At least two kidney epithelial cell lines, the Madin-Darby canine kidney (MDCK) and the murine inner medullary collecting duct line mIMCD-3, can be induced to form branching tubular structures when cultured with hepatocyte growth factor (HGF) plus serum in collagen I gels. In our studies, whereas MDCK cells remained unable to form tubules in the presence of serum alone, mIMCD-3 cells formed impressive branching tubular structures with apparent lumens, suggesting the existence of specific factors in serum that are tubulogenic for mIMCD-3 cells but not for MDCK cells. Since normal serum does not contain enough HGF to induce tubulogenesis, these factors appeared to be substances other than HGF. This was also suggested by another observation: when MDCK cells or mIMCD-3 cells were cocultured under serum-free conditions with the embryonic kidney, both cell types formed branching tubular structures similar to those induced by HGF; however, only in the case of MDCK cells could this be inhibited by neutralizing antibodies against HGF. Thus, the embryonic kidney produces growth factors other than HGF capable of inducing tubule formation in the mIMCD-3 cells. Of a number of growth factors examined, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) were found to be tubulogenic for mIMCD-3 cells. Whereas only HGF was a potent tubulogenic factor for MDCK cells, HGF, TGF-alpha, and EGF were potent tubulogenic factors for mIMCD-3 cells. Nevertheless, there were marked differences in the capacity of these tubulogenic factors to induce tubulation as well as branching events in those tubules that did form (HGF>>) TGF-alpha>> EGF). Thus, at least three different growth factors can induce tubulogenesis and branching in a specific epithelial cell in vitro (though to different degrees), and different epithelial cells that are capable of forming branching tubular structures demonstrate vastly different responses to tubulogenic growth factors. The results are discussed in the context of branching morphogenesis during epithelial tissue development.

Anti-angiogenic treatment of glioblastoma with Vascular Endothelial Growth Factor (VEGF)- or VEGF Receptor 2 (VEGFR2) inhibitors normalizes tumor vessels, resulting in a profound radiologic response and improved quality of life. This approach however does not halt tumor progression by diffuse infiltration, as this phenotype is less angiogenesis dependent. Combined inhibition of angiogenesis and diffuse infiltrative growth would therefore be a more effective treatment approach in these tumors. The HGF/c-MET axis is important in both angiogenesis and cell migration in several tumor types including glioma. We therefore analyzed the effects of the c-MET- and VEGFR2 tyrosine kinase inhibitor cabozantinib (XL184, Exelixis) on c-MET positive orthotopic E98 glioblastoma xenografts, which routinely present with angiogenesis-dependent areas of tumor growth, as well as diffuse infiltrative growth. In in vitro cultures of E98 cells, cabozantinib effectively inhibited c-MET phosphorylation, concomitant with inhibitory effects on AKT and ERK1/2 phosphorylation, and cell proliferation and migration. VEGFR2 activation in endothelial cells was also effectively inhibited in vitro. Treatment of BALB/c nu/nu mice carrying orthotopic E98 xenografts resulted in a significant increase in overall survival. Cabozantinib effectively inhibited angiogenesis, resulting in increased hypoxia in angiogenesis-dependent tumor areas, and induced vessel normalization. Yet, tumors ultimately escaped cabozantinib therapy by diffuse infiltrative outgrowth via vessel co-option. Of importance, in contrast to the results from in vitro experiments, in vivo blockade of c-MET activation was incomplete, possibly due to multiple factors including restoration of the blood-brain barrier resulting from cabozantinib-induced VEGFR2 inhibition. In conclusion, cabozantinib is a promising therapy for c-MET positive glioma, but improving delivery of the drug to the tumor and/or the surrounding tissue may be needed for full activity.

Hepatocyte growth factor (HGF) has been shown to protect renal epithelial cells against apoptosis. To define the mechanism by which HGF inhibits apoptosis, we investigated the effect of HGF on the phosphorylation and expression of the Bcl-2 family proteins. Using a human proximal tubular epithelial cell (HKC) line as a model, we demonstrated that constitutive expression of HGF conveyed marked resistance to apoptotic death induced by serum withdrawal. HGF induced rapid phosphorylation of Akt in HKC cells, which was immediately followed by phosphorylation and resultant inactivation of Bad, a pro-apoptotic member of the Bcl-2 family. Pretreatment of the HKC cells with 10 nM wortmannin completely abolished HGF-induced phosphorylation of Akt and Bad, suggesting that this pathway is dependent on phosphoinositide (PI) 3-kinase. Overexpression of Bad increased apoptotic death in wild-type HKC cells but not in HGF-producing H4 cells. Immunoblotting confirmed that the Bad protein over-expressed in H4 cells was fully phosphorylated at both Ser(112) and Ser(136) sites. Prolonged incubation of HKC cells with HGF also dramatically induced expression of Bcl-xL, an anti-apoptotic member of the Bcl-2 family. These results suggest that the anti-apoptotic effect of HGF in renal epithelial cells is mediated by dual mechanisms involving two distinct Bcl-2 family proteins. HGF triggers Bad phosphorylation via the PI 3-kinase/Akt pathway, thereby inactivating this pro-apoptotic protein, while simultaneously inducing expression of anti-apoptotic Bcl-xL.

Hepatocyte growth factor (HGF) has been shown to reduce renal injury in a variety of animal models of chronic renal disease. Suggested mechanisms to explain this action include prevention of tubular cell apoptosis, blocking epithelial-to-mesenchymal transition, and promotion of extracellular matrix degradation. Inflammation is another common finding in kidneys that progress to end-stage renal failure; however, the effect of HGF on inflammation has hardly been investigated. For examining this issue, beginning 2 wk after subtotal nephrectomy, rats received a continuous infusion of recombinant HGF, neutralization of endogenous HGF by daily injection of an anti-HGF antibody, or preimmune IgG for an additional 2 wk. HGF infusion halted the progression of proteinuria and decreased renal collagen accumulation. Renal inflammation in both glomeruli and tubulointerstitium was significantly attenuated, associated with reductions in the tubular expression of the chemokines macrophage chemoattractant protein-1 (MCP-1) and RANTES (regulated upon expression normal T cell expressed and secreted). In contrast, HGF neutralization worsened renal fibrosis, aggravated renal inflammation, and enhanced tubular expression of MCP-1 and RANTES. In vitro, HGF suppressed basal and TNF-alpha-induced expression of these chemokines at both the mRNA and protein levels in a time- and dose-dependent manner in proximal tubular epithelial cells. HGF also blunted TNF-alpha-induced nuclear translocation and activation of NF-kappaB, a pivotal transcription factor that regulates chemokine expression. Immunohistochemistry showed that activated NF-kappaB was evident in tubules in remnant kidneys and increased remarkably with anti-HGF treatment. HGF infusion markedly suppressed expression of activated NF-kappaB in remnant kidneys. These findings suggest that the beneficial effect of HGF in chronic renal disease is attributable, at least in part, to a direct anti-inflammatory action, likely via NF-kappaB, on tubular epithelial cells.

The mammalian small GTPase ADP-ribosylation factor 6 (ARF6) plays important roles in a wide variety of cellular events, including endocytosis, actin cytoskeletal reorganization, and phosphoinositide metabolism. However, physiological functions for ARF6 have not previously been examined. Here, we described the consequence of ARF6 ablation in mice, which manifests most obviously in the context of liver development. Livers from ARF6-/- embryos are smaller and exhibit hypocellularity, due to the onset of midgestational liver cell apoptosis. Preceding the apoptosis, however, defective hepatic cord formation is observed; the liver cells migrate abnormally upon exiting the primordial hepatic epithelial sheet and clump rather than becoming dispersed. Consistent with this observation, the ability of hepatocyte growth factor/scatter factor (HGF) to induce hepatic cord-like structures from ARF6-/- fetal hepatocytes cultured in vitro in collagen gel matrix is impaired. Finally, we show that endogenous ARF6 in wild-type fetal hepatocytes is activated in response to HGF stimulation. These results provide evidence that ARF6 is an essential component in the signaling pathway coupling HGF signaling to hepatic cord formation.

Human umbilical cord-derived mesenchymal stem cells (hucMSCs) are particularly attractive cells for cellular and gene therapy in acute kidney injury (AKI). Adenovirus-mediated gene therapy has been limited by immune reaction and target genes selection. However, in the present study, we investigated the therapeutic effects of hepatocyte growth factor modified hucMSCs (HGF-hucMSCs) in ischemia/reperfusion-induced AKI rat models. In vivo animal models were generated by subjecting to 60 min of bilateral renal injury by clamping the renal pedicles and then introduced HGF-hucMSCs via the left carotid artery. Our results revealed that serum creatinine and urea nitrogen levels decreased to the baseline more quickly in HGF-hucMSCs-treated group than that in hucMSCs- or green fluorescent protein-hucMSCs-treated groups at 72 h after injury. The percent of proliferating cell nuclear antigen-positive cells in HGF-hucMSCs-treated group was higher than that in the hucMSCs or green fluorescent protein-hucMSCs-treated groups. Moreover, injured renal tissues treated with HGF-hucMSCs also exhibited less hyperemia and renal tubule cast during the recovery process. Immunohistochemistry and living body imaging confirmed that HGF-hucMSCs localize to areas of renal injury. Real-time polymerase chain reaction result showed that HGF-hucMSCs also inhibited caspase-3 and interleukin-1β mRNA expression in injured renal tissues. Western blot also showed HGF-hucMSCs-treated groups had lower expression of interleukin-1β. Terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate (dUTP) nick end labeling method indicated that HGF-hucMSCs-treated group had the least apoptosis cells. In conclusion, our findings suggest that HGF modification promotes the amelioration of ischemia/reperfusion-induced rat renal injury via antiapoptotic and antiinflammatory mechanisms; thus, providing a novel therapeutic application for hucMSCs in AKI.

Scatter factor (SF) (also known as hepatocyte growth factor [HGF]) is a cytokine that induces cell motility in vitro and angiogenesis in vivo. SF appears to be a determinant of the malignant phenotype in certain systemic cancers. We detected SF in extracts prepared from human gliomas, with the highest levels found in malignant tumors. Human glioblastoma cells expressed both SF and its receptor (c-met protein) in vivo, as demonstrated by immunohistochemistry. Consistent with these observations, we found moderate to high levels of production of immunoreactive and biologically active SF by cultured human glioblastoma cells (3 of 8 lines) and by neural microvascular endothelial cells (NMVEC) (3 of 3 lines). SF stimulated the proliferation of glioblastoma and NMVEC cell lines by paracrine or autocrine mechanisms. Conditioned medium (CM) from both glioblastoma and NMVEC cells contained SF-inducing factor (SF-IF) activity, defined by its ability to stimulate SF production in an indicator cell line (MRC5 human fibroblasts). This activity consisted of a high-molecular-weight >> 30 kDa), heat-sensitive component and a low-molecular weight (< 30 kDa), heat-stable component. Furthermore, glioblastoma CM stimulated NMVEC SF production, and NMVEC CM stimulated glioblastoma cell SF production, by 3- to 6-fold in each case. Our findings demonstrate that SF-dependent interactions between glioma cells, and between glioma cells and endothelium, can contribute to the heterogeneous proliferative and angiogenic phenotypes of malignant gliomas in vivo.

c-Met is a receptor tyrosine kinase that upon binding of its ligand, hepatocyte growth factor (HGF), activates downstream pathways with diverse cellular functions that are important in organ development and cancer progression. Anomalous c-Met signalling has been described in a variety of cancer types, and the receptor is regarded as a novel therapeutic target. In breast cancer there is a need to develop new treatments, particularly for the aggressive subtypes such as triple-negative and basal-like cancer, which currently lack targeted therapy. Over the last two decades, much has been learnt about the functional role of c-Met signalling in different models of breast development and cancer. This work has been complemented by clinical studies, establishing the prognostic significance of c-Met in tissue samples of breast cancer. While the clinical trials of anti-c-Met therapy in advanced breast cancer progress, there is a need to review the existing evidence so that the potential of these treatments can be better appreciated. The aim of this article is to examine the role of HGF/c-Met signalling in in vitro and in vivo models of breast cancer, to describe the mechanisms of aberrant c-Met signalling in human tissues, and to give a brief overview of the anti-c-Met therapies currently being evaluated in breast cancer patients. We will show that the HGF/c-Met pathway is associated with breast cancer progression and suggest that there is a firm basis for continued development of anti-c-Met treatment, particularly for patients with basal-like and triple-negative breast cancer.

Liver regeneration is known to be a process involving highly organized and ordered tissue growth triggered by the loss of liver tissue, and remains a fascinating topic. A large number of genes are involved in this process, and there exists a sequence of stages that results in liver regeneration, while at the same time inhibitors control the size of the regenerated liver. The initiation step is characterized by priming of quiescent hepatocytes by factors such as TNF-α, IL-6 and nitric oxide. The proliferation step is the step during which hepatocytes enter into the cell cycle's G1 phase and are stimulated by complete mitogens including HGF, TGF-α and EGF. Hepatic stimulator substance, glucagon, insulin, TNF-α, IL-1 and IL-6 have also been implicated in regulating the regeneration process. Inhibitors and stop signals of hepatic regeneration are not well known and only limited information is available. Furthermore, the effects of other factors such as VEGF, PDGF, hypothyroidism, proliferating cell nuclear antigen, heat shock proteins, ischemic-reperfusion injury, steatosis and granulocyte colony-stimulating factor on liver regeneration are also systematically reviewed in this article. A tissue engineering approach using isolated hepatocytes for in vitro tissue generation and heterotopic transplantation of liver cells has been established. The use of stem cells might also be very attractive to overcome the limitation of donor liver tissue. Liver-specific differentiation of embryonic, fetal or adult stem cells is currently under investigation.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the third leading cause of cancer-related deaths worldwide. Limitations in HCC treatment result due to poor prognosis and resistance against traditional radiotherapy and chemotherapies. The multikinase inhibitor sorafenib is the only FDA approved drug available for advanced HCC patients, and development of second-line treatment options for patients who cannot tolerate or develop resistance to sorafenib is an urgent medical need. In this study, we established sorafenib-resistant cells from Huh7 and Mahlavu cell lines by long-term sorafenib exposure. Sorafenib-resistant HCC cells acquired spindle-shape morphology, upregulated mesenchymal markers, and showed significant increase in both migration and invasion abilities compared to their parental counterparts. Moreover, after long-term sorafenib treatment, HCC cells showed induction of hepatocyte growth factor (HGF) synthesis and secretion along with increased levels of c-Met kinase and its active phosphorylated form, indicating autocrine activation of HGF/c-Met signaling. Importantly, the combined treatment of the resistant cells with c-Met kinase inhibitor SU11274 and HGF neutralizing antibody significantly reversed the increased invasion ability of the cells. The combined treatment also significantly augmented sorafenib-induced apoptosis, suggesting restoration of sorafenib sensitivity. These results describe, for the first time, compensatory upregulation of HGF synthesis leading to autocrine activation of HGF/c-Met signaling as a novel cellular strategy in the acquisition of sorafenib resistance. Therefore, we suggest that combinatorial therapeutic strategies with HGF and c-Met inhibitors comprise promising candidates for overcoming sorafenib resistance.

BACKGROUND

An increasing number of basic, translational and clinical studies demonstrate the importance of the protein tyrosine kinase receptor, c-Met, in the progression of prostate cancer. c-Met is overexpressed in primary prostate cancers, further increased in expression in bone metastases and is associated with the development of castrate-resistant disease. Because of its importance as a target, c-Met inhibitors have reached clinical trial for advanced, castrate-resistant prostate cancer.

METHODS

In this review, altered expression of c-Met and hepatocyte growth factor in prostate tumors and the microenvironment and how they contribute to growth and invasion of prostate cancer cells is described. Next, preclinical studies providing the support for use of c-Met inhibitors are discussed. Finally, early promising results from c-Met inhibitors in clinical trial, and future prospects for c-Met inhibitors in the treatment of advanced stage prostate cancer, are discussed.

CONCLUSIONS

An emerging theme in treating metastatic prostate cancer is the requirement to target both the epithelial and stromal compartments. Results from clinical trials suggest that inhibitors of c-Met that block stromal-mediated c-Met activation in prostate tumors may be important therapeutic agents in at least a subset of patients with metastatic prostate cancer. However, as many of the inhibitors have multiple targets, the efficacy of targeting c-Met alone remains to be determined.

To understand the molecular mechanisms underlying the regulation of hepatocyte growth factor (HGF) gene expression and to define the DNA sequences essential for its cell-type specific and inducible expression, we have isolated and characterized the 5'-flanking region of the HGF gene. A genomic clone containing 2.8 kilobases of the 5'-flanking region of the HGF gene has been isolated from a mouse liver genomic library. Sequence analysis showed that the promoter region of the mouse HGF gene contains a noncanonical TATA box (ATAAA). Further analysis of the 5'-flanking region revealed a number of putative regulatory elements, such as four interleukin-6 response elements (IL-6 RE), two potential binding sites for NF-IL6, a TGF-beta inhibitory element (TIE), a cAMP response element (CRE), two estrogen response elements (ERE) including one located in the first intron, a potential vitamin D response element (VDRE) which overlaps a chicken ovalbumin upstream promoter (COUP) transcription factor binding element, two liver-specific transcription factor (C/EBP) binding sites, and a B cell- and macrophage-specific transcriptional factor binding site (PU.1/ETS). To determine the location of sites that may be critical for the function of the HGF promoter, we constructed a series of chimeric genes containing variable regions of the 5'-flanking sequence of HGF gene and the coding region for chloramphenicol acetyltransferase (CAT). Transient transfection of chimeric plasmids demonstrated that the mouse HGF gene promoter containing 70 base pairs of the 5'-flanking sequences were active in mouse fibroblast NIH 3T3 cells and in human endometrial carcinoma RL95-2 cells. This basal transcription activity of the HGF promoter was modulated in NIH 3T3 and RL95-2 cells by multiple upstream elements. Three positive elements were identified at positions -2848 to -2674, -1386 to -1231, and -699 to -274, and three negative candidate elements were mapped to positions -1652 to -1386, -964 to -699, and -274 to -70, respectively. By the combination of a series of 5'-end deletion and internal deletion, a cell type-specific negative regulatory element in RL95-2 cells was localized to the nucleotide position -964 to -699. Moreover, the reporter plasmid containing interleukin 6 (IL-6) response element was responsive to IL-6 stimulation in stably transfected NIH 3T3 cells. Our findings revealed a complex pattern of transcriptional regulation of the mouse HGF gene expression.

Rac1-regulated reactive oxygen species (ROS) production has been implicated in apoptosis. In contrast, pleiotropic protein kinase Akt protects against apoptosis. However, the pro- and antiapoptotic mechanisms of rac1 and Akt, respectively, and the intersection between these mechanisms are incompletely understood. In a model of oxidative stress and apoptosis induced by hypoxia/reoxygenation (H/R) in primary hepatocytes, activation of the PI3-K Akt axis by the prosurvival hepatocyte growth factor (HGF) inhibited H/R-stimulated rac1 activation and intracellular ROS production, and suppressed apoptosis. Suppression of PI3-K or Akt activity abrogated the inhibitory effect of HGF on rac1 activity and rac1-regulated oxidative stress. Furthermore, constitutive activation of Akt or PI3-K in the absence of HGF was sufficient to phosphorylate rac1, inhibit rac1 activation, and suppress rac1-regulated ROS production. These findings demonstrate that growth factor-stimulated activation of PI3-K-Akt is necessary and sufficient to suppress intracellular oxidative stress and apoptosis by inhibiting activation of pro-apoptotic, prooxidative rac1 GTPase.

BACKGROUND

Breast tumor kinase (Brk/protein tyrosine kinase 6 (PTK6)) is a nonreceptor, soluble tyrosine kinase overexpressed in the majority of breast tumors. Previous work has placed Brk downstream of epidermal growth factor receptor (ErbB) activation and upstream of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein (MAP) kinases. Herein we investigate the regulation of Brk kinase activity and cell migration in response to treatment of keratinocytes (HaCaT cells) and breast cancer cell lines (MDA-MB-231 and T47D cells) with hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), peptide ligands for Met and Ron receptors, respectively.

METHODS

In vitro kinase assays were performed to directly measure Brk kinase activity in response to MET and RON ligands. Transfection of Brk-targeted RNAi was used to knock down endogenous Brk or ERK5 in multiple cell lines. Kinase activities (downstream of MET signaling) were assayed by Western blotting using total and phospho-specific antibodies. Boyden chamber assays were used to measure cell migration in response to manipulation of Brk and downstream MET effectors. Rescue experiments were performed by knock down of endogenous Brk using RNAi (targeting the untranslated region (3'-UTR)) and transient transfection (re-expression) of either wild-type or kinase-inactive Brk.

RESULTS

Brk gene silencing revealed that HGF, but not MSP, induced robust Brk-dependent cell migration. Brk and ERK5 copurified in HGF-induced protein complexes, and Brk/ERK5 complexes formed independently of Brk kinase activity. ERK5 was required for breast cancer cell but not keratinocyte cell migration, which became ERK1/2-dependent upon ERK5 knockdown. Notably, rescue experiments indicated that the kinase activity of Brk was not required for HGF-induced cell migration. Further, expression of either wild-type or kinase-inactive Brk in Brk-null MDA-MB-435 cells activated ERK5 and conferred increased HGF-induced cell migration.

CONCLUSIONS

These results have identified Brk and ERK5 as important downstream effectors of Met signaling to cell migration. Targeting ERK5 kinase activity or inhibiting the formation of Brk/ERK5 complexes may provide an additional means of blocking cell migration associated with breast cancer progression to metastasis.

Circulating endothelial progenitor cells (EPCs) play a pivotal role in angiogenesis. Hepatocyte growth factor (HGF) is known to induce proliferation and motility in endothelial cells, and to play a role in mitogenic and morphogenic actions. However, the role of HGF in EPC mobilization has not been clearly described yet. We investigated the effect of HGF on mobilizing EPCs and on angiogenesis in elastase-induced lung injury. HGF significantly increased the triple-positive (Sca-1(+), Flk-1(+), and c-kit(+)) fraction in peripheral mononuclear cells in mice. The bone marrow-derived cells were recruited into the injured lungs, where they differentiated to capillary endothelial cells. HGF induced proliferation of both bone marrow-derived and resident endothelial cells in the alveolar wall. In conclusion, the present study suggests that HGF induces EPC mobilization from the bone marrow and enhances the proliferation of endothelial cells in vivo. These complex effects induced by HGF orchestrate pulmonary regeneration in emphysematous lung parenchyma.

BACKGROUND

CD151, c-Met, and integrin alpha3/alpha6 are all involved in the hepatocyte growth factor (HGF)/c-Met signal pathway, which plays an important role in the malignant progression of tumors.

OBJECTIVE

The purpose of this study was to explore the expression and prognostic significance of these proteins in pancreatic ductal adenocarcinoma (PDAC).

METHODS

We used immunohistochemical methods to investigate the expression patterns of CD151, c-Met, and integrin alpha3/alpha6proteins in 71 patients with PDAC and in ten samples of normal pancreatic tissue. We also assessed correlations between these proteins and clinicopathological parameters and survival of PDAC patients using various statistical methods.

RESULTS

CD151, c-Met, and integrin alpha3/alpha6 were all overexpressed in PDAC. CD151 and c-Met overexpressions were significantly associated with TNM stage (p=0.001 and p=0.038, respectively) and lymph node invasion (p=0.000, p=0.012, respectively). A significant positive linear correlation was found between CD151 and c-Met (r=0.583; p=0.000), integrin alpha3 (r=0.457; p=0.000), and integrin alpha6 (r=0.671; p=0.000). Overexpression of CD151, c-Met, integrin alpha3, or integrin alpha6 was related to poor survival of PDAC patients (p=0.000, p=0.000, p=0.005, and p=0.003, respectively), and CD151 and c-Met were independent factors in prognosis of PDAC.

CONCLUSIONS

CD151, c-Met, and integrin alpha3/alpha6 were all overexpressed in PDAC. CD151 and c-Met might be new molecular markers to predict the prognosis of PDAC patients.

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is an integral membrane protein expressed on epithelial cells and contains two extracellular Kunitz domains (N-terminal KD1 and C-terminal KD2) known to inhibit trypsin-like serine proteases. In tumorigenesis and tissue regeneration, HAI-1 regulates the hepatocyte growth factor (HGF)/c-Met pathway by inhibiting the activity of HGF activator (HGFA) and matriptase, two serine proteases that convert pro-HGF into its biologically active form. By screening a placental cDNA library, we discovered a new splice variant of HAI-1 designated HAI-1B that contains an extra 16 amino acids adjacent to the C terminus of KD1. To investigate possible consequences on Kunitz domain function, a soluble form of HAI-1B (sHAI-1B) comprising the entire extracellular domain was produced. First, we found that sHAI-1B displayed remarkable enzyme specificity by potently inhibiting only HGFA (IC50 = 30.5 nm), matriptase (IC50 = 16.5 nm), and trypsin (IC50 = 2.4 nm) among 16 serine proteases examined, including plasminogen activators (urokinase- and tissue-type plasminogen activators), coagulation enzymes thrombin, factors VIIa, Xa, XIa, and XIIa, and activated protein C. Relatively weak inhibition was found for plasmin (IC50 = 399 nm) and plasma kallikrein (IC50 = 686 nm). Second, the functions of the KD1 and KD2 domains in sHAI-1B were investigated using P1 residue-directed mutagenesis to show that inhibition of HGFA, matriptase, trypsin, and plasmin was due to KD1 and not KD2. Furthermore, analysis by reverse transcription-PCR demonstrated that HAI-1B and HAI-1 were co-expressed in normal tissues and various epithelial-derived cancer cell lines. Both isoforms were up-regulated in eight examined ovarian carcinoma specimens, three of which had higher levels of HAI-1B RNA than of HAI-1 RNA. Therefore, previously demonstrated roles of HAI-1 in various physiological and pathological processes likely involve both HAI-1B and HAI-1.

Human gingival fibroblasts (HGFs), a predominant cell type in tooth-supporting structure, are presently recognized for their active role in the innate immune response. They produce a variety of inflammatory cytokines in response to microbial components such as LPS from the key periodontal pathogen, Porphyromonas gingivalis. In this study, we demonstrated that HGFs expressed mRNA of TLRs 1, 2, 3, 4, 5, 6, and 9, but not TLRs 7, 8, and 10. Stimulation of HGFs with highly purified TLR2 ligand (P. gingivalis LPS), TLR3 ligand (poly(I:C)), TLR4 ligand (Escherichia coli LPS), and TLR5 ligand (Salmonella typhimurium flagellin) led to expression of IL-8 and IDO. A potent TLR 9 ligand, CpG oligodeoxynucleotide 2006 had no effect, although HGFs showed a detectable TLR9 mRNA expression. No significant enhancement on IL-8 or IDO expression was observed when HGFs were stimulated with various combinations of TLR ligands. Surprisingly, the TLR9 ligand CpG oligodeoxynucleotide 2006 was able to specifically inhibit poly(I:C)-induced IL-8 and IDO expression. TNF-alpha enhanced TLR ligand-induced IL-8 production in HGFs, whereas IFN-gamma enhanced TLR ligand-induced IDO expression. HGF production of IDO in response to P. gingivalis LPS, IFN-gamma, or the two in combination inhibited T cell proliferation in MLRs. The observed T cell inhibition could be reversed by addition of either 1-methyl-dl-tryptophan or l-tryptophan. Our results suggest an important role of HGFs not only in orchestrating the innate immune response, but also in dampening potentially harmful hyperactive inflammation in periodontal tissue.

Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are among the most potent mitogens identified for alveolar type II epithelial cells and may have other important functions in repair of the alveolar epithelium in acute lung injury (ALI). However, neither growth factor has been identified in the distal air spaces or plasma of patients with ALI. The goals of this study were to determine: (1) whether HGF and KGF are present in pulmonary edema fluid from patients with ALI and control patients with hydrostatic pulmonary edema; (2) whether HGF and KGF are biologically active in pulmonary edema; and (3) whether HGF or KGF levels are associated with clinical outcome. Pulmonary edema and plasma samples were obtained within 48 h of onset of acute pulmonary edema requiring mechanical ventilation in 26 patients with ALI and 11 control patients with hydrostatic edema. HGF and KGF concentrations were measured with enzyme-linked immunosorbent assays (ELISAs). The median (25th to 75th percentiles) concentration of HGF in pulmonary edema fluid was 21.4 (8.3 to 41.3) ng/ml in ALI and 6.6 (4.8 to 11.4) ng/ml in hydrostatic edema fluid (p < 0.01). The HGF concentration was 7-fold higher in the edema fluid than in the plasma of patients with ALI. In contrast, KGF was detected in low concentrations in edema fluid of patients with ALI and hydrostatic pulmonary edema; the concentration of KGF did not differ in ALI edema (0.6 [0.3 to 2.1] ng/ml) and hydrostatic edema fluid (0.2 [0.0 to 2.6] ng/ml) (p = NS). HGF and KGF were partly purified from four edema-fluid samples by heparin-Sepharose chromatography. Partly purified edema fluids were potent stimuli of DNA synthesis in cultured rat type II alveolar cells; addition of neutralizing antibodies to HGF and KGF attenuated this increase in DNA synthesis by 66% and 53%, respectively. Interestingly, higher edema-fluid levels of HGF were associated with higher mortality in patients with ALI. These novel results show that HGF and KGF are active in the alveolar space early in ALI, probably mediating early events in lung repair, and that increased levels of HGF in edema fluid may have prognostic value early in ALI.

Salivary gland stem/progenitor cells are thought to be present in intercalated ductal cells, but the fact is unclear. In this study, we sought to clarify if stem/progenitor cells are present in submandibular glands using colony assay, which is one of the stem cell assay methods. Using a low-density culture of submandibular gland cells of neonatal rats, we developed a novel culture system that promotes single cell colony formation. Average doubling time for the colony-forming cells was 24.7 (SD=+/-7.02)h, indicating high proliferative potency. When epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were added to the medium, the number of clonal colonies increased greater than those cultured without growth factors (13.2+/-4.18 vs. 4.5+/-1.73). The RT-PCR and immunostaining demonstrated expressing acinar, ductal, and myoepithelial cell lineage markers. This study demonstrated the presence of the salivary gland stem/progenitor cells that are highly proliferative and multipotent in salivary glands.

Effect of hepatocyte growth factor (HGF) on normal human epidermal keratinocytes cultured under conditions of low Ca2+ (0.1 mM, growth-promoting condition) and physiological Ca2+ (1.8 mM, differentiation-promoting condition) was investigated. In low Ca2+, HGF markedly enhanced the migration of keratinocytes while it suppressed cell growth and DNA synthesis in a dose-dependent manner. In contrast, HGF enhanced the migration, cell growth, and DNA synthesis of keratinocytes cultured under conditions of physiological Ca2+. The maximal stimulation of DNA synthesis (2.4-fold stimulation) in physiological Ca2+ was seen at 2.5-5 ng/ml HGF and the stimulatory effect of HGF was suppressed by transforming growth factor-beta 1. Analysis of the HGF receptor using 125I-HGF as a ligand showed that human keratinocytes expressed a single class of specific, saturable receptor for HGF in both low and physiological Ca2+ conditions, exhibiting a Kd = 17.3 pM and approximately 690 binding sites/cell under physiological Ca2+. Thus, HGF is a potent factor which enhances growth and migration of normal human keratinocytes under conditions of physiological Ca2+. HGF may play an important role in epidermal tissue repair as it enhances both the migration and growth of keratinocytes.

The lethality of pancreatic adenocarcinoma stems from an elevated incidence of tumor cell invasion and metastasis that are mediated by mechanisms not yet understood. Recent studies indicate that the proinvasive integrin alpha 6 beta 4 is highly upregulated in pancreatic adenocarcinomas. To assess the importance of this integrin in pancreatic cancer cell migration and invasion, cell lines were screened for integrin alpha 6 beta 4 expression by immunoblotting and fluorescence-activated cell sorting and their ability to migrate and invade toward hepatocyte growth factor (HGF). We found that cell surface expression of the alpha 6 beta 4 integrin correlated with the cells' ability to migrate and invade toward HGF. When cells expressing high levels of integrin alpha 6 beta 4 were treated with small interfering RNA targeting alpha 6 or beta 4 integrin subunits, we observed a reduction in cell migration and invasion. Furthermore, the activity of the small GTPase Rac1 was stimulated by alpha 6 beta 4 integrin expression and was necessary for HGF-stimulated chemotaxis. We discovered that expression of the Rac-specific nucleotide exchange factor, Tiam1 (T-lymphoma invasion and metastasis), was upregulated in cells overexpressing the integrin alpha 6 beta 4 and required for the elevated Rac1 activity in these cells. We conclude that the integrin alpha 6 beta 4 promotes the migratory and invasive phenotype of pancreatic carcinoma cells through the Tiam1-Rac1 pathway in part through the upregulation of Tiam1.

Netrin-4, a laminin-related secreted protein is an axon guidance cue recently shown essential outside of the nervous system, regulating mammary and lung morphogenesis as well as blood vascular development. Here, we show that Netrin-4, at physiologic doses, induces proliferation, migration, adhesion, tube formation and survival of human lymphatic endothelial cells in vitro comparable to well-characterized lymphangiogenic factors fibroblast growth factor-2 (FGF-2), hepatocyte growth factor (HGF), vascular endothelial growth factor-A (VEGF-A), and vascular endothelial growth factor-C (VEGF-C). Netrin-4 stimulates phosphorylation of intracellular signaling components Akt, Erk and S6, and their specific inhibition antagonizes Netrin-4-induced proliferation. Although Netrin receptors Unc5B and neogenin, are expressed by human lymphatic endothelial cells, suppression of either or both does not suppress Netrin-4-promoted in vitro effects. In vivo, Netrin-4 induces growth of lymphatic and blood vessels in the skin of transgenic mice and in breast tumors. Its overexpression in human and mouse mammary carcinoma cancer cells leads to enhanced metastasis. Finally, Netrin-4 stimulates in vitro and in vivo lymphatic permeability by activating small GTPases and Src family kinases/FAK, and down-regulating tight junction proteins. Together, these data provide evidence that Netrin-4 is a lymphangiogenic factor contributing to tumor dissemination and represents a potential target to inhibit metastasis formation.

OBJECTIVE

To evaluate the efficacy, safety, biomarkers, and pharmacokinetics of rilotumumab, a fully human, monoclonal antibody against hepatocyte growth factor (HGF)/scatter factor, combined with mitoxantrone and prednisone (MP) in patients with castration-resistant prostate cancer (CRPC).

METHODS

This double-blinded phase II study randomized (1:1:1) patients with progressive, taxane-refractory CRPC to receive MP (12 mg/m(2) i.v. day 1, 5 mg twice a day orally days 1-21, respectively) plus 15 mg/kg rilotumumab, 7.5 mg/kg rilotumumab, or placebo (i.v. day 1) every 3 weeks. The primary endpoint was overall survival (OS).

RESULTS

One hundred and forty-four patients were randomized. Median OS was 12.2 versus 11.1 months [HR, 1.10; 80% confidence interval (CI), 0.82-1.48] in the combined rilotumumab versus control arms. Median progression-free survival was 3.0 versus 2.9 months (HR, 1.02; 80% CI, 0.79-1.31). Treatment appeared well tolerated with peripheral edema (24% vs. 8%) being more common with rilotumumab. A trend toward unfavorable OS was observed in patients with high tumor MET expression regardless of treatment. Soluble MET levels increased in all treatment arms. Total HGF levels increased in the rilotumumab arms. Rilotumumab showed linear pharmacokinetics when co-administered with MP.

CONCLUSIONS

Rilotumumab plus MP had manageable toxicities and showed no efficacy improvements in this estimation study. High tumor MET expression may identify patients with CRPC with poorer prognosis.