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Multiple biological responses are induced by glycosylation-deficient hepatocyte growth factor.

ABSTRACT: HGF (hepatocyte growth factor), a heterodimeric glycoprotein composed of alpha- and beta-chains, exerts biological activities through the c-Met receptor tyrosine kinase. The alpha-chain has three glycosylation sites, while the beta-chain has two; however, the role of sugar chains on HGF is still unknown. To address the significance of glycosylation of HGF, three different types of glycosylation-deficient HGFs, i.e. non-glycosylated in the alpha-chain, the beta-chain, and in both the alpha- and beta-chains, were respectively expressed in COS-7 cells and then purified from culture supernatants. Unexpectedly, glycosylation-deficient HGFs induced tyrosine phosphorylation of the c-Met receptor and subsequent phosphorylation of ERK (extracellular-signal-regulated kinase) and Akt in rat hepatocytes with the same potency as glycosylated HGF. Consistent with this, glycosylation-deficient HGFs strongly stimulated DNA synthesis of hepatocytes equal to glycosylated HGF. Likewise, glycosylation-deficient HGFs induced cell scattering and branching tubulogenesis in MDCK (Madin-Darby canine kidney) cells, and thus were indistinguishable from glycosylated HGF in biological activities. Glycosylation also did not affect stability, protease sensitivity and tissue distribution, although the plasma clearance of HGF was slightly prolonged by glycosylation deficiency. Glycosylation deficiency resulted in a decrease in post-transcriptional biosynthesis of HGF in the cells, whereas extracellularly secreted HGFs were efficiently activated to a two-chain form. These results indicate that glycosylation influences post-transcriptional biosynthesis of HGF, whereas biological activities and basic physicochemical characteristics are retained, even in completely non-glycosylated HGF. Hence, non-glycosylated HGF is promising as an alternative for glycosylated HGF in clinical applications.

SUBMITTER: Fukuta K

PROVIDER: S-EPMC1138963 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Fibroblasts are major cellular components of the tumor microenvironment, regulating tumor cell behavior in part through secretion of extracellular matrix proteins, growth factors, and angiogenic factors. In previous studies, conditional deletion of the type II transforming growth factor-beta (TGF-beta) receptor in fibroblasts (Tgfbr2FspKO) was shown to promote mammary tumor metastasis in fibroblast-epithelial cell cotransplantation studies in mice, correlating with increased expression of hepatocyte growth factor (HGF). Here, we advance our findings to show that Tgfbr2(FspKO) fibroblasts enhance HGF/c-Met and HGF/Ron signaling to promote scattering and invasion of mammary carcinoma cells. Blockade of c-Met and Ron by small interfering RNA silencing and pharmacologic inhibitors significantly reduced mammary carcinoma cell scattering and invasion caused by Tgfbr2FspKO fibroblasts. Moreover, neutralizing antibodies to c-Met and Ron significantly inhibited HGF-induced cell scattering and invasion, correlating with reduced Stat3 and p42/44MAPK phosphorylation. Investigation of the signal transducer and activator of transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) signaling pathways by pharmacologic inhibition and small interfering RNA silencing revealed a cooperative interaction between the two pathways to regulate HGF-induced invasion, scattering, and motility of mammary tumor cells. Furthermore, whereas c-Met was found to regulate both the Stat3 and MAPK signaling pathways, Ron was found to regulate Stat3 but not MAPK signaling in mammary carcinoma cells. These studies show a tumor-suppressive role for TGF-beta signaling in fibroblasts, in part by suppressing HGF signaling between mammary fibroblasts and epithelial cells. These studies characterize complex functional roles for HGF and TGF-beta signaling in mediating tumor-stromal interactions during mammary tumor cell scattering and invasion, with important implications in the metastatic process.

Project description:ObjectivesTo investigate the role of hepatocyte growth factor (HGF)/c-Met signaling in oral malignant transformation.MethodsWe used immunohistochemistry to investigate HGF and c-Met expression in 53 oral squamous cell carcinoma (OSCC) specimens and 21 adjacent nontumor specimens and evaluated the associations between HGF and c-Met expression and clinicopathological parameters. Additionally, HGF-overexpression transgenic (HGF-Tg) and wild-type (Wt) mice were treated with 4-nitroquinoline-1-oxide (4NQO) to induce oral carcinogenesis for 16 weeks. At 16, 20, and 24 weeks, tongue lesions were collected for clinical observation; estimation of HGF, c-Met, and PCNA expression; apoptosis (TUNEL) assays; and RNA sequencing (RNA-seq).ResultsHGF and c-Met were positively expressed in 92.5% and 64% of OSCC samples, respectively. High HGF expression was significantly associated with smaller tumor size (p = 0.006) and inferior TNM stage (p = 0.032). No correlation between HGF and c-Met levels and other clinical parameters or prognosis was noted. In addition, HGF and c-Met expression was elevated in 4NQO-induced lesions of Wt mice. Compared with Wt mice, HGF-Tg mice have lower tumor incidence, number, volume, and lesion grade. In addition, the percentage of PCNA-positive cells in Wt mice was significantly higher than that in HGF-Tg mice at different time points. At 16 weeks, HGF-Tg mice exhibited less apoptotic cells compared with Wt mice (p < 0.000), and these levels gradually increased until the levels were greater than that of Wt mice at 24 weeks (p < 0.000). RNA-seq data revealed that 140 genes were upregulated and 137 genes were downregulated in HGF-Tg mice. KEGG enrichment analysis showed that upregulated differentially expressed genes (DEGs) are highly correlated with oxidative and metabolic signaling and that downregulated DEGs are related to MAPK and PI3K-AKT signaling.ConclusionsHGF and c-Met expression is upregulated in OSCC tissues and is associated with the occurrence and development of OSCC. HGF overexpression in normal oral epithelial tissue can inhibit 4NQO-induced tumorigenesis potentially through inhibiting proliferation and accelerating apoptosis via MAPK and PI3K-AKT signaling.

Project description:BACKGROUND:Hepatocyte growth factor (HGF) is a pleiotropic cytokine which can lead to cancer cell proliferation, migration and metastasis. In multiple myeloma (MM) patients it is an abundant component of the bone marrow. HGF levels are elevated in 50% of patients and associated with poor prognosis. Here we aim to investigate its source in myeloma. METHODS:HGF mRNA levels in bone marrow core biopsies from healthy individuals and myeloma patients were quantified by real-time PCR. HGF gene expression profiling in CD138+ cells isolated from bone marrow aspirates of healthy individuals and MM patients was performed by microarray analysis. HGF protein concentrations present in peripheral blood of MM patients were measured by enzyme-linked immunosorbent assay (ELISA). Cytogenetic status of CD138+ cells was determined by fluorescence in situ hybridization (FISH) and DNA sequencing of the HGF gene promoter. HGF secretion in co-cultures of human myeloma cell lines and bone marrow stromal cells was measured by ELISA. RESULTS:HGF gene expression profiling in both bone marrow core biopsies and CD138+ cells showed elevated HGF mRNA levels in myeloma patients. HGF mRNA levels in biopsies and in myeloma cells correlated. Quantification of HGF protein levels in serum also correlated with HGF mRNA levels in CD138+ cells from corresponding patients. Cytogenetic analysis showed myeloma cell clones with HGF copy numbers between 1 and 3 copies. There was no correlation between HGF copy number and HGF mRNA levels. Co-cultivation of the human myeloma cell lines ANBL-6 and JJN3 with bone marrow stromal cells or the HS-5 cell line resulted in a significant increase in secreted HGF. CONCLUSIONS:We here show that in myeloma patients HGF is primarily produced by malignant plasma cells, and that HGF production by these cells might be supported by the bone marrow microenvironment. Considering the fact that elevated HGF serum and plasma levels predict poor prognosis, these findings are of particular importance for patients harbouring a myeloma clone which produces large amounts of HGF.

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Multiple biological responses are induced by glycosylation-deficient hepatocyte growth factor.

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