Heparin Modulates the Receptor-Binding and Mitogenic Activity of Hepatocyte Growth Factor on Hepatocytes

doi:10.1006/excr.1993.1316

Experimental Cell Research

Volume 209, Issue 2, December 1993, Pages 317-324

https://doi.org/10.1006/excr.1993.1316 Get rights and content

Abstract

Hepatocyte growth factor (HGF) is a mitogen for hepatocytes, having high affinity for heparin. In this study, we examined the function of cell surface heparin-like molecules in HGF/receptor interaction and HGF-induced mitogenic activity using hepatocytes. Binding studies using ¹²⁵I-HGF showed that more than 85% of bound HGF was released from the cell surface by washing with heparin. This procedure also released HGF from the c-Met protein, which is a component of the high-affinity receptor. In addition, heparitinase digestion of hepatocytes reduced the HGF bound to c-Met protein. Furthermore, excess heparin added during the binding of ¹²⁵I-HGF to hepatocytes, significantly diminished HGF bound to c-Met protein. Moreover, when DNA synthesis of hepatocytes was repressed and retarded by excess HGF, exogenous heparin restored it. These results suggest that HGF is bound to c-_Met protein and that its mitogenic activity is regulated by heparin-like molecules on hepatocytes.

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Cited by (69)

Myeloma-derived extracellular vesicles mediate HGF/c-Met signaling in osteoblast-like cells
2019, Experimental Cell Research
Citation Excerpt :
We argue, however, that heparin displacement of HGF is a more likely explanation for reduced IL-11 secretion than general inhibition of EV-cell interactions, especially considering the differences in methodology, where we have attempted a thorough removal of excess heparin. Additionally, heparin displacement of HGF from proteoglycans is well documented [29,30], as demonstrated several times in the same HGF bioassay [28,31,32]. Characterization of EVs from BMP and cultured primary myeloma cells has to our knowledge previously not been performed.
Multiple myeloma is an incurable cancer of antibody-producing plasma cells. Hepatocyte growth factor (HGF), a cytokine aberrantly expressed in half of myeloma patients, is involved in myeloma pathogenesis by enhancing myeloma growth and invasiveness, and may play a role in myeloma bone disease by inhibiting osteoblastogenesis. In this study, we investigated whether extracellular vesicles (EVs) may play a role in HGF signaling between myeloma cells and osteoblast-like target cells. EVs from the HGF-positive cell line JJN-3 and the HGF-negative cell line INA-6, and from bone marrow plasma and primary human myeloma cells, were isolated using sequential centrifugation techniques and the presence of HGF on the EV-surface was investigated with ELISA. EVs from both cell lines were added to an established bioassay where HGF is known to induce interleukin-11 secretion in osteoblast-like cells. Our results show that HGF was bound to the surface of JJN-3-derived EVs, while INA-6-derived EVs were negative for HGF. Only JJN-3-derived EVs induced IL-11 secretion in osteoblast-like recipient cells. When osteoblast-like cells were preincubated with a specific HGF-receptor (c-Met) inhibitor, no induction of interleukin-11 was observed. Downstream c-Met phosphorylation was demonstrated by immunoblotting. EVs isolated from bone marrow plasma and primary myeloma cells were HGF-positive for a subset of myeloma patients. Taken together, this work shows for the first time that HGF bound on the surface of myeloma-derived EVs can effectuate HGF/c-Met signaling in osteoblast-like cells. Myeloma-derived EVs may play a role in myeloma bone disease by induction of the osteoclast-activating cytokine interleukin-11 in osteoblasts.
Low molecular weight heparin prevents hepatic fibrogenesis caused by carbon tetrachloride in the rat
2007, Journal of Hepatology
Citation Excerpt :
It has been reported that heparin directly stimulates HGF production in various cell types [31]. HGF is produced as a single-chain inactive precursor (pro-HGF), which is a matrix-associated form, and proteolytic cleavage is required for its activation [32,33], and heparin has also been demonstrated to facilitate the release of HGF from cell surface/ECM [34]. Taken together, it is postulated that dalteparin enhances regenerative responses in the liver through up-regulation of HGF.
In this study, we investigated the effect of dalteparin sodium, a low molecular weight (LMW)-heparin, on hepatic fibrogenesis caused by chronic carbon tetrachloride (CCl₄) administration in the rat.
Female Wistar rats were given a single, or repeated intraperitoneal injections of CCl₄ (1 ml/kg, twice per week) and dalteparin (50 IU/kg, daily) for 7 weeks.
Dalteparin did not prevent acute CCl₄-induced hepatic necrosis and elevation in serum aminotransferases levels; however, proliferating cell nuclear antigen (PCNA)-positive hepatocytes were dramatically increased 24 h after simultaneous administration of CCl₄ and dalteparin. Interestingly, serum hepatocyte growth factor (HGF) levels 12 h after injection of CCl₄ were almost doubled when dalteparin was given simultaneously. Hepatic fibrosis following 7-week CCl₄ treatment was markedly ameliorated by daily co-administration of dalteparin. Indeed, dalteparin largely inhibited CCl₄-induction of smooth muscle α-actin expression, α1(I)procollagen and transforming growth factor (TGF)-β1 mRNA levels in the liver. Further, dalteparin blunted platelet-derived growth factor (PDGF)-induced increases in 5-bromo-2′deoxyuridine (BrdU) uptake in 3-day cultured hepatic stellate cells (HSCs) in a dose-dependent manner.
Dalteparin enhances hepatic regeneration and minimizes hepatic fibrogenesis caused by chronic CCl₄ treatment. The mechanism underlying these effects most likely involves both up-regulation of HGF and inhibition of HSC proliferation.
Modulation of hepatocyte growth factor plasma levels in relation to the dose of exogenous heparin administered: An experimental study in rats
2005, Transplantation Proceedings
Partial liver transplantation has been consolidated to be a valid treatment option. We sought to understand the factors that modulate and may be harnessed to accelerate hepatocyte regeneration. We sought to determine the impact of heparin on m-hepatocyte growth factor (HGF) plasma concentrations.
Sixteen rats were assigned to four groups of four animals each: group A, without heparin; group B, 600 IU/kg; group C, 1000 IU/kg, group D, 1400 IU/kg. Blood samples (0.5 mL) were obtained from each rat at baseline and at 30, 60, 120, and 240 minutes. After the samples were centrifuged to separate supernates from the cell phase they were stored at −20°C in the m-HGF reagent and subsequently tested using enzyme-linked immunosorbent assay. Results were analyzed using SPSS 11.5 statistical software.
Among the 16 rats, one died at 110 minutes, just prior to the last extraction. The remaining rats were sacrificed. Mean weight was: 466 ± 64.24 g with no intergroup differences (P = .149). The comparative results (using Student t test) were: baseline A_1–4 versus A_1–4 30 minutes: P < .05; baseline A_1–4 versus A_1–4 60 minutes: P < .05; baseline A_1–4 versus A_1–4 120 minutes: P = .10 (NS); baseline A_1–4 versus A_1–4 240 minutes: P = .15 (NS). No significant differences were found among group B: baseline C_1–4 versus C_1–4 30 minutes and 60 minutes: NS; baseline C_1–4 versus C_1–4 120 minutes: P < .001; baseline C_1–4 versus C_1–4 240 minutes: P < .10 (NS). Finally, the results in group D were: baseline D_1–4 versus D_1–4 30 minutes: NS; baseline D_1–4 versus D_1–4 60 minutes and 120 minutes: P < .05; baseline D_1–4 versus D_1–4 240 minutes: P < .0005. When we compared group A to C and D, we detected differences (albeit not when compared to B) with P values = .01. Peak values were obtained at 120 and 240 minutes (225.21 pg/mL and 221.78 pg/mL) among groups C and D.
Heparin has a positive effect to increase serum HGF concentrations among rats. The effect was dependent on the administered dose and the time elapsed.
Zonal differences in effects of HGF/SF and EGF on DNA synthesis in hepatocytes at different times post-hepatectomy
2003, Hepatology Research
This study was done for investigation of the DNA synthesis induced by HGF/SF and EGF in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) isolated at different times post-hepatectomy. Preparation, culture, and DNA synthesis of PPH and PVH were done as previously described. In both PPH and PVH isolated at 6 and 12 h post-hepatectomy, the maximum DNA synthesis induced by both HGF/SF and EGF was observed from 24 to 48 h post-hepatectomy. In contrast, in both types of hepatocytes isolated at 36, 72 and 96 h, 1 and 2 weeks, and 1 month post-hepatectomy, maximum DNA synthesis was observed from 48 to 72 h. However, maximum DNA synthesis induced by HGF/SF and EGF was at its lowest in PPH isolated at 12 h and PVH isolated at 6 h post-hepatectomy. Maximum DNA synthesis returned to the control level in PPH and PVH isolated at 72 and 96 h post-hepatectomy, respectively. Specific binding studies using [ $^{125} I$ ] HGF/SF and [ $^{125} I$ ] EGF supported the above data. From these data, it was demonstrated that first, the PPH and PVH cell cycles have the same post-hepatectomy, but are dependent on the preparation time. Secondly, differences in DNA synthesis in PPH and PVH post-hepatectomy suggest that liver regeneration may occur unevenly.
Heparan sulfate is required for bone morphogenetic protein-7 signaling
2003, Biochemical and Biophysical Research Communications
Citation Excerpt :
Regarding other HS-binding molecules, exogenous application of heparin or chlorate treatment inhibits glial cell line-derived neurotrophic factor-mediated signaling [34]. Moreover, exogenous heparin disrupts hepatocyte growth factor-mediated signaling [35] and HS-associated retinal axon targeting in Xenopus[36], suggesting that membrane-anchored HSPGs play roles in several signaling systems. Possible roles of HS in BMP-7 signaling are in concentrating BMP ligands on the plasma membrane and facilitating the ligand–receptor interaction.
Although genetic studies have suggested that heparan sulfate (HS) is involved in bone morphogenetic protein (BMP)-mediated embryonic morphogenesis, it is unclear whether HS is directly involved in BMP-mediated signaling. Here, we investigate the involvement of HS in BMP-7 signaling. We show that HS and heparin chains specifically bind to BMP-7. Digestion of cell-surface HS with heparitinase interferes with BMP-7-mediated Smad phosphorylation in ROS 17/2.8 osteoblastic cells. Inhibiting sulfation of cell-surface HS with chlorate also causes interruption of Smad phosphorylation. Addition of exogenous heparin to ROS 17/2.8 cells prevents BMP-7-mediated Smad phosphorylation rather than enhances the BMP-7 signal, suggesting that HS should be anchored on the plasma membrane for BMP signaling. Moreover, BMP-7 binding to ROS 17/2.8 cells is inhibited by chlorate treatment and exogenous application of heparin. These results demonstrate that BMP-7 specifically binds to cell-surface HS and the BMP-7–HS interaction is required for BMP-7 signaling.
Betaglycan expression is transcriptionally up-regulated during skeletal muscle differentiation: Cloning of murine betaglycan gene promoter and its modulation by MyoD, retinoic acid, and transforming growth factor-β
2003, Journal of Biological Chemistry
Betaglycan is a membrane-anchored proteoglycan co-receptor that binds transforming growth factor β (TGF-β) via its core protein and basic fibroblast growth factor through its glycosaminoglycan chains. In this study we evaluated the expression of betaglycan during the C₂C₁₂ skeletal muscle differentiation. Betaglycan expression, as determined by Northern and Western blot, was up-regulated during the conversion of myoblasts to myotubes. The mouse betaglycan gene promoter was cloned, and its sequence showed putative binding sites for SP1, Smad3, Smad4, muscle regulatory factor elements such as MyoD and MEF2, and retinoic acid receptor. Transcriptional activity of the mouse betaglycan promoter reporter was also up-regulated in differentiating C₂C₁₂ cells. We found that MyoD, but not myogenin, stimulated this transcriptional activity even in the presence of high serum. Betaglycan promoter activity was increased by RA and inhibited by the three isoforms of TGF-β. On the other hand, basic fibroblast growth factor, BMP-2, and hepatocyte growth factor/scatter factor, which are inhibitors of myogenesis, had little effect. In myotubes, up-regulated betaglycan was also detectable by TGF-β affinity labeling and immunofluorescence microscopy studies. The latter indicated that betaglycan was localized both on the cell surface and in the ECM. Forced expression of betaglycan in C₂C₁₂ myoblasts increases their responsiveness to TGF-β2, suggesting that it performs a TGF-β presentation function in this cell lineage. These results indicate that betaglycan expression is up-regulated during myogenesis and that MyoD and RA modulate its expression by a mechanism that is independent of myogenin.

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Regular ArticleHeparin Modulates the Receptor-Binding and Mitogenic Activity of Hepatocyte Growth Factor on Hepatocytes

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Regular Article
Heparin Modulates the Receptor-Binding and Mitogenic Activity of Hepatocyte Growth Factor on Hepatocytes