Hepatocyte growth factor (HGF) receptor expression and role of HGF during embryonic mouse testis development

The hepatocyte growth factor (HGF) receptor, c-met, transduces the HGF multiple biological activities. During embryonic development the system HGF/c-met regulates the morphogenesis of different organs and tissues. In this study we examined c-met gene expression during mouse testis development and, by means of Northern blot and in situ hybridization, we report the receptor expression pattern. C-met expression is not detectable in male genital ridges isolated from embryos at 11.5 days postcoitum (dpc). In testes isolated from 12.5 and 13.5 dpc, c-met expression is detectable and essentially localized in the developing cords. Male genital ducts do not express c-met at the reported ages, whereas female ducts appear c-met positive. Moreover, we report that HGF is able to induce testicular morphogenesis in vitro. Male genital ridges isolated from embryos at 11.5 dpc are morphologically nonorganized. Culturing 11.5 dpc urogenital ridges in the presence of HGF we obtained testis organization and testicular cord formation. Our data demonstrate that c-met is expressed during the beginning period of testis differentiation and that HGF is able to support testicular differentiation in vitro. All these data indicate that this growth factor, besides its role as mitogenic factor, plays a fundamental role during testicular cord formation probably inducing cell migration and/or cell differentiation.     

Effect of hepatocyte growth factor on methionine adenosyltransferase genes and growth is cell density-dependent in HepG2 cells

Hepatocyte growth factor (HGF) is a potent hepatocyte mitogen but its effect in liver cancer is conflicting. Methionine adenosyltransferase (MAT) is an essential enzyme encoded by two genes (MAT1A and MAT2A), while a third gene (MAT2beta) encodes for a subunit that regulates the MAT2A-encoded isoenzyme. MAT1A is silenced while MAT2A and MAT2beta are induced in hepatocellular carcinoma (HCC). The current work examined expression of HGF/c-met in HCC and whether HGF regulates MAT genes and growth in HepG2 cells. We found the mRNA levels of HGF and c-met are markedly increased in HCC. To study the influence of cell density, HepG2 cells were plated under high-density (HD) or low-density (LD) and treated with HGF (10 ng/ml). Cell density had a dramatic effect on MAT1A expression, being nearly undetectable at LD to a ninefold induction under HD. Cell density also determined the effect of HGF. At HD, HGF increased the mRNA levels of p21 and p27, while lowering the levels of MAT genes, cyclin A, and c-met. At LD, HGF increased the mRNA levels of cyclin A, MAT2A, MAT2beta, and c-met. Consistently, HGF inhibits growth under HD but stimulates growth under LD. HGF induced sustained high ERK activation under HD as compared to LD. In summary, HGF induces genes favoring growth and is mitogenic when HepG2 cells are plated under LD; however, the opposite occurs under HD. This involves cell density-dependent differences in HGF-induced ERK activation. This may explain why HGF is mitogenic only when there is loss of cell-cell contact in vivo.     

Developmental changes in hepatocyte growth factor mRNA and its receptor in rat liver, kidney and lung.

Hepatocyte growth factor (HGF) is a mesenchymal-derived factor which induces mitosis, cell movement and morphogenesis of tissue-like structure. We analyzed changes in HGF mRNA and its receptor, the c-met proto-oncogene product, in the liver, kidney and lung during late fetal and postnatal development in rats. In the liver, the HGF-mRNA level was very low during late gestation and in neonates, it increased remarkably and reached a maximum two weeks postnatally, to be followed by a decrease to 33% of the maximum. HGF mRNA in the kidney and lung was either undetectable or very low during late gestation and the neonatal period and increased markedly to reach a maximum, respectively, 3-4 weeks postnatally. HGF-mRNA level in the adult rat lung was fivefold higher than that in the liver and kidney. The number of HGF receptors on plasma membranes of these tissues was low in neonates but there was a rapid increase after birth and a maximum was reached within three weeks. The number of HGF receptors/ng plasma membrane protein at the maximal level was highest in the liver and lowest in the lung. c-met/HGF-receptor mRNA in the liver was also low during late-gestation or in early neonatal periods and increased postnatally. Since HGF-mRNA and HGF-receptor levels changed differently in liver, kidney and lung, the expression of HGF and its receptor may be independently regulated in each organ. However, in these organs, HGF mRNA and the HGF receptor increased within a few weeks of birth, HGF may play roles in organ growth, organ maturation and the maintenance of tissue homeostasis during the postnatal period, presumably through its potential to act as mitogen, motogen and morphogen.     

Expressions of receptor gene for hepatocyte growth factor in kidney after unilateral nephrectomy and renal injury.

The renal expressions of the receptor gene (c-met) for hepatocyte growth factor (HGF) were examined in unilateral nephrectomy (UNX), renal ischemia or folic acid administration. The levels of c-met mRNA were increased rapidly in all rat models at 6h after the operations. On the other hand, the expression of c-met mRNA in a kidney cell line (MDCK cells) was down-regulated for 8 h after HGF addition, indicating that c-met mRNA induction in rat models may be independent of the stimulated production of HGF. The stimulated expression of c-met in these models suggest that HGF may play an important role in renal hypertrophy after UNX and regeneration after ischemic or nephrotoxic injury.     

Pleiotropic activity of hepatocyte growth factor during embryonic mouse testis development

The hepatocyte growth factor (HGF) is a pleiotropic cytokine whose action is mediated by c-met, a glycoproteic receptor with tyrosine kinase activity which transduces its multiple biological activities including cell proliferation, motility and differentiation. During embryonic development HGF acts as a morphogenetic factor as previously demonstrated for metanephric and lung development. Recently, culturing male genital ridges, we demonstrated that HGF is able to support in vitro testicular cord formation. In the present paper we report the expression pattern of the HGF gene during embryonic testis development and the multiple roles exerted by this factor during the morphogenesis of this organ. Northern blot analysis reveals a positive signal in urogenital ridges isolated from 11.5 days post coitum (dpc) embryos and in testes isolated from 13.5 and 15.5 dpc male embryos. On the contrary HGF mRNA is undetectable in ovaries isolated from 13.5 and 15.5 dpc embryos. Moreover, we demonstrate that HGF is synthesized and secreted by the male gonad and is biologically active. These data indicate a male specific biological function of HGF during embryonic gonadal development. This hypothesis is supported by the in vitro demonstration that HGF acts as a migratory factor for male mesonephric cells which is a male specific event. In addition we demonstrate that during testicular development, HGF acts as a morphogenetic factor able to reorganize dissociated testicular cells which, under HGF stimulation, form a tridimensional network of cord-like structures. Finally, we demonstrate that HGF induces testicular cell proliferation in this way being responsible for the size increase of the testis. All together the data presented in this paper demonstrate that HGF is expressed during the embryonic development of the testis and clarify the multiple roles exerted by this factor during the morphogenesis of the male gonad.     

In vivo transfer of hepatocyte growth factor gene accelerates proliferation of hepatic oval cells in a 2-acetylaminofluorene/partial hepatectomy model in rats

To clarify the effect of hepatocyte growth factor (HGF) on proliferation of hepatic oval cells, we transferred HGF gene into liver of the Solt-Farber rat model. Male Fisher 344 rats were infected with a recombinant adenovirus carrying the cDNA for HGF (pAxCAHGF) from tail vein. HGF mRNA showed its peak at 4 days, and diminished thereafter. The total and proliferating cell nuclear antigen-positive hepatic oval cells were significantly elevated in HGF-transferred rats, in which stem cell factor and c-kit mRNA increased at each time point. Our results suggest that in vivo transfer of the HGF gene into liver accelerates proliferation of hepatic oval cells in the Solt-Farber model in rats.     

Hepatocyte growth factor promotes colonic epithelial regeneration via Akt signaling

Hepatocyte growth factor (HGF) can promote the regeneration of injured organs, including HGF gene therapy by electroporation (EP) for liver injury. In this study, we investigated the effect of HGF on dextran sulfate sodium-induced colitis and tried to clarify the regenerative mechanisms of colonic epithelial cells and the signaling pathway involved. Colitis was induced by dextran sulfate sodium in mice, together with HGF gene transfer by EP. On day 10, the colitis was evaluated histologically and by Western blot analysis. The colonic epithelial cell line MCE301 was exposed to HGF protein, and its proliferation and activated signaling pathway were analyzed. In vivo, the histological score improved and the number of Ki-67-positive epithelial cells increased in the HGF-treated mice compared with the controls. Western blot analysis showed enhanced expression of phospho-Akt in the HGF-treated mice compared with the controls. In vitro, HGF stimulated the proliferation of MCE301 cells. There was enhanced phospho-Akt expression for more than 48 h after HGF stimulation, although phospho-ERK1/2 was enhanced for only 10 min. LY-294002 or Akt small interfering RNA suppressed cell proliferation induced by HGF. Thus HGF induces the proliferation of colonic epithelial cells via the phosphatidylinositol 3-kinase/Akt signaling pathway. HGF gene therapy can attenuate acute colitis via epithelial cell proliferation through the PI3K/Akt pathway. These data suggested that HGF gene therapy by EP may be effective for the regeneration and repair of injured epithelial cells in inflammatory bowel disease.     

Loss of TGF-beta dependent growth control during HSC transdifferentiation

Liver injury induces activation of hepatic stellate cells (HSCs) comprising expression of receptors, proliferation, and extracellular matrix synthesis triggered by a network of cytokines provided by damaged hepatocytes, activated Kupffer cells and HSCs. While 6 days after bile duct ligation in rats TGF-beta inhibited DNA synthesis in HSCs, it was enhanced after 14 days, indicating a switch from suppression to DNA synthesis stimulation during fibrogenesis. To delineate mechanisms modulating TGF-beta function, we analyzed crosstalk with signaling pathways initiated by cytokines in damaged liver. Lipopolysaccharide and tumor necrosis factor-alpha enhanced proliferation inhibition of TGF-beta, whereas interleukin-6, oncostatin M, interleukin-1alpha, and interleukin-1beta did not. Hepatocyte growth factor (HGF) counteracted TGF-beta dependent inhibition of DNA synthesis in quiescent HSCs. Since expression of c-met is induced during activation of HSCs and HGF is overrepresented in damaged liver, crosstalk of HGF and TGF-beta contributes to loss of TGF-beta dependent inhibition of DNA synthesis in HSCs.     

Urokinase-type plasminogen activator gene therapy in liver cirrhosis is mediated by collagens gene expression down-regulation and up-regulation of MMPs, HGF and VEGF

Human urokinase-type plasminogen activator (uPA) gene administration via an adenoviral (Ad)-vector induced cirrhosis regression and ameliorated hepatic dysfunction in a model of experimental liver cirrhosis. The administration of a single dose of 6 x 10(11) viral particles per kilogram of a clinical-grade Ad-vector was evaluated after the onset of rat liver cirrhosis via degradation of deposited collagen and a substantial decrease of alpha-sma-positive cells. Also, gene expression for pro-fibrogenic molecules (Col I, III, IV, TIMP-1 and PAI-1) was clearly down-regulated. In contrast, gene expression for collagen-degrading enzymes such as MMP-13 and MMP-2 was up-regulated. These events correlated with increased amounts of proteic free-TIMP-1, i.e. non-complexed with metalloproteinases (MMPs), indicating the presence of higher amounts of active MMPs inside the liver of cirrhotic animals treated with Ad-huPA. The harmonized and concerted expression of HGF and c-met resulted in exacerbated hepatocyte proliferation, although these events did not induce an abnormal liver growth. Angiogenesis, i.e. formation of new blood vessels, was evaluated by vascular endothelial growth factor (VEGF) expression which was notably detected to be 10 times higher during the first 6 days after Ad-huPA-treatment in cirrhotic animals as compared with controls. These events provide a clearer rationale as to how Ad-huPA-induced liver regeneration on CCl(4)-induced liver fibrosis takes place.     

HGF and Direct Mesenchymal Stem Cells Contact Synergize to Inhibit Hepatic Stellate Cells Activation through TLR4/NF-kB Pathway

Aims

Bone marrow-derived mesenchymal stem cells (BMSCs) can reduce liver fibrosis. Apart from the paracrine mechanism by which the antifibrotic effects of BMSCs inhibit activated hepatic stellate cells (HSCs), the effects of direct interplay and juxtacrine signaling between the two cell types are poorly understood. The purpose of this study was to explore the underlying mechanisms by which BMSCs modulate the function of activated HSCs.

Methods

We used BMSCs directly and indirectly co-culture system with HSCs to evaluate the anti-fibrosis effect of BMSCs. Cell proliferation and activation were examined in the presence of BMSCs and HGF. c-met was knockdown in HSCs to evaluate the effect of HGF secreted by BMSCs. The TLR4 and Myeloid differentiation primary response gene 88(MyD88) mRNA levels and the NF-kB pathway activation were determined by real-time PCR and western blotting analyses. The effect of BMSCs on HSCs activation was investigated in vitro in either MyD88 silencing or overexpression in HSCs. Liver fibrosis in rats fed CCl₄ with and without BMSCs supplementation was compared. Histopathological examinations and serum biochemical tests were compared between the two groups.

Results

BMSCs remarkably inhibited the proliferation and activation of HSCs by interfering with LPS-TLR4 pathway through a cell–cell contact mode that was partially mediated by HGF secretion. The NF-kB pathway is involved in HSCs activation inhibition by BMSCs. MyD88 over expression reduced the BMSC inhibition of NF-kB luciferase activation. BMSCs protected liver fibrosis in vivo.

Conclusion

BMSCs modulate HSCs in vitro via TLR4/MyD88/NF-kB signaling pathway through cell–cell contact and secreting HGF. BMSCs have therapeutic effects on cirrhosis rats. Our results provide new insights into the treatment of hepatic fibrosis with BMSCs.     

Generation of a mouse expressing a conditional knockout of the hepatocyte growth factor gene: demonstration of impaired liver regeneration

Hepatocyte growth/scatter factor (HGF/SF) is a pleiotropic cytokine originally identified as a potent mitogen for rat hepatocytes. Two HGF/SF knockout mouse models have been reported, both of which exhibit developmental abnormalities causing embryonic lethality. To circumvent this limitation, we created a mouse conditionally deficient in liver expression of HGF/SF to specifically investigate the role of this mitogen in the process of adult liver regeneration. Gene targeting technology was used to generate a mouse with loxP sites flanking exon 5 of the HGF/SF gene (ex5-flox). In the absence of cre recombinase activity, mice homozygous for ex5-flox were indistinguishable from wild-type littermates. To ablate HGF/SF gene expression in vitro, primary hepatocytes established from homozygous HGF(ex5-flox) mice were infected with a recombinant adenoviral vector coding for cre recombinase (AdCre1). PCR analyses of genomic DNA demonstrated greater than 90% ablation of the ex5-floxed gene sequence. In vivo, HGF(ex.5-flox) mice were administered AdCre1 vector and the ablation of the HGF gene confirmed by Southern blot analysis. To induce liver regeneration, mice were injected with the hepatotoxin carbon tetrachloride. The regenerative capacity of hepatocytes in mice administered cre recombinase was shown to be significantly reduced when compared with mice injected with an adenovirus expressing LacZ. A similar reduction in hepatocyte regeneration was observed in HGF(ex.5.flox) mice carrying the cre transgene under the control of the interferon-inducible (pI:pC) Mx1 promoter, as an alternative strategy to ablate the HGF/SF gene in liver. Our results confirm the mitogenic role of HGF/SF in liver regeneration.     

Stanniocalcin 1 is an autocrine modulator of endothelial angiogenic responses to hepatocyte growth factor

Stanniocalcin 1 (STC1) is a secreted glycoprotein originally described as a hormone involved in calcium and phosphate homeostasis in bony fishes. We recently identified the mammalian homolog of this molecule to be highly up-regulated in an in vitro model of angiogenesis, as well as focally and intensely expressed at sites of pathological angiogenesis (e.g. tumor vasculature). In the present study, we report that STC1 is a selective modulator of hepatocyte growth factor (HGF)-induced endothelial migration and morphogenesis, but not proliferation. STC1 did not inhibit proliferative or migratory responses to vascular endothelial growth factor or basic fibroblast growth factor. The mechanism of STC1 inhibitory effects on HGF-induced endothelial migration seem to occur secondary to receptor activation because STC1 did not inhibit HGF-induced c-met receptor phosphorylation, but did block HGF-induced focal adhesion kinase activation. In the mouse femoral artery ligation model of angiogenesis, STC1 expression closely paralleled that of the endothelial marker CD31, and the peak level of STC1 expression occurred after an increase in HGF expression. We propose that STC1 may play a selective modulatory role in angiogenesis, possibly serving as a "stop signal" or stabilizing factor contributing to the maturation of newly formed blood vessels. HGF is a mesenchyme-derived pleiotropic factor with mitogenic, motogenic, and morphogenic activities on a number of different cell types. HGF effects are mediated through a specific tyrosine kinase, c-met, and aberrant HGF and c-met expression are frequently observed in a variety of tumors. Recent studies have shown HGF to be a potent growth factor implicated in wound healing, tissue regeneration, and angiogenesis.     

c-met 在大鼠胰岛β细胞INS-1 胰岛素分泌中的作用

目的:从c-met对胰岛β细胞增殖,细胞周期、糖耐受和对GLUT2的表达影响三个方面探讨c-met在胰岛β细胞功能的影响及相关机制。方法:在大鼠胰岛β细胞系INS-1中运用RNA干扰技术(RNAi)抑制HGF的特异性受体c-met蛋白的表达,检测其在正常的生理状况下对成熟的胰岛β细胞增殖以及功能维持的作用。结果:c-met蛋白对成熟的胰岛β细胞的增殖与周期并没有显著影响,但对于β细胞的功能维持具有重要意义。结论:通过调节GLUT2蛋白来维持β细胞的胰岛素分泌功能,有助于进一步阐明HGF/c-met通路在胰岛β细胞功能损伤的分子机制,从而为糖尿病的预防和治疗提供新的理论依据。