Impaired autoregulation in chronic kidney disease can result in elevation of glomerular capillary pressure and progressive glomerular damage; however, the factors linking chronic glomerular disorders to impaired autoregulation have not been identified. We tested the hypothesis that the cytokine most closely associated with progressive glomerular disease, transforming growth factor (TGF)-beta, may also attenuate autoregulation. Kidneys from normal rats were prepared for videomicroscopy, using the blood-perfused juxtamedullary nephron technique. Autoregulatory responses were measured under control conditions and during superfusion with TGF-beta1 (10 ng/ml). Control afferent arteriolar diameter averaged 18.4 +/- 1 microm and significantly decreased to 16.3 +/- 0.9 and 13.2 +/- 0.8 microm at perfusion pressures of 130 and 160 mmHg, respectively. In the presence of TGF-beta1, autoregulatory responses were completely blocked. In similar experiments performed using PDGF-BB (10 ng/ml) and HGF (25 ng/ml), the normal autoregulatory response was not affected. In vitro studies, using isolated preglomerular vascular smooth muscle cells, revealed that exposure to TGF-beta1 stimulated a rapid increase in reactive oxygen species (ROS) that was inhibited by NADPH oxidase inhibitors. In situ studies, with dihydroethidium staining, revealed a marked increase in renal vessel ROS production on exposure to TGF-beta1. Pretreatment of the juxtamedullary afferent arterioles with tempol, a ROS scavenger, or with apocynin, a NADPH oxidase inhibitor, prevented the impaired autoregulation induced by TGF-beta1. These data reveal a novel hemodynamic pathway by which TGF-beta could lead to progressive glomerular injury by impairing normal renal microvascular function.

Therapeutic angiogenesis using angiogenic growth factors is expected to be a new treatment for patients with critical limb ischemia (CLI). Because hepatocyte growth factor (HGF) has potent angiogenic activity, we investigated the safety and efficiency of HGF plasmid DNA in patients with CLI as a prospective open-labeled clinical trial. Intramuscular injection of naked HGF plasmid DNA was performed in ischemic limbs of 6 CLI patients with arteriosclerosis obliterans (n=3) or Buerger disease (n=3) graded as Fontaine III or IV. The primary end points were safety and improvement of ischemic symptoms at 12 weeks after transfection. Severe complications and adverse effects caused by gene transfer were not detected in any patients. Of particular importance, no apparent edema was observed in any patient throughout the trial. In addition, serum HGF concentration was not changed throughout the therapy period in all patients. In contrast, a reduction of pain scale of more than 1 cm in visual analog pain scale was observed in 5 of 6 patients. Increase in ankle pressure index more than 0.1 was observed in 5 of 5 patients. The long diameter of 8 of 11 ischemic ulcers in 4 patients was reduced >25%. Intramuscular injection of naked HGF plasmid is safe, feasible, and can achieve successful improvement of ischemic limbs. Although the present data are conducted to demonstrate the safety as phase I/early phase IIa, the initial clinical outcome with HGF gene transfer seems to indicate usefulness as sole therapy for CLI.

OBJECTIVE

Diabetes results from a deficiency of functional beta-cells. Previous studies have identified hepatocyte growth factor (HGF) and parathyroid hormone-related protein (PTHrP) as two potent beta-cell mitogens. The objective of this study is to determine 1) whether HGF and PTHrP have additive/synergistic effects on beta-cell growth and proliferation; 2) the signaling pathways through which these growth factors mediate beta-cell mitogenesis; and 3) whether activation of this/these signaling pathway(s) enhances human beta-cell replication.

METHODS

We generated and phenotypically analyzed doubly transgenic mice overexpressing PTHrP and HGF in the beta-cell. INS-1 and primary mouse and human islet cells were used to identify mitogenic signaling pathways activated by HGF and/or PTHrP.

RESULTS

Combined overexpression of HGF and PTHrP in the beta-cell of doubly transgenic mice did not result in additive/synergistic effects on beta-cell growth and proliferation, suggesting potential cross-talk between signaling pathways activated by both growth factors. Examination of these signaling pathways in INS-1 cells revealed atypical protein kinase C (PKC) as a novel intracellular target activated by both HGF and PTHrP in beta-cells. Knockdown of PKC zeta, but not PKC iota/lambda, expression using specific small-interfering RNAs blocked growth factor-induced INS-1 cell proliferation. Furthermore, adenovirus-mediated delivery of kinase-dead PKC zeta completely inhibited beta-cell proliferation in primary islet cells overexpressing PTHrP and/or HGF. Finally, adenovirus-mediated delivery of constitutively active PKC zeta in mouse and human primary islet cells significantly enhanced beta-cell proliferation.

CONCLUSIONS

PKC zeta is essential for PTHrP- and HGF-induced beta-cell proliferation. PKC zeta activation could be useful in therapeutic strategies for expanding beta-cell mass in vitro and in vivo.

The pathogenesis of both ulcerative colitis and Crohn's disease is unknown but these forms of inflammatory bowel disease (IBD) may be associated with an inability of the intestinal mucosa to protect itself from luminal challenges and/or inappropriate repair following intestinal injury. Numerous cell populations regulate these broad processes through the expression of a complex array of peptides and other agents. Growth factors can be distinguished by their actions regulating cell proliferation. These factors also mediate processes such as extracellular matrix formation, cell migration and differentiation, immune regulation, and tissue remodeling. Several families of growth factors may play an important role in IBD including: epidermal growth factor family (EGF) [transforming growth factor alpha (TGF alpha), EGF itself, and others], the transforming growth factor beta (TGF beta) super family, insulin-like growth factors (IGF), fibroblast growth factors (FGF), hepatocyte growth factor (HGF), trefoil factors, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and others. Collectively these families may determine susceptibility of IBD mucosa to injury and facilitate tissue repair.

Growing three-dimensional scaffolds that contain more than a few layers of seeded cells is crucial for the creation of thick and viable cardiac tissues. To achieve this goal, a bioengineered cardiac patch (the MSC patch) composed of a sliced porous biological scaffold inserted with multilayered mesenchymal stem cells (MSCs) was developed for myocardial repair in a syngeneic rat model. After culture, sliced layers of the scaffold were stuck together and seeded MSCs were redistributed throughout the scaffold. Immunofluorescence analyses indicated that cells were viable and tightly adhered to a robust fibronectin meshwork within the scaffold. Results of echocardiography and heart catheterization revealed that the MSC-patch group had a superior heart function to the infarct group. Cells together with neo-muscle fibers and neo-microvessels were clearly observed in the entire MSC patch to fill its original pores, indicating that the implanted patch became well integrated into the host. The thickness of the retrieved MSC patch increased significantly as compared to that before implantation. When compared with the infarct group, expressions of angiogenic cytokines (bFGF, vWF and PDGF-B) and cardioprotective factors (IGF-1 and HGF) were significantly increased in the MSC-patch group. The aforementioned results indicated that transplantation of the MSC patch could restore the dilated LV and preserve cardiac functions after infarction.

Excessive activity of the Fas system in the liver is an essential event and contributor to fulminant hepatic failure, whose prognosis is extremely poor with high mortality due to lack of effective therapy. Administration of agonistic anti-Fas antibody to mice rapidly led to massive liver apoptosis and fulminant hepatic failure. In contrast, administration of human recombinant hepatocyte growth factor (HGF) abrogated Fas-induced massive liver apoptosis and the lethal hepatic failure. Addition of anti-Fas antibody to hepatocytes in primary culture induced cell death, but Fas-mediated cell death was potently suppressed by HGF. HGF strongly induced Bcl-xL expression and subsequently blocked Fas-mediated signaling pathway upstream of CPP32 in the liver. These results implicate a potential therapeutic usage of HGF for treatment of fulminant hepatic failure.

Hepatocyte Growth Factor, also known as Scatter Factor, is a polypeptide that shows structural homology with enzymes of the blood coagulation cascade. It is a biologically inactive single chain precursor that is then cleaved by specific serine proteases to a fully active alphabeta heterodimer. All the biological responses induced by HGF/SF are elicited by binding to its receptor, a transmembrane tyrosine kinase encoded by the MET proto-oncogene. The signaling cascade triggered by HGF begins with the autophosphorylation of the receptor and is mediated by concomitant activation of different cytoplasmic effectors that bind to the same multifunctional docking site. During development, HGF function is essential: knock-out mice for both ligand and receptor show an embryonic lethal phenotype. HGF/SF displays a unique feature in inducing "branching morphogenesis", a complex program of proliferation and motogenesis in a number of different cell types. Moreover, HGF is involved in the invasive behaviour of several tumor cells both in vivo and in vitro. The role of HGF as putative therapeutical agent in pathologies characterized by massive cell loss or deregulated cell proliferation is under investigation.

The hepatocyte growth factor (HGF)-MET pathway supports several hallmark cancer traits, and it is frequently activated in a broad spectrum of human cancers (http://www.vai.org/met/). With the development of many cancer drugs targeting this pathway, there is a need for relevant in vivo model systems for preclinical evaluation of drug efficacy. Here, we show that production of the human HGF ligand in transgenic severe combined immunodeficient mice (hHGF(tg)-SCID mice) enhances the growth of many MET-expressing human carcinoma xenografts, including those derived from lung, breast, kidney, colon, stomach, and pancreas. In this model, the MET-specific small-molecule kinase inhibitor SGX523 partially inhibits the HGF-dependent growth of lung, breast, and pancreatic tumors. However, much greater growth suppression is achieved by combinatorial inhibition with the epidermal growth factor receptor (EGFR) kinase inhibitor erlotinib. Together, these results validate the hHGF(tg)-SCID mouse model for in vivo determination of MET sensitivity to drug inhibition. Our findings also indicate that simultaneously targeting the MET and EGFR pathways can provide synergistic inhibitory effects for the treatment of cancers in which both pathways are activated.

Osteopontin (OPN) is a secreted, integrin-binding protein which has been implicated in cancer, as well as other pathologies and some aspects of normal development. Here we focus on the role of OPN in breast cancer. We describe studies that have shown that OPN plays a role in normal mammary gland development as well as in progression of breast cancer. We also summarize studies that have shown that OPN can play a functional role in malignancy of breast cancer. At least some of these effects are mediated by specific cell surface integrins (alpha(v)beta3 vs. alpha(v)beta1 and alpha(v)beta5) and lead to increased cell migration, activation of growth factor/receptor pathways (e.g. HGF and EGF), and increased proteolytic enzyme activity (e.g. uPA). We also summarize clinical studies that show that OPN levels in tumors and blood are elevated in women with metastatic breast cancer and may offer promise as prognostic markers in breast cancer.

Liver metastasis is one of the poor prognostic factors for gastric cancer. Hepatocyte growth factor (HGF) and its receptor, c-Met, have been reported to be related to the proliferation of carcinoma cells. We examined c-Met and HGF expression in stage IV gastric cancers (n = 121) and compared the results in groups with liver metastasis (n = 47, LM group) and without liver metastasis (n = 74, no-LM group). The survival rate for the LM group was significantly poorer than for the no-LM group (p < 0.01). We found a high frequency of c-Met expression in the LM group compared with the no-LM group at protein level detected by immunohistochemistry (p = 0.0005) and at mRNA level detected by semiquantitative reverse transcriptase-polymerase chain reaction (p = 0.0386) in primary gastric tumors. Furthermore, we evaluated HGF expression in both carcinoma cells and stromal cells in gastric cancers. There was no significant difference in the HGF expression between the LM and no-LM groups. The labeling index of proliferating cell nuclear antigen for the carcinomas in the LM group was higher than that in the no-LM group (47.1 +/- 24.5 vs. 26.2 +/- 24.5%, p < 0.0001). Thus, the high frequency of c-Met overexpression in carcinoma cells may be involved in the mechanism of liver metastasis in gastric cancers. Moreover, the evaluation of c-Met expression might be a useful indicator of liver metastasis in patients with gastric cancer.

The pleiotropic effects (mitogenesis, motogenesis, and morphogenesis) elicited by hepatocyte growth factor/scatter factor (HGF/SF) are mediated by the activation of the tyrosine kinase receptor encoded by the MET proto-oncogene. Following autophosphorylation, the receptor associates with the p85/110 phosphatidylinositol (PI) 3-kinase complex in vivo and in vitro. By a combination of two complementary approaches, competition with synthetic phosphopeptides and association with Tyr-Phe receptor mutants, we have identified Y-1349 and Y-1356 in the HGF/SF receptor as the binding sites for PI 3-kinase. Y-1349VHV and Y-1356VNV do not conform to the canonical consensus sequence YXXM for PI 3-kinase binding and thus define YVXV as a novel recognition motif. Y-1349 and Y-1356 are located within the C-terminal portion of the HGF/SF receptor and are phosphorylation sites. The affinity of the N- and C-terminal src homology region 2 (SH2) domains of p85 for the phosphopeptides including Y-1349 and Y-1356 is 2 orders of magnitude lower than that measured for Y-751 in the platelet-derived growth factor receptor binding site. However, the closely spaced duplication of the novel recognition motif in the native HGF/SF receptor may allow binding with both SH2 domains of p85, thus generating an efficient docking site for PI 3-kinase. In agreement with this model, we have observed that a phosphopeptide including both Y-1349 and Y-1356 activates PI 3-kinase in vitro.

OBJECTIVE

Circulating platelet counts gradually decrease in parallel with progression of chronic liver disease. Thrombocytopenia is a common complication of advanced liver fibrosis and is thought to be a consequence of the destruction of circulating platelets that occurs during secondary portal hypertension or hypersplenism. It is not clear whether thrombocytopenia itself affects liver fibrosis.

METHODS

Thrombocytopenic mice were generated by disruption of Bcl-xL, which regulates platelet life span, specifically in thrombocytes. Liver fibrosis was examined in thrombocytopenic mice upon bile duct ligation. Effect of platelets on hepatic stellate cells (HSCs) was investigated in vitro.

RESULTS

Thrombocytopenic mice developed exacerbated liver fibrosis, with increased expression of type I collagen alpha1 and alpha2, during cholestasis. In vitro experiments revealed that, upon exposure to HSCs, platelets became activated, released hepatocyte growth factor (HGF), and then inhibited HSC expression of the type I collagen genes in a Met signal-dependent manner. In contrast to the wild-type mice, the thrombocytopenic mice did not accumulate hepatic platelets or phosphorylate Met in the liver following bile duct ligation. Administration of recombinant HGF to thrombocytopenic mice reduced liver fibrosis to the levels observed in wild-type mice and attenuated hepatic expression of the type I collagen genes.

CONCLUSIONS

Thrombocytopenia exacerbates liver fibrosis; platelets have a previously unrecognized, antifibrotic role in suppressing type I collagen expression via the HGF-Met signaling pathway.

Hepatocyte growth factor (HGF) modulates cell adhesion, migration, and branching morphogenesis in cultured epithelial cells, events that require regulation of cell-matrix interactions. Using mIMCD-3 epithelial cells, we studied the effect of HGF on the focal adhesion proteins, focal adhesion kinase (FAK) and paxillin and their association. HGF was found to increase the tyrosine phosphorylation of paxillin and to a lesser degree FAK. In addition, HGF induced association of paxillin and activated ERK, correlating with a gel retardation of paxillin that was prevented with the ERK inhibitor U0126. The ability of activated ERK to phosphorylate and induce gel retardation of paxillin was confirmed in vitro in both full-length and amino-terminal paxillin. Several potential ERK phosphorylation sites in paxillin flank the paxillin-FAK association domains, so the ability of HGF to regulate paxillin-FAK association was examined. HGF induced an increase in paxillin-FAK association that was inhibited by pretreatment with U0126 and reproduced by in vitro phosphorylation of paxillin with ERK. The prevention of the FAK-paxillin association with U0126 correlated with an inhibition of the HGF-mediated FAK tyrosine phosphorylation and inhibition of HGF-dependent cell spreading and adhesion. An examination of cellular localization of FAK and paxillin demonstrated that HGF caused a condensation of focal adhesion complexes at the leading edges of cell processes and FAK-paxillin co-localization in these large complexes. Thus, these data suggest that HGF can induce serine/threonine phosphorylation of paxillin most probably mediated directly by ERK, resulting in the recruitment and activation of FAK and subsequent enhancement of cell spreading and adhesion.

Pancreatic carcinoma cells overexpress the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and the hepatocyte growth factor (HGF) receptor, c-Met, which are both known to mediate tumor cell migration and invasion. We hypothesized that IGF-IR and c-Met cooperate to induce migration and invasion of human pancreatic carcinoma cells and that IGF-I-mediated migration and invasion depend on c-Met. Migration and invasion assays were done with the human pancreatic cancer cell line L3.6pl treated with PBS, IGF-I, HGF, or IGF-I plus HGF. To determine if c-Met is necessary for IGF-IR-mediated migration and invasion, c-Met was down-regulated in L3.6pl cells via adenoviral infection with a c-Met ribozyme before IGF-I treatment. IGF-I and HGF increased cell migration and invasion. Furthermore, IGF-I plus HGF had a greater than additive effect on cell migration and invasion compared with either growth factor alone. Down-regulation of c-Met nearly completely inhibited IGF-I-mediated migration and invasion. Our findings suggest that IGF-IR and c-Met cooperate to induce migration and invasion of human pancreatic carcinoma cells. Furthermore, c-Met is required for both HGF- and IGF-I-mediated migration and invasion. Elucidation of the signaling pathways that contribute to tumor progression and metastasis should provide a foundation for the development of targeted therapies for pancreatic carcinoma.

Metastasis is the leading cause of death in patients with hepatocellular carcinoma (HCC) and microRNAs have been implicated to influence this process. Emerging evidence indicates that miR-198 is down-regulated in HCC compared to normal liver parenchyma, but the functional roles of miR-198 in HCC cells remains unexplored. Herein, we show that miR-198 directly targets c-MET via its 3'UTR. Forced expression of miR-198 decreased c-MET expression at both mRNA and protein levels and consequently diminished HGF induced phosphorylation of p44/42 MAPK in HCC cells. Forced expression of miR-198 inhibited HGF promotion of HCC cell migration and invasion in a c-MET dependent manner. In conclusion, we have identified miR-198 as a novel suppressor of HCC cell invasion by negative regulation of the HGF/c-MET pathway.

The forkhead box f1 (Foxf1) transcription factor is expressed in the visceral (splanchnic) mesoderm, which is involved in mesenchymal-epithelial signaling required for development of organs derived from foregut endoderm such as lung, liver, gall bladder, and pancreas. Our previous studies demonstrated that haploinsufficiency of the Foxf1 gene caused pulmonary abnormalities with perinatal lethality from lung hemorrhage in a subset of Foxf1+/- newborn mice. During mouse embryonic development, the liver and biliary primordium emerges from the foregut endoderm, invades the septum transversum mesenchyme, and receives inductive signaling originating from both the septum transversum and cardiac mesenchyme. In this study, we show that Foxf1 is expressed in embryonic septum transversum and gall bladder mesenchyme. Foxf1+/- gall bladders were significantly smaller and had severe structural abnormalities characterized by a deficient external smooth muscle cell layer, reduction in mesenchymal cell number, and in some cases, lack of a discernible biliary epithelial cell layer. This Foxf1+/- phenotype correlates with decreased expression of vascular cell adhesion molecule-1 (VCAM-1), alpha(5) integrin, platelet-derived growth factor receptor alpha (PDGFRalpha) and hepatocyte growth factor (HGF) genes, all of which are critical for cell adhesion, migration, and mesenchymal cell differentiation.

OBJECTIVE

To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I.

METHODS

RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agilent Technologies, Palo Alto, CA). Gene expression in each diabetic corneal cRNA was assessed against pooled cRNA from 7 to 9 normal corneas. Select differentially expressed genes were validated by quantitative real-time RT-PCR (QPCR) and immunohistochemistry. Organ cultures were treated with a cathepsin inhibitor, cystatin C, or MMP-10.

RESULTS

More than 100 genes were upregulated and 2200 were downregulated in DR corneas. Expression of cathepsin F and hepatocyte growth factor (HGF) genes was increased in ex vivo and organ-cultured DR corneas compared with normal corneas. HGF receptor c-met, fibroblast growth factor (FGF)-3, its receptor FGFR3, tissue inhibitor of metalloproteinase (TIMP)-4, laminin alpha4 chain, and thymosin beta(4) genes were downregulated. The data were corroborated by QPCR and immunohistochemistry analyses; main changes of these components occurred in corneal epithelium. In organ-cultured DR corneas, cystatin C increased laminin-10 and integrin alpha(3)beta(1), whereas in normal corneas MMP-10 decreased laminin-10 and integrin alpha(3)beta(1) expression.

CONCLUSIONS

Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy.

Invasion and subsequent establishment of metastasis are devastating events for patients with cancer, but past therapeutic approaches have paid relatively little attention to these important issues. Hepatocyte growth factor (HGF) and its receptor, the c-Met tyrosine kinase, play roles in cancer invasion and metastasis in a wide variety of tumor cells. Activation of the c-Met receptor integrates multiple signal transduction pathways involved in cell-cell and cell-matrix interactions, cellular migration, and breakdown of the extracellular scaffold. Paracrine activation of the c-Met receptor by stromal-derived HGF mediates tumor-stromal interactions that facilitate invasion and metastasis. Likewise, aberrant expression of the c-Met receptor and autocrine or mutational activation of c-Met receptor tyrosine kinase are closely associated with the progression of malignant tumors. Based on this background, NK4, a competitive antagonist of HGF-c-Met association was prepared so as to block cancer invasion and metastasis. NK4, an internal fragment of HGF, binds to but does not activate the c-Met receptor, thereby competitively antagonizing the biological activities of HGF. Unexpectedly, NK4 was subsequently shown to be an angiogenesis inhibitor as well, and this angioinhibitory activity is independent of its action as an HGF-antagonist. Importantly, NK4 protein or NK4 gene therapy have been shown to inhibit tumor invasion, metastasis and angiogenesis, effectively converting malignant tumors into benign tumors. Targeting tumor invasion-metastasis and angiogenesis with NK4 seems to have considerable therapeutic potential for cancer patients.

Scatter factor/hepatocyte growth factor (SF/HGF) has various biological effects upon different cells, i.e. induces increased motility and proliferation as well as invasiveness and morphogenesis. The signals given to epithelial cells by SF/HGF are all mediated through the Met receptor tyrosine kinase (Weidner, K. M., Sachs, M., and Birchmeier, W. (1993) J. Cell Biol. 111, 145-154) suggesting that signal diversity is due to the interplay of different downstream pathways. It has also been shown that SF/HGF activates the protooncogene product Ras, i.e. stimulates guanine nucleotide exchange. In order to examine whether Ras is involved in mediating the dissociation and motility signal of SF/HGF to epithelial cells, we have expressed in Madin-Darby canine kidney cells the dominant-negative N17Ras under the control of a modified metallothionein promoter. Induced expression of N17Ras by the addition of Zn2+ clearly prevented dissociation of the cells by SF/HGF. These data indicate that the Ras pathway is indeed essential to mediate the motility signal of SF/HGF-Met to the cell-cell adhesion system and the cytoskeleton of epithelial cells.

HGF (hepatocyte growth factor) is a pleiotropic cytokine homologous to the serine protease zymogen plasminogen that requires canonical proteolytic cleavage to gain functional activity. The activating proteases are key components of its regulation, but controversy surrounds their identity. Using quantitative analysis we found no evidence for activation by uPA (urokinase plasminogen activator), despite reports that this is a principal activator of pro-HGF. This was unaffected by a wide range of experimental conditions, including the use of various molecular forms of both HGF and uPA, and the presence of uPAR (uPA receptor) or heparin. In contrast the catalytic domains of the TTSPs (type-II transmembrane serine proteases) matriptase and hepsin were highly efficient activators (50% activation at 0.1 and 3.4 nM respectively), at least four orders of magnitude more efficient than uPA. PS-SCL (positional-scanning synthetic combinatorial peptide libraries) were used to identify consensus sequences for the TTSPs, which in the case of hepsin corresponded to the pro-HGF activation sequence, demonstrating a high specificity for this reaction. Both TTSPs were also found to be efficient activators at the cell surface. Activation of pro-HGF by PC3 prostate carcinoma cells was abolished by both protease inhibition and matriptase-targeting siRNA (small interfering RNA), and scattering of MDCK (Madin-Darby canine kidney) cells in the presence of pro-HGF was abolished by inhibition of matriptase. Hepsin-transfected HEK (human embryonic kidney)-293 cells also activated pro-HGF. These observations demonstrate that, in contrast with the uPA/uPAR system, the TTSPs matriptase and hepsin are direct pericellular activators of pro-HGF, and that together these proteins may form a pathway contributing to their involvement in pathological situations, including cancer.

Dynamic remodeling of the actin cytoskeleton is required for cell spreading, motility, and migration and can be regulated by tyrosine kinase activity. Phosphotyrosine proteomic screening revealed phosphorylation of the lipid-, calcium-, and actin-binding protein annexin A2 (AnxA2) at Tyr23 as a major event preceding ts-v-Src kinase-induced cell scattering. Expression of the phospho-mimicking mutant Y23E-AnxA2 itself was sufficient to induce actin reorganization and cell scattering in MDCK cells. While Y23E-AnxA2, but not Y23A-AnxA2, enhanced Src- or hepatocyte growth factor (HGF)-induced cell scattering, short hairpin RNA-mediated knockdown of AnxA2 inhibited both v-Src- and HGF-induced cell scattering. Three-dimensional branching morphogenesis was induced in wild-type-AnxA2-expressing cells only in the presence of HGF, while Y23E-AnxA2 induced HGF-independent branching morphogenesis. Knockdown of AnxA2 prevented lumen formation during cystogenesis. The Y23E-AnxA2-induced scattering was associated with dephosphorylation/activation of the actin-severing protein cofilin. Likewise, inactive S3E-cofilin and constitutively active LIM kinase, a direct upstream kinase of cofilin, inhibited Y23E-AnxA2-induced scattering. Together, our studies indicate an essential role for AnxA2 phosphorylation in regulating cofilin-dependent actin cytoskeletal dynamics in the context of cell scattering and branching morphogenesis.

The multifunctional cytokine interleukin 6 (IL-6) is expressed in a wide variety of disease states and pathologic processes. Mice deficient in IL-6 display abnormal and delayed liver regeneration and repair. Currently, IL-6 is thought to influence liver growth indirectly by priming hepatocytes to respond to growth factors such as hepatocyte growth factor (HGF) by inducing expression of HGF and by inhibiting hepatocyte apoptosis, as distinct from the direct mitotic effects of IL-6 on myeloid and other cell types. Here, we show that systemic administration of IL-6 using CHO cell tumors in nude mice results in dramatic hepatomegaly and hepatocyte hyperplasia in the absence of liver injury. Liver mass and liver to body mass ratios increased to 2 to 3 times normal because of proliferation of hepatocytes. Liver growth was associated with high levels of serum IL-6 and with activation of the IL-6-signaling pathway, including increased expression of IL-6 receptor-alpha/gp80, activation of the signal transducer and activator of transcription-3 (STAT-3), and mitogen-activated protein kinase (MAPK/ERK)-signaling pathways and induction of downstream target genes, including c-myc. HGF receptor and transforming growth factor alpha (TGF-alpha)/epidermal growth factor (EGF) receptor activation were decreased in hypertrophied livers, suggesting that IL-6-induced liver growth was independent of these known hepatocyte mitotic pathways. In conclusion, we suggest that IL-6 may function as a direct hepatic mitogen in vivo and, furthermore, that IL-6 warrants closer examination as a potent liver growth factor with potential clinical utility for increasing liver mass following injury.

OBJECTIVE

Uveal melanoma is the most common primary intraocular malignancy in adult humans. Unlike cutaneous melanoma, uveal melanoma disseminates preferentially to the liver through the hematogenous system. To date, the mechanism underlying this metastatic homing is largely unknown. This study investigated the effect of hepatocyte growth factor (HGF)-triggered signaling pathways to identify the role of HGF and its downstream effectors in inducing the migration of uveal melanoma cells.

METHODS

Migration of uveal melanoma cells was measured by in vitro wound healing and transwell migration assays. The expression and translocation of c-Met were detected using indirect immunofluorescence. The activation of extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathways was analyzed using specific antibodies against phospho-ERK1/2 and phospho-Akt. The impact of HGF treatment on the expression of cell adhesion molecules was measured using Western blotting.

RESULTS

HGF was found to enhance cell migration, and that HGF-induced migration depends on PI3K/Akt pathway. The activation of PI3K/Akt pathway induced by the HGF/c-Met axis is involved in the downregulation of cell adhesion molecules E-cadherin and beta-catenin, contributing to the attenuation of cell-cell adhesion and promoting the enhanced motility and migration of uveal melanoma cells. On HGF stimulation, receptor c-Met is translocated to the nucleus in a ligand-dependent manner, suggesting that c-Met may modulate the expression of genes involved in melanoma cell migration.

CONCLUSIONS

Data from this study directly linked the central PI3K/Akt pathway to uveal melanoma migration and pointed to new avenues for therapeutic intervention in hepatic metastasis.

The utility of neural stem cells (NSCs) has extended beyond regenerative medicine to targeted gene delivery, as NSCs possess an inherent tropism to solid tumors, including invasive gliomas. However, for optimal clinical implementation, an understanding of the molecular events that regulate NSC tumor tropism is needed to ensure their safety and to maximize therapeutic efficacy. We show that human NSC lines responded to multiple tumor-derived growth factors and that hepatocyte growth factor (HGF) induced the strongest chemotactic response. Gliomatropism was critically dependent on c-Met signaling, as short hairpin RNA-mediated ablation of c-Met significantly attenuated the response. Furthermore, inhibition of Ras-phosphoinositide 3-kinase (PI3K) signaling impaired the migration of human neural stem cells (hNSCs) toward HGF and other growth factors. Migration toward tumor cells is a highly regulated process, in which multiple growth factor signals converge on Ras-PI3K, causing direct modification of the cytoskeleton. The signaling pathways that regulate hNSC migration are similar to those that promote unregulated glioma invasion, suggesting shared cellular mechanisms and responses. Disclosure of potential conflicts of interest is found at the end of this article.