI response as a function acquired by differentiated macrophages in humans and as a priming mechanism for ALD.

Gene profiling and pathway analysis of neuroendocrine transdifferentiated prostate cancer cells.

Neuroendocrine (NE) cells are present in both normal prostate and prostate cancer. In addition, NE differentiation can be induced by various factors, such as IL‐6, in vitro and in vivo. However, the mechanism of this differentiation and the role of NE cells in prostate cancer are not well understood. In this study, we evaluated the gene expression and analyzed the pathways in prostate cancer cells exposed to various NE differentiation inducing factors in vitro.