High-affinity Receptor For Hepatocyte Growth Factor Research Articles

Induction of tumor initiation is dependent on CD44s in c-Met⁺ hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) patients with active hepatocyte growth factor (HGF)/c-Met signaling have a significantly worse prognosis. c-Met, a high affinity receptor for HGF, plays a critical role in cancer growth, invasion and metastasis. c-Met and CD44 have been utilized as cell surface markers to identify mesenchymal tumor-initiating stem-like cells (TISC) in several cancers including HCC. In this work, we examine the complex relationship between c-Met and CD44s (standard form), and investigate the specific role of CD44s as a tumor initiator and stemness marker in HCC.MethodsGene and protein expression assays were utilized to investigate the relationship between CD44s and c-Met in HCC cell lines. Tumor-sphere assays and in vivo tumor assays were performed to investigate the role of CD44+ cells as TISCs. Student’s t-test or one-way ANOVA with Tukeys post-hoc test was performed to test for differences amongst groups with a p < .05 as significant.ResultsIn an immunohistochemical and immunoblot analysis of human HCC samples, we observed that more than 39% of human HCC samples express c-Met and CD44. To study the relationship between c-Met and CD44, we used MHCC97-H cells, which are CD44+/c-Met+. The knockdown of c-Met in MHCC97-H cells decreased CD44s, reduced TISC characteristics and decreased tumorsphere formation. Furthermore, we demonstrate that the inhibition of PI3K/AKT signaling decreased CD44s expression and subsequently decreased tumorsphere formation. The down-regulation of CD44s leads to a significant loss of a TISC and mesenchymal phenotype. Finally, the down-regulation of CD44s in MHCC97-H cells decreased tumor initiation in vivo compared with the scrambled control.ConclusionsIn summary, our data suggest that CD44s is modulated by the c-Met-PI3K-AKT signaling cascade to support a mesenchymal and TISC phenotype in HCC cells. Moreover, c-Met could be a potential therapeutic drug for targeting HCC cells with TISC and mesenchymal phenotypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1166-4) contains supplementary material, which is available to authorized users.

Efficacy of HGF Gene Transfer for Various Nervous Injuries and Disorders

Hepatocyte growth factor (HGF) was originally identified as a molecule that could stimulate DNA synthesis in rat and human hepatocytes by autophosphorylation of the proto-oncogene c-met, which is a high-affinity receptor for HGF. Although it was at first considered that HGF could exert biological effects only on specific target cells, it has since been demonstrated that HGF mediates inflammatory responses to tissue injury and also regulates cell growth, cell motility, and morphogenesis in a wide variety of cell types, including cells within the nervous system. In the nervous system, HGF plays a role as a potent neurotrophic and angiogenetic factor. This factor promotes both the survival of neurons and the regeneration of injured nerves, and may also function as target-derived axonal chemoattractants, guiding axons to their target. These observations raised hopes that HGF protein might be useful for the clinical treatment of nervous system disorders. However, administration of HGF as a recombinant protein is still beset by a number of problems, such as a short serum half-life and poor access to the central nervous system by the systemic route because of the presence of the blood-brain barrier. These problems can be major obstacles to the clinical use of this factor in a recombinant protein form, and has highlighted the need to develop innovative therapeutic strategies for more efficient delivery into the nervous system. Gene transfer into the nervous system has enormous therapeutic potential for a wide variety of disorders. It appears to have advantages over the administration of single or multiple bolus doses of a recombinant protein because gene transfer can achieve an optimally high, local concentration within the nervous system. In this article, we demonstrate the efficacy of HGF gene transfer and provide an overview of ideal treatment regimes for various nervous injuries and disorders.

COX‐2 is induced by HGF stimulation in Met‐positive thyroid papillary carcinoma cells and is involved in tumour invasiveness

Thyroid papillary carcinoma (TPC) cells express high levels of cytoplasmic cyclo-oxygenase 2 protein. Analysis of microdissected samples of the tumour and of the paired normal thyroid tissue confirmed that mRNA transcripts for cyclo-oxygenase 2 (COX-2) were significantly more numerous in the tumour (7.6 +/- 13-fold; p = 0.01). High levels of COX-2 mRNA were not associated with age, sex, tumour size or lymph node metastasis. COX-2 was not homogeneously expressed throughout the tumour, but was significantly higher at the tumour invasion front. Hepatocyte growth factor (HGF) can up-regulate the expression of COX-2 mRNA. A marked increase in COX-2 mRNA levels was observed in 8/8 primary TPC cultures after HGF stimulation (6.3 +/- 6-fold) and in two papillary carcinoma cell lines (TPC-1 and NPA). Specific involvement of the high-affinity HGF receptor (Met protein) was suggested by the observation that PHA-665752, an inhibitor of the catalytic activity of c-Met kinase, caused a 54% reduction of the hepatocyte growth factor-induced COX-2 up-regulation. The possibility that HGF-Met interactions also had a causative role in the up-regulation of COX-2 in vivo was investigated in 30 tumour samples, where it was found that there was a statistically significant correlation (p = 0.001, r = 0.85) in the levels of expression of MET and COX-2 RNAs. The biological role of COX-2 in TPC cells was investigated by treating the TPC cell lines with the specific COX-2 inhibitor NS-398. It was found that NS-398 treatment significantly reduced the migration (50-75%) and invasiveness (47-92%) of tumour cells, but did not alter cell proliferation. Our data suggest that the increased expression of Met protein in TPC cells has a role in up-regulating the expression of COX-2, which in turn contributes to the invasive capacity of TPC cells.

Hepatocyte growth factor (HGF) downregulates thrombospondin 1 (TSP‐1) expression in thyroid papillary carcinoma cells

This study investigates the expression of thrombospondin-1 in papillary carcinoma of the thyroid and the role of Met-HGF interaction in TSP-1 regulation. In tissue sections, immunostaining for TSP-1 was associated with the fibrous tumour stroma, and showed areas of marked intensity adjacent to the basal membrane of tumour cells. Investigation of TSP-1 RNA expression showed that, in 10 of 14 cases, TSP-1 mRNA levels were significantly lower in tumour tissue (20-100% reduction; mean = 55% +/- 20; p = 0.001) than in the corresponding normal thyroid. Since it has been reported that HGF can downregulate the expression of TSP-1 mRNA, TSP-1 mRNA levels were measured in 7 primary cultures, established from thyroid papillary carcinomas (TPC), and in 1 TPC cell line prior to, or after, stimulation with HGF. A marked decrease in TSP-1 mRNA levels was observed after HGF stimulation in 6/7 primary cultures (60-100% decrease (mean = 79 +/- 15%; p = 0.006) and in the TPC cell line; moreover, the decrease in TSP-1 mRNA in cell extracts was associated with a decrease in TSP-1 protein in culture supernatants. The HGF activity was dose dependent and the downregulation lasted for at least 48 h after stimulation. The high-level expression of Met protein, the high-affinity receptor for HGF, in most cases of papillary carcinoma of the thyroid is consistent with the possibility that HGF-Met interaction plays a crucial role in regulating the expression of TSP-1 in this tumour type.

Synergistic effects of hepatocyte growth factor on human cord blood CD34+ progenitor cells are the result of c-met receptor expression.

Hepatocyte growth factor (HGF) is a pleiotropic growth factor which, in addition to its mitogenic potency for primary hepatocytes, also has a role in the regulation of cell motility, cell growth and morphogenesis. In the present study, we show that c-met, the high-affinity receptor for HGF, is expressed on human cord blood (CB) CD34+ progenitor cells and CD34+Thy-1+ Lin-(lin-) cells. We have investigated the capacity of HGF to synergize with other growth factors to induce colony formation by CB CD34+ progenitor cells. CD34+ cells were cultured in semisolid medium containing serum with increasing concentrations of GM-CSF, G-CSF, macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), interleukin 3 (IL-3) and IL-11 alone or in combination with HGF. HGF acted as a potent synergist and enhanced, up to fourfold, colony formation induced by GM-CSF, G-CSF or M-CSF. HGF in combination with SCF, IL-3 or IL-11 did not induce proliferation of colony forming units-granulocyte macrophage (CFU-GM) above control levels. In serum-deprived cultures, HGF was only detectably synergistic with IL-11, and all other culture combinations showed no proliferation. To determine whether the stimulatory effect of IL-11 and the synergistic effect of HGF in the absence of serum could be attributed to the effect of these two cytokines on stem cells, IL-11-stimulated and unstimulated lin- cells were analyzed for expression of c-met. CD34+Thy-1+Lin- cells were positive for c-met, both in the presence and absence of IL-11 stimulation, and Northern analysis indicated that c-met RNA expression was upregulated in response to IL-11 compared to unstimulated controls. These results provide strong evidence for upregulation of the HGF receptor on primitive hematopoietic cells by IL-11, and for the synergistic role of HGF in colony formation by hematopoietic stem cells.

Hepatocyte Growth Factor as a Potent Survival Factor for Rat Pheochromocytoma PC12 Cells

Hepatocyte growth factor (HGF), a natural ligand for the c-met protooncogene product, is a multipotent polypeptide which elicits mitogenic, motogenic, and morphogenic activities for various types of cells. To better understand the biological activity of HGF, as related to neuroectodermal-derived cells, we investigated the effects of HGF on rat pheochromocytoma PC12 cells. HGF increased the number of PC12 cells during long culture, but elicited no direct mitogenic activity, as determined by DNA synthesis. When the cells were cultured in medium containing lower concentrations of fetal calf serum, HGF prolonged the survival of PC12 cells; the number of cells did not decrease during 13 days when the cells were cultured in the presence of HGF, but the cells were completely withdrawn when cultured in the absence of HGF. Nerve growth factor but not HGF induced the differentiation of PC12 cells. High affinity receptor for HGF with Kd values of 20-40 pM was expressed in PC12 cells and other types of cells derived from the central nervous tissue: T98G cells (human glioblastoma), GOTO, and SCCH-26 cells (human neuroblastoma). HGF stimulated motility of T98G cells, while it induced weak mitogenic response in GOTO cells. We suggest that HGF is a potent survival factor for PC12 cells, without exerting any direct mitogenic activity and inducing the cell differentiation, and that this factor may have a distinct biological activity for neuroectoderm-derived cells.

Regulation of mitogenesis, motogenesis, and tubulogenesis by hepatocyte growth factor in renal collecting duct cells.

Hepatocyte growth factor (HGF) has been implicated in branching tubulogenesis of the developing kidney and in response to renal injury. We therefore examined the effects of response to renal injury. We therefore examined the effects of HGF on a recently described murine inner medullary collecting duct epithelial cell line (mIMCD-3 cells) in comparison with Madin-Darby canine kidney (MDCK) cells. HGF induced mitosis, scattering, and tubulogenesis in both mIMCD-3 cells and MDCK cells. However, mIMCD-3 cells underwent branching tubulogenesis under matrix conditions that did not support these morphogenetic changes in MDCK cells, suggesting substantial differences in regulation of tubulogenesis in these two cell types. In quiescent mIMCD-3 cells, the high-affinity receptor for HGF, c-met, was expressed in a nonphosphorylated state. After stimulation with HGF, there was a > 10-fold increase in receptor tyrosine phosphorylation and selective association with at least two intracellular proteins, including the phosphatidylinositol-3-kinase. Thus mIMCD-3 cells, which undergo HGF-dependent mitosis, scattering, and branching tubulogenesis, express the c-met receptor in a highly regulated state and therefore should make an excellent model for examining the mechanisms of HGF-dependent tubulogenesis in the renal collecting duct.

Hepatocyte Growth Factor Stimulates Growth of Hematopoietic Progenitor Cells

Hepatocyte growth factor (HGF), a mesenchymal-derived polypeptide, stimulates growth, motility and morphogenesis of various types of cells, most predominantly of epithelial cells. We have now identified an additional biological activity of HGF; this factor probably has a role in early hematopoiesis. We examined the effects of HGF on the growth of various murine hematopoietic progenitor tumor cell lines and found that HGF stimulated DNA synthesis in the myeloid leukemia cell line, NFS-60. The mitogenic effect of HGF on NFS-60 cells was additive with the effect of interleukin 3 (IL-3). On the other hand, HGF had no apparent effect on other myeloid leukemia cell lines examined, such as DA-3 and FDC-P1 cells. Scatchard analysis of specific binding of [ 125I]HGF revealed expression of a high-affinity HGF receptor on NFS-60 and DA-3 cells, but not on FDC-P1 cells. Expression of c-met mRNA correlated well with the existence of a high-affinity HGF receptor. Since the myeloid leukemia cell lines used are cells in the early stage of myeloid differentiation, HGF may play a role in early hematopoiesis.

Regulation of cell growth and motility by hepatocyte growth factor and receptor expression in various cell species

Hepatocyte growth factor (HGF), a humoral mediator for regeneration of liver and kidney, possesses multiple biological activities. To investigate target cell specificity and to examine whether multiple actions of HGF are related to properties of the HGF receptor on target cells, we examined the effects of HGF on cell growth and motility and analyzed the HGF receptor in various species of cells. HGF stimulated growth and DNA synthesis of PAM212 (naturally immortalized mouse keratinocytes), Mv1Lu (mink lung epithelia), and A431 (human epidermoid carcinoma) cells, as well as mature hepatocytes, but inhibited those of IM-9 (human B-lymphoblasts). Conversely, HGF had a marked stimulatory effect on cell motility of MDCK (Mardin-Darby canine kidney epithelia) cells, but not on their growth. Also, HGF enhanced the motility of various species of cells, including A431, PAM212, HepG2 (human hepatoma), KB (human epidermoid carcinoma), and J-111 (human monocytes) cells. Scatchard analysis of 125I-HGF binding to hepatocytes indicated that the cells expressed both high- and low-affinity binding sites for HGF with K d values of 23 and 260 pM, respectively. High-affinity HGF receptor with K d values of 20–25 p M was detected at 40–720 sites/cell in MDCK, A431, PAM212, Lu99, and IM-9 cells, but not in fibroblasts and hematopoietic cells. In contrast, low-affinity binding sites were detected in all cell lines examined, even in those not responsive to HGF. Nothern blots revealed that cells possessing a high-affinity HGF receptor expressed c- MET/HGF receptor mRNA. Therefore, HGF probably regulates both cell growth and motility of various types of epithelial cells and some types of mesenchymal cells. The multiple biological activities of HGF may be exerted through a high-affinity HGF receptor linked to multiple distinct intracellular signaling pathways.

Tissue distribution of hepatocyte growth factor receptor and its exclusive down-regulation in a regenerating organ after injury.

Using 125I-labeled hepatocyte growth factor (HGF) as a ligand, we examined the tissue distribution of the HGF receptor in adult rats. Specific binding of 125I-HGF was detected in the plasma membranes of liver, spleen, kidney, lung, adrenal gland, pituitary, and thyroid. Scatchard analysis of HGF binding in liver, spleen, kidney, lung, and adrenal gland revealed the presence of a single class of high affinity receptor with a dissociation constant (Kd) of 20-30 pM. The maximum number of binding sites (Bmax) was determined to be 400-3,000 sites per ng of plasma membrane protein, the highest number being in the liver. Such a wide distribution of a high affinity HGF receptor indicates that HGF may be a multifunctional growth factor, targeting to a variety of organs, and not restricted to liver. After 70% partial hepatectomy, specific binding of 125I-HGF to membranes of the residual liver rapidly decreased, but there was no change in the kidney, lung, and spleen. On the other hand, after unilateral nephrectomy rapid down-regulation of the HGF receptor was clearly evident in the remaining kidney, but not in other organs including the liver. These findings suggest the presence of control mechanisms governing HGF receptor function only in a regenerating organ after injury.