Regulation Of Hepatocyte Growth Factor Research Articles

HGF Regulation in the Tumor-Supportive Microenvironment Combined with Pan-PIM Kinases Inhibition in Malignant Cells As a Multi-Target Approach for Acute Myeloid Leukemia

Background: In acute myeloid leukemia (AML), persistent clonal proliferation of malignant myeloid cells results in high severity and low cure potential. Resistance to currently available chemotherapeutics and disease relapse remains an important concern worldwide. Several molecular targets play key roles in impairing therapeutic response, including PIM-kinases, a serine/threonine kinase family associated with the progression of hepatocyte growth factor (HGF)-driven cancers through its receptor, MET. We reported that enhanced HGF secretion by bone marrow (BM) mesenchymal stromal cells (MSC) correlated with low levels of the serine protease inhibitor kunitz-type2 (SPINT2) in AML (Roversi et al., 2019), which are affected by immunosuppressive properties of the tumor microenvironment (TME). However, the interplay between SPINT2, HGF, and PIM-kinases in the TME is yet to be elucidated and could represent an important multi-target therapeutic strategy in hematological cancers. Aims: Given the importance of HGF to AML prognosis and progression, we analyzed the interplay between SPINT2, HGF, and pan-PIM kinases by evaluating the gene expression and the effects of a novel pan-PIM kinase inhibitor, PIM447 (Novartis), in leukemia cells and in the TME. Methods: SPINT2, HGF, PIM1, PIM2 and PIM3 mRNA expression data were obtained from OHSU (2018) AML study and TCGA (cBio Portal). Correlation analysis was obtained by Spearman's coefficient (r). A total of 5 healthy donors (HD) and 5 de novo AML patients BMMSC (CD90+CD105+CD73+CD45-CD34-CD31-HLA-DR-) were treated with 5-Azacitidine (Aza), and evaluated for SPINT2 gene expressions. Myeloid (OCI-AML3, MOLM13, U937) and lymphoblastic (RS4;11, Jurkat) leukemia cells were treated with increasing concentrations of PIM447, and the 101nd apoptosis rates were determined. Peripheral blood and BM mononuclear cells (PBMC/BMMC) from HD were tested for cytotoxicity. Leukemia cells were also examined for reactive oxygen species (ROS), nitric oxide (NO) production, and mitochondrial membrane potential (ΔΨm). To verify PIM447 action onto an immunosuppressive TME, cocultures of leukemia cells and monocytes or monocyte-derived macrophages (MDMs) were performed. To understand the interplay between SPINT2 and PIM-kinases, cocultures of SPINT2-overexpressed MSC and leukemia cells, were performed. Statistical analyzes were performed using Kruskal Wallis, ANOVA or Mann-Whitney tests ( P<.05). Results: The analysis of gene expression performed in MC from 188 AML patients (age median: 57.5 years [range: 18-88]) showed that levels of HGF correlated positively with PIM1, PIM2, and PIM3 ((r) =.8727;.9094;.9093, respectively, P<.0001) and negatively with SPINT2 (r=-.06402). We also verified that SPINT2 mRNA expression was upregulated in AML-BMMSC ( P<.05) after Aza treatment when compared to HD-BMMSC, suggesting that this gene is methylated in AML. Our in vitro results showed PIM447 treatment (2-20μM) yielded reduced cell viability and increased apoptosis in leukemia cells [IC50 range: 12.2-22.2μM, P<.01] without affecting HD cells. PIM447 reduced ROS, NO production, and ΔΨm (all P<.0001) in all cell lines. PIM447 treatment of macrophages differentiated from HD-PBMC revealed a shorter population of M2 macrophages (HLA-DR-CD206+) ( P<.001) and an increased population of M1 macrophages (HLA-DR+CD80+) ( P<.05). Moreover, PIM447 promoted apoptosis of leukemia cells co-cultured with MDMs ( P<.0001). Interestingly, co-culture analyses of SPINT2-overexpressed MSC enhanced PIM447 cytotoxic activity towards leukemia cells (P<.05) and reduced cell-cell adhesion by diminishing CD49b, CD49d, and CD49e (P<.05), suggesting an interplay between SPINT2 and pan-PIM kinases Conclusion: Collectively, our data identify a novel pan-PIM kinase inhibitor, PIM447, which effectively induces leukemia cell apoptosis through reducing MET receptor activation as well as acts on their tumor-supportive microenvironment by inducing a phenotypic shift from immunosuppressive M2 to anti-tumor M1 macrophages. PIM447 also interplays with SPINT2/HGF pathway in the TME, reducing AML progression. These findings suggest that a pivotal strategy to treat AML could be a multi-target therapy, acting both on leukemia cells and in the tumor microenvironment. Funding: FAPESP #2017/21801-2, #2019/25247-5, #2021/05320-0; CNPq #303405/2018-0.

CircATIC Contributes to Multiple Myeloma Progression via miR-324-5p-Dependent Regulation of HGF.

Circular RNA (circRNA) 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (circATIC; hsa_circ_0058058) was observed to be upregulated in multiple myeloma (MM) by former article. However, the function and exact mechanism of circATIC in MM development remain barely known. CircRNA-microRNA (miRNA)-messenger RNA (mRNA) axis was established through using bioinformatic databases (starbase, Circinteractome, and microT-CDS). Dual-luciferase reporter assay, RNA immunoprecipitation assay, and RNA-pull down assay were utilized to verify the target relationship between microRNA-324-5p (miR-324-5p) and circATIC or hepatocyte growth factor (HGF). CircATIC expression was upregulated in MM patients and cell lines. CircATIC interference notably hampered cell proliferation, migration, invasion, and glycolysis and induced cell apoptosis of MM cells. MiR-324-5p was a target of circATIC. CircATIC silencing-mediated effects in MM cells were largely overturned by the knockdown of miR-324-5p. HGF was a target of miR-324-5p, and circATIC upregulated the expression of HGF partly through sponging miR-324-5p in MM cells. MiR-324-5p suppressed the malignant behaviors of MM cells, which were largely counteracted by the overexpression of HGF in MM cells. CircATIC accelerated the proliferation, migration, invasion, and glycolysis and suppressed the apoptosis of MM cells through mediating miR-324-5p/HGF signaling.

SIPA1 Is a Modulator of HGF/MET Induced Tumour Metastasis via the Regulation of Tight Junction-Based Cell to Cell Barrier Function.

Simple SummaryThe role of Signal Induced Proliferation Associated 1 (SIPA1) in lung cancer remains largely unknown. This study aimed to evaluate the importance of SIPA1 in the development and progression of lung cancer, to demonstrate the cellular functions of SIPA1 and the molecular mechanisms involved.Background: Lung cancer is the leading cause of cancer death. SIPA1 is a mitogen induced GTPase activating protein (GAP) and may hamper cell cycle progression. SIPA1 has been shown to be involved in MET signaling and may contribute to tight junction (TJ) function and cancer metastasis. Methods: Human lung tumour cohorts were analyzed. In vitro cell function assays were performed after knock down of SIPA1 in lung cancer cells with/without treatment. Quantitative polymerase chain reaction (qPCR) and western blotting were performed to analyze expression of HGF (hepatocyte growth factor), MET, and their downstream markers. Immunohistochemistry (IHC) and immunofluorescence (IFC) staining were performed. Results: Higher expression of SIPA1 in lung tumours was associated with a poorer prognosis. Knockdown of SIPA1 decreased invasiveness and proliferation of in vitro cell lines, and the SIPA1 knockdown cells demonstrated leaky barriers. Knockdown of SIPA1 decreased tight junction-based barrier function by downregulating MET at the protein but not the transcript level, through silencing of Grb2, SOCS, and PKCμ (Protein kinase Cµ), reducing the internalization and recycling of MET. Elevated levels of SIPA1 protein are correlated with receptor tyrosine kinases (RTKs), especially HGF/MET and TJs. The regulation of HGF on barrier function and invasion required the presence of SIPA1. Conclusions: SIPA1 plays an essential role in lung tumourigenesis and metastasis. SIPA1 may be a diagnostic and prognostic predictive biomarker. SIPA1 may also be a potential therapeutic target for non-small cell lung cancer (NSCLC) patients with aberrant MET expression and drug resistance.

Age-related elevation of HGF is driven by the reduction of fibroblast size in a YAP/TAZ/CCN2 axis-dependent manner

BackgroundAged human skin is primarily attributable to the loss of collagen. Hepatocyte growth factor (HGF) acts as an anti-fibrotic factor by suppression of collagen production. In aged human skin, HGF is elevated in dermal fibroblasts and thus contributes to dermal aging (thin dermis) by suppression of collagen production. ObjectiveWe aimed to investigate the underlying mechanisms of age-related elevation of HGF expression. MethodsCollagen fibrils in the aged skin dermis are fragmented and disorganized, which impairs collagen-fibroblast interaction, resulting in reduced fibroblast spreading and size. To explore the connection between reduced dermal fibroblast size and age-related elevation of HGF expression, we manipulate dermal fibroblast size, and cell-size dependent regulation of HGF was investigated by laser capture microdissection, immunostaining, capillary electrophoresis immunoassay, and quantitative RT-PCR. ResultsWe found that reduced fibroblast size is responsible for age-related elevation of HGF expression. Further investigation indicated that cell size-dependent upregulation of HGF expression was mediated by impeded YAP/TAZ nuclear translocation and their target gene, CCN2. Conversely, restoration of dermal fibroblast size rapidly reversed cell-size-dependent upregulation of HGF in a YAP/TAZ-dependent manner. Finally, we confirmed that elevated HGF expression is accompanied by the reduced expression of YAP/TAZ and CCN2 in the aged human skin in vivo. ConclusionAge-related elevation of HGF is driven by the reduction of fibroblast size in a YAP/TAZ/CCN2 axis-dependent manner. These data reveal a novel mechanism by which reduction of fibroblast size upregulates HGF expression, which in turn contributes to loss of collagen, a prominent feature of aged human skin.

HGFA Is an Injury-Regulated Systemic Factor that Induces the Transition of Stem Cells into GAlert

The activation of quiescent stem cells into the cell cycle is a key step in initiating the process of tissue repair. We recently reported that quiescent stem cells can transition into GAlert, a cellular state in which they have an increased functional ability to activate and participate in tissue repair. However, the precise molecular signals that induce GAlert in stem cells have remained elusive. Here, we show that the injury-induced regulation of hepatocyte growth factor (HGF) proteolytic processing via the systemic protease, hepatocyte growth factor activator (HGFA), stimulates GAlert in skeletal muscle stem cells (MuSCs) and fibro-adipogenic progenitors (FAPs). We demonstrate that administering active HGFA to animals is sufficient to induce GAlert in stem cells throughout the body and to significantly accelerate the processes of stem cell activation and tissue repair. Our data suggest that factors that induce GAlert will have broad therapeutic applications for regenerative medicine and wound healing.

Regulation of HGF and c-MET Interaction in Normal Ovary and Ovarian Cancer.

Binding of hepatocyte growth factor (HGF) to the c-MET receptor has mitogenic, motogenic, and morphogenic effects on cells. The versatile biological effects of HGF and c-MET interactions make them important contributors to the development of malignant tumors. We and others have demonstrated a therapeutic value in targeting the interaction of c-MET and HGF in epithelial ovarian cancer (EOC). However, both HGF and c-MET are expressed in the normal ovary as well. Therefore, it is important to understand the differences in mechanisms that control HGF signaling activation and its functional role in the normal ovary and EOC. In the normal ovary, HGF signaling may be under hormonal regulation. During ovulation, HGF-converting proteases are secreted and the subsequent activation of HGF signaling enhances the proliferation of ovarian surface epithelium in order to replenish the area damaged due to expulsion of the ovum. In contrast, EOC cells that exhibit epithelial characteristics constitutively express both c-MET and HGF-converting proteases such as urokinase-type plasminogen activator. In EOC, mechanisms to control the activation of HGF signaling are absent since HGF is provided locally from the tissue microenvironment as well as remotely throughout the body. Potential incessant HGF signaling in EOC may lead to an increase in proliferation, invasion through the stroma, and migration to other tissues of cancer cells. Therefore, targeting the interaction of c-MET and HGF would be beneficial in treating EOC.

Effect of colon cancer metastasis associated gene-1 on biological function of gastric cancer cell line MGC-803 by regulating hepatocyte growth factor/c-Met signaling pathway

Objective To study the effect of colon cancer metastasis associated gene-1 (MACC-1) expression on the proliferation and apoptosis of gastric cancer cell line MGC-803 in vitro. Methods Cultured gastric cancer MGC-803 cells and passaged cells were divided into three groups: group A [MACC-1-short hairpin RNA (shRNA) and PSV2neo3 co transfection group]; group B (shNC and PSV2neo co transfection group); group C (non transfection group). By Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR), the expression of MACC-1 protein and mRNA was detected in the three groups, and that of of c-Met, E-cadherin and N-cadherin in 3 groups of MGC-803 cells were detected. Flow cytometry and scratch test were used to analyze migration, proliferation and apotosis of MGC-803 cells transfected with MACC-1-shRNA. Results (1) Western blotting and Real-time PCR test results showed that as compared with groups B and C, MACC-1 and c-Met protein and mRNA expression in group A were significantly down-regulated (P<0.01). (2) Western blotting and Real-time PCR detection results showed as compared with groups B and C, E-cadherin protein and mRNA expression levels were significantly increased (P<0.01), c-Met and N-cadherin mRNA and protein levels were significantly decreased (P<0.01) in group A. There was no significant difference between groups B and C. (3) Scratch assay showed the scratch space was wide, and healing was inhibited in group A. the migration ability of MGC-803 cells in group A was reduced as compared with groups B and C (P<0.01), but there was no statistically significant difference in the scratch space between groups B and C. (4) Flow cytometry revealed that the apoptosis rate in groups A, B and C was (21.06±1.12)%, (2.18±0.14)% and (2.04±0.11%) respectively. The apoptosis rate in group A was significantly higher than in group C (P<0.01), and there was no significant difference between groups B and C. Conclusion The transfection of MACC-1 gene can promote the proliferation and inhibit the apoptosis of gastric cancer cell line MGC-803 cells, which may play a role in the regulation of hepatocyte growth factor (HGF)/c-Met signal transduction pathway. Key words: Gastric cancer; Colon cancer metastasis associated gene-1; Apoptosis; Biological function

PAX2 promoted prostate cancer cell invasion through transcriptional regulation of HGF in an in vitro model

Elucidating the mechanism of prostate cancer cell invasion may lead to the identification of novel therapeutic strategies for its treatment. Paired box 2 (PAX2) and hepatocyte growth factor (HGF) proteins are promoters of prostate cancer cell invasion. We found that PAX2 protein activated the HGF gene promoter through histone H3 acetylation and upregulated HGF gene expression. Deletion analysis revealed that the region from −637 to −314 of the HGF gene was indispensable for HGF promoter activation by PAX2. This region contains consensus PAX2 binding sequences and mutations of the sequences attenuated HGF promoter activation. Using an in vitro invasion model, we found that PAX2 and HGF promoted prostate cancer cell invasion in the same pathway. Knockdown of HGF expression attenuated the cells' invasive capacity. Moreover, in tissue samples of human prostate cancers, HGF and PAX2 expression levels were positively correlated. These results suggested that upregulation of HGF gene expression by PAX2 enhanced the invasive properties of prostate cancer cells. The PAX2/HGF pathway in prostate cancer cells may be a novel therapeutic target in prostate cancer patients.

Increased miR-16 expression induced by hepatitis C virus infection promotes liver fibrosis through downregulation of hepatocyte growth factor and Smad7.

Hepatitis C virus (HCV) is involved in the initiation and progression of liver fibrosis by regulating genes encoding host proteins. However, the underlying mechanism of HCV-induced liver fibrosis is still to be determined. Reverse transcription polymerase chain reaction (RT-PCR) and western blot were performed to investigate the effect of HCV infection on the expression of the cellular microRNA miR-16 and its target genes encoding hepatocyte growth factor (HGF) and Smad7 in patients infected with HCV and in a liver cell line, QSG-7701, transfected with Ad-HCV, a recombinant adenovirus construct for expression of the HCV core protein. Regulation of HGF and Smad7 expression by miR-16 was assessed using luciferase reporter construct assays and miR-16 mimic transfection. Interferon-α (IFN-α) was used to verify the alteration of gene expression induced by HCV in QSG-7701 cells. Here, we found that miR-16 levels were increased in patients with HCV infection and were correlated with HGF and Smad7 expression levels in patients with HCV infection. Furthermore, HGF and Smad7 were predicted by bioinformatics analysis to be targets of miR-16. Upregulation of miR-16 and decreased HGF and Smad7 expression were still shown in QSG-7701 cells infected with Ad-HCV. Additionally, interferon-α (IFN-α) could reverse the changes in gene expression induced by HCV infection. These results suggest that the upregulation of miR-16 expression induced by HCV infection is a novel mechanism that contributes to downregulation of HGF and Smad7 in the development of liver fibrosis.

Regulation of hepatocyte growth factor in mice with pneumonia by peptidases and trans-alveolar flux.

Hepatocyte growth factor (HGF) promotes lung epithelial repair after injury. Because prior studies established that human neutrophil proteases inactivate HGF in vitro, we predicted that HGF levels decrease in lungs infiltrated with neutrophils and that injury is less severe in lungs lacking HGF-inactivating proteases. After establishing that mouse neutrophil elastase cleaves mouse HGF in vitro, we tested our predictions in vivo by examining lung pathology and HGF in mice infected with Mycoplasma pulmonis, which causes neutrophilic tracheobronchitis and pneumonia. Unexpectedly, pneumonia severity was similar in wild type and dipeptidylpeptidase I-deficient (Dppi-/-) mice lacking neutrophil serine protease activity. To assess how this finding related to our prediction that Dppi-activated proteases regulate HGF levels, we measured HGF in serum, bronchoalveolar lavage fluid, and lung tissue from Dppi+/+ and Dppi-/- mice. Contrary to prediction, HGF levels were higher in lavage fluid from infected mice. However, serum and tissue concentrations were not different in infected and uninfected mice, and HGF lung transcript levels did not change. Increased HGF correlated with increased albumin in lavage fluid from infected mice, and immunostaining failed to detect increased lung tissue expression of HGF in infected mice. These findings are consistent with trans-alveolar flux rather than local production as the source of increased HGF in lavage fluid. However, levels of intact HGF from infected mice, normalized for albumin concentration, were two-fold higher in Dppi-/- versus Dppi+/+ lavage fluid, suggesting regulation by Dppi-activated proteases. Consistent with the presence of active HGF, increased expression of activated receptor c-Met was observed in infected tissues. These data suggest that HGF entering alveoli from the bloodstream during pneumonia compensates for destruction by Dppi-activated inflammatory proteases to allow HGF to contribute to epithelial repair.

Pentoxifylline Prevents Driamycin-Induced Myocardial Fibrosis and Apoptosis in Rats.

Adriamycin (ADR) is a potent antineoplastic agent, but long-term treatment is limited by its cumulative, life-threatening cardiomyopathy. Recently, a few reports have shown that pentoxifylline (PTX) might produce cardioprotection in cardiac dysfunction. Here, we investigated the protective effects of PTX on ADR-induced cardiomyopathy in rats. Male rats were randomly assigned either to saline, ADR (adriamycin, 5 mg/kg/week), or A (adriamycin, 5 mg/kg/week) + PTX (pentoxifylline, 50 mg/kg/day) groups. After 3 weeks, these animals were sacrificed and the heart tissue was harvested for histological analysis and assessment of hepatocyte growth factor (HGF) and caspase-3 expression. Histopathological findings showed that PTX can alleviate myocardial damage caused by ADR. Cardiac fibrosis was significantly suppressed in the A+PTX group compared to that in the ADR group. The HGF gene expression was decreased significantly in the ADR group compared with the control group, but was increased in the A+PTX group. Caspase-3 was up-regulated in the ADR group, and down-regulated in the A+PTX group. These results show that treatment with PTX exerts a protective effect against ADR-induced myocardial fibrosis via regulation of HGF and caspase-3 gene expression. PTX may thus represent a useful new clinical tool for the treatment of ADR-induced cardiomyopathy.

Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells

BackgroundVia the hepatocyte growth factor receptor (Met), hepatocyte growth factor (HGF) exerts key roles involving skeletal muscle development and regeneration. Heparan sulfate proteoglycans (HSPGs) are critical modulators of HGF activity, but the role of specific HSPGs in HGF regulation is poorly understood. Glypican-1 is the only HSPG expressed in myoblasts that localize in lipid raft membrane domains, controlling cell responses to extracellular stimuli. We determined if glypican-1 in these domains is necessary to stabilize the HGF-Met signaling complex and myoblast response to HGF.MethodsC2C12 myoblasts and a derived clone (C6) with low glypican-1 expression were used as an experimental model. The activation of Met, ERK1/2 and AKT in response to HGF was evaluated. The distribution of Met and its activated form in lipid raft domains, as well as its dependence on glypican-1, were characterized by sucrose density gradient fractionation in both cell types. Rescue experiments reexpressing glypican-1 or a chimeric glypican-1 fused to the transmembrane and cytoplasmic domains of mouse syndecan-1 or myoblast pretreatment with MβCD were conducted. In vitro and in vivo myoblast migration assays in response to HGF were also performed.ResultsGlypican-1 localization in membrane raft domains was required for a maximum cell response to HGF. It stabilized Met and HGF in lipid raft domains, forming a signaling complex where the active phospho-Met receptor was concentrated. Glypican-1 also stabilized CD44 in a HGF-dependent manner. In addition, glypican-1 was required for in vitro and in vivo HGF-dependent myoblast migration.ConclusionsGlypican-1 is a regulator of HGF-dependent signaling via Met in lipid raft domains.

Transforming growth factor-β1 selectively inhibits hepatocyte growth factor expression via a micro-RNA-199–dependent posttranscriptional mechanism

Hepatocyte growth factor (HGF) is a multipotent endogenous repair factor secreted primarily by mesenchymal cells with effects on cells expressing its receptor, Met. HGF promotes normal tissue regeneration and inhibits fibrotic remodeling in part by promoting proliferation and migration of endothelial and epithelial cells and protecting these cells from apoptosis. HGF also inhibits myofibroblast proliferation. The profibrotic cytokine transforming growth factor beta 1 (TGF-β1) suppresses HGF expression but not the expression of NK2, an HGF splice variant that antagonizes HGF-induced proliferation. We investigated the mechanism for differential regulation of HGF and NK2 by TGF-β1. TGF-β1 down-regulated HGF in primary human adult pulmonary fibroblasts (HLFb) and increased the expression of miR-199a-3p, a microRNA (miRNA) associated with fibrotic remodeling. HGF and NK2 contain completely different 3' untranslated regions (UTRs), and we determined that miR-199a-3p targeted HGF mRNA for suppression but not NK2. A pre-miR-199 mimic inhibited the expression of a luciferase reporter harboring the HGF 3' UTR but not a pmirGLO reporter containing the NK2 3' UTR. In contrast, an anti-miRNA inhibitor specific for miR-199a-3p prevented TGF-β1-induced reduction of both HGF mRNA and HGF protein secretion. Taken together, these findings demonstrate that HGF is distinctly regulated at the posttranscriptional level from its antagonist NK2.

Expression of Factors in the Hepatocyte Growth Factor (HGF) Pathway in Whole Bone Marrow Biopsies in Association to the Osteolytic Bone Disease of Multiple Myeloma

AbstractAbstract 3977Osteolytic bone disease (OBD) in multiple myeloma (MM) is known to be caused by a combination of osteoclast hyperactivation and osteoblast inhibition. One of the pathways known to be involved in osteoblast inhibition from in vitro studies is the HGF pathway consisting of HGF, its receptor MET, the co-receptor Syndecan-1 (SDC-1), the partial MET antagonist Decorin and HGF activator responsible for HGF processing to its active form. So far, gene expression studies in MM have been performed on isolated MM plasma cells or bone marrow (BM) aspirates, which are not completely representative of the cell composition in the BM micro-environment. We used a novel strategy, whereby gene expression of factors associated with the HGF pathway was evaluated in snap-frozen BM biopsies, and moreover we determined the protein levels in matched BM plasma samples.An additional BM core biopsy obtained during the diagnostic procedure of MM patients was snap-frozen. Biopsies were cut, homogenized and RNA was purified and analyzed by qRT-PCR using low density arrays (Applied Biosystems). The relative quantitative gene expression was calculated using 3 internal reference genes (ABL, GAPDH and GUS). OBD was evaluated using standard radiographs. All patients were untreated and did not receive medicine that could influence bone remodeling. We examined 10 healthy volunteers (HV), 35 monoclonal gammopathy of unknown significance (MGUS) and 65 untreated MM patients, which according to radiographic findings were divided into NO/LOW and advanced OBD, i.e. OBD in ≥2 regions.ELISA was performed on a total of 31 matched BM plasma samples of HV, MGUS and MM obtained at the same time point as the biopsies. In addition, extra samples without gene data (N=52) were analyzed. Commercial kits for SDC-1 (Diaclone), HGF (RnD, Quantikine) and Decorin (RnD, Duoset) were run in duplicates according to manufacturer's instructions.Gene expression of HGF, SDC1, and MET were significantly different comparing HV, MGUS, no/low and advanced OBD (p<0.05) (For HGF, see figure 1). Decorin was not associated to OBD. HGF activator was not expressed in any of our samples, but only in the positive control.A significant correlation between gene and protein expression levels measured by ELISA was found for SDC-1 (Spearman's rho= 0.463, p=0.0058) and HGF (Spearman's rho=0.45, p=0.01). No correlation was found between Decorin gene levels and BM plasma levels (Spearman's rho =-0.24, p=0.22).The protein level of SDC-1 and HGF in BM plasma were both upregulated in MM and associated significantly to OBD level (p<0.05), while Decorin were significantly downregulated in MGUS and MM samples compared to HVs (p<0.05). A significant difference in HGF BM plasma levels were found between patients with no/limited OBD (median: 1.7ng/mL) and advanced OBD (median: 6.2ng/mL) in BM plasma.In our expression study reflecting the in vivo situation in MM patients, genes in the HGF pathway and proteins were significantly associated to OBD. The use of whole snap-frozen BM biopsies is a novel strategy for evaluation of gene expression in MM making it possible to investigate patients independent of degree of MM plasma cell infiltration. In addition to the dys-regulated gene expression levels alteration of SDC-1 and HGF was also observed at protein level, supporting the gene expression findings, and underscoring the usefulness of BM biopsies for gene expression studies in MM. Furthermore, our study for the first time shows up regulation of HGF in association with OBD at both gene and protein level in a large clinical material. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Acute kidney injury: Effect of hemodialysis membrane on Hgf and recovery of renal function

ObjectivesAcute kidney injury (AKI) is associated with a high mortality and morbidity rate. In this study we investigated whether dialysis membranes influence the recovery of renal function, through the regulation of hepatocyte growth factor (HGF). Design and methods21 patients were enrolled and assigned to hemodialysis (HD) with cellulose (CE, N=11) versus polymethylacrylate (PMMA, N=10) membranes in alternating order.HGF and IL-1 were measured in serum and in peripheral blood mononuclear cells (PBMC) supernatants collected immediately before the first HD session (T0), at 15minutes (T15), at 240minutes (T240) and after the last HD, when renal recovery occurred. Eight healthy volunteers were the controls (CON). ResultsTime to renal function recovery was lower in CE than in PMMA patients. Serum HGF in HD patients was significantly higher than in CON. HGF levels were higher in CE than in PMMA patients at T15 (13.4±2.7 vs 8.9±3.0ng/mL, P=0.004) and T240.At recovery, HGF levels decreased. IL-1 serum levels showed a similar trend (at T15 CE: 20.5±2.9 vs PMMA: 16.9±3.2pg/mL, P=0.005). HGF release significantly increased in the course of HD, resulting in higher levels in CE than that in PMMA patients.Considering all the patients, basal HGF release negatively correlated with time to renal recovery (r2=0.42, P<0.01). ConclusionsHere we demonstrated that dialysis membranes influence the cytokine profile in AKI patients, HGF release being higher in patients treated with the CE membrane, in comparison to PMMA. Our results suggest that treatment with CE might improve clinical outcomes, possibly through increased release of HGF.

Integrin-linked kinase (ILK) modulates wound healing through regulation of hepatocyte growth factor (HGF)

Integrin-linked kinase (ILK) is an intracellular effector of cell–matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The present work analyzes the role of ILK in wound healing in adult animals using a conditional knock-out of the ILK gene generated with the tamoxifen-inducible Cre-lox system (CRE-LOX mice). Results show that ILK deficiency leads to retarded wound closure in skin. Intracellular mechanisms involved in this process were analyzed in cultured mouse embryonic fibroblast (MEF) isolated from CRE-LOX mice and revealed that wounding promotes rapid activation of phosphatidylinositol 3-kinase (PI3K) and ILK. Knockdown of ILK resulted in a retarded wound closure due to a decrease in cellular proliferation and loss of HGF protein expression during the healing process, in vitro and in vivo. Alterations in cell proliferation and wound closure in ILK-deficient MEF or mice could be rescued by exogenous administration of human HGF. These data demonstrate, for the first time, that the activation of PI3K and ILK after skin wounding are critical for HGF-dependent tissue repair and wound healing.

Self-Control of HGF Regulation on Human Trophoblast Cell Invasion via Enhancing c-Met Receptor Shedding by ADAM10 and ADAM17

Hepatocyte growth factor (HGF)/c-Met signaling has been implicated in mammalian placental development. Integral c-Met can be released from endothelial cell membrane by proteolysis to form a soluble, truncated protein [soluble Met (sMet)], which is biochemically able to bind HGF and may disrupt HGF/c-Met signaling. By far, production of sMet in human placenta has not been reported, and the shedding mechanism remains unclear. In this study, production of sMet in healthy pregnant placenta and preeclamptic ones was compared, and the role of sMet on trophoblast cell invasion as well as the regulation of c-Met shedding by HGF were investigated in an immortal trophoblast cell line, B6Tert-1. Placenta productions of sMet, pro- and active forms of a disintegrin and metalloprotease 10 (ADAM10) and ADAM17 in preeclamptic patients were significantly higher than those in normal pregnant women. In B6Tert-1 cells, the HGF-induced promotion on cell invasion and activation of MAPK and AKT could be extensively blocked by sMet. ADAM10 and ADAM17, but not ADAM12, were explored to be sheddases of c-Met. HGF down-regulated c-Met receptor expression, whereas it up-regulated pro- and active/mature forms of ADAM10 and ADAM17 expression, which resulted in enhanced sMet production. Stimulation of H(2)O(2) caused an increase in active ADAM10, pro-ADAM17, and active ADAM17 levels and thus excessive c-Met shedding. HGF could negatively self-control its regulatory effect on trophoblast cell invasion via enhancing proteolysis of its receptor. Unbalancing of HGF self-control by oxidative stress may lead to impeding placentation in relevance to preeclampsia.

Human bone marrow-derived stem cell proliferation is inhibited by hepatocyte growth factor via increasing the cell cycle inhibitors p53, p21 and p27

Human bone marrow-derived stem cells (hMSCs) are a major source of osteoprogenitors. Hepatocyte growth factor (HGF), a glycoprotein constitutively produced by hMSCs, is reported to act on differentiated osteoblasts and also osteoclasts. Moreover, HGF has been shown by us and others to enhance osteoblastic differentiation from hMSCs. Typically, the pro-differentiation effects of HGF have required cooperative action with regulatory factors such as vitamin D or bone matrix material. Here, we have pursued the molecular mechanisms underlying the osteogenic effect of HGF on hMSCs, the principal precursors to bone forming cells. HGF treatment of hMSCs reduced the cell number over time and increased G1/S cell-cycle arrest compared to control (non-treated) cells. RT-qPCR showed treatment with HGF increased gene expression of the cell-cycle inhibitors p53, p21, and p27, possibly explaining the cell growth inhibition and G1 arrest, a step critical to phenotypic differentiation. Transfection of siRNA specific for cMet, the HGF receptor, eliminated the HGF anti-proliferation effect on hMSCs and the HGF-mediated increase in p53, p21, and p27, strongly supporting a role for these cell-cycle inhibitors in HGF's regulation of hMSCs. HGF in combination with a known inducer of osteogenic differentiation, 1,25-dihydroxyvitamin D, significantly increased cell maturation/differentiation as indicated by an increase in several osteoblast markers. Taken together these results demonstrate that HGF significantly enhances hMSC osteoblast differentiation by 1,25-dihydroxyvitamin D.

Ganglioside GD1a negatively regulates hepatocyte growth factor expression through caveolin-1 at the transcriptional level in murine osteosarcoma cells

Background Hepatocyte growth factor (HGF) is a mesenchyme-derived, multifunctional protein that is implicated in tumor growth and invasive behavior. Some tumor cells express both HGF and its receptor MET, forming an autocrine loop that permanently activates it. Ganglioside GD1a suppresses metastatic capacity in murine FBJ osteosarcoma cells and MET phosphorylation activated by HGF binding, but the signaling pathway controlling HGF production has not been fully explored. Methods Expression of HGF, caveolins, or MET of the cells that had been transfected with siRNA or cDNA directed to GM2/GD2 synthase, caveolin-1 or HGF was determined by semi-quantitative RT-PCR and Western blots. Results HGF expression in highly metastatic, GD1a-deficient FBJ-LL cells was higher than that in the poorly metastatic, GD1a-rich FBJ-S1 cells. Transfection with GM2/GD2 synthase cDNA increased GD1a levels in FBJ-LL cells and suppressed HGF expression. Treatment with siRNAs directed toward GM2/GD2 synthase in FBJ-S1 cells reduced gangliosides and augmented HGF expression. GD1a was found to be the only ganglioside species suppressing HGF expression upon addition to FBJ-LL cells. HGF expression was decreased by GD1a addition to FBJ-LL cells after 48 h, enough to induce caveolin-1 expression. Silencing caveolin-1 up-regulated HGF, and the re-introduction of caveolin-1 cDNA decreased HGF expression. Caveolin-1 suppressed MET phosphorylation. We also found GD1a regulation of HGF in Lewis lung carcinoma cells. Conclusions HGF expression was negatively regulated by GD1a through caveolin-1 at the transcriptional level via the suppression of MET phosphorylation. General significance This is the first report that ganglioside GD1a negatively regulates HGF expression through caveolin-1.

Correlation between serum hepatocyte growth factor level and percutaneous coronary balloon angioplasty: An alternative mechanism of reduction in restenosis with citostazol

Hepatocyte growth factor (HGF) is a potent endothelium specific growth factor and cilostazol reduces restenosis after percutaneous transluminal coronary balloon angioplasty (PTCA). To investigate the correlation between HGF and PTCA, values of serum HGF were serially examined by angiographic follow-up for 3 months in 100 patients who underwent PTCA, and for 1 week in 49 patients who underwent coronary angiography (CAG). Of the PTCA group, 36 patients received cilostazol (200 mg/day) and 64 were given aspirin (250 mg/day). Angiographic restenosis was defined as ≧50% diameter stenosis (DS) at follow-up angiography. Although HGF values did not change after CAG, they increased 2 days after PTCA (0.37±0.12 ng/mL; p<0.0001 vs. 0.32±0.11 at baseline, p=0.0004 vs. 0.30±0.09 ng/mL of the CAG group). The HGF values until 1 week after PTCA was similar between the cilostazol and aspirin sub-groups. However, the values 1 month and 3 months after PTCA were significantly lower in the cilostazol (0.29±0.08 vs. 0.34±0.10 ng/mL; p=0.012, and 0.31±0.09 vs. 0.35±0.10 ng/mL; p=0.037), than in the aspirin sub-group. Follow-up angiography revealed a significantly lower DS (37.2±16.2% vs. 45.6±18.5%; p=0.009) associated with a reduced restenosis rate (16.0% vs. 37.5%; p=0.008) in the cilostazol sub-group. These data suggest systemic HGF regulation is provoked after PTCA and the subsequent change in the serum HGF level is associated with restenosis.