Expression of transforming growth factor β receptors in normal human colon and sporadic adenocarcinomas

Background & Aims: An absence or a presence of mutated transforming growth factor (TGF)-β receptors is a possible hypothesis explaining the resistance of cancer cells to the growth-inhibitory effect of TGF-β. Mutations involving microsatellite-like regions of the type II TGF-β receptor have been described in subgroups of colorectal cancers. The aim of this study was to investigate the expression and distribution of TGF-β receptors in sporadic colorectal cancers and normal tissues. Methods: Thirty-three sporadic colorectal cancers and 20 normal colonic tissues were explored by immunohistochemistry for the expression of type I and type II TGF-β receptors. Eighteen tumor and 20 normal samples were used for radioactive thermocycling and sequencing of the two microsatellite-like regions of the type II receptor. Results: Both receptors were overexpressed in tumors compared with normal samples. There was a relationship between the abundance of type II receptor expression and the degree of differentiation of the tumors but not the Dukes' staging or the localization of the neoplasias. No mutation was observed in the microsatellite-like regions of receptor II in any of the samples. Conclusions: Sporadic colorectal cancers do not show an absence or a presence of mutated TGF-β receptors that could explain a resistance to TGF-β–mediated growth inhibition. The pathways to tumorigenesis of sporadic colorectal cancers may be different from those of some hereditary ones.GASTROENTEROLOGY 1998;114:1211-1220     

Hepatocyte growth factor stimulates cell growth and enhances the expression of transforming growth factor α mRNA in AsPC-1 human pancreatic cancer cells

We investigated the effects of hepatocyte growth factor (HGF) and transforming growth factor α (TGF α) on cell growth in four human pancreatic cancer cell lines. Changes in the expression of mRNAs of HGF, c-met, TGF α, and epidermal growth factor receptor (EGFR) by treatment with HGF and TGF α were observed. Cell growth with growth factors was assessed with the MTT assay and compared with basal growth without growth factors. Although HGF stimulated cell growth in AsPC-1, COLO-357, and T3M4 cells, Panc-1 cells showed no response to HGF. TGF α stimulated the growth of all the above cells. The expression of c-met mRNA under nonstimulated conditions was detected with Northern blotting in all cells. Treatment with HGF slightly enhanced the expression of c-met mRNA only in COLO-357 cells. The intensity of EGFR expression was consistent, and HGF mRNA was not detected during induction experiments in any cell type. Concomitant treatment with HGF and TGF α exerted an effect that was additive or less on the growth of all cells. Expression of TGF α was enhanced by HGF treatment only in AsPC-1 cells. These results suggested that HGF and TGF α stimulated cell growth through a final common pathway of signal transduction. Received: November 11, 1998 / Accepted: December 18, 1998     

Fibroblast growth factor 2 and transforming growth factor β1 interactions in human liver myofibroblasts

During liver fibrogenesis, myofibroblastic liver cells proliferate and synthesize components of fibrosis. Fibroblast growth factor 2 (FGF-2) is expressed in vivo in myofibroblastic liver cells (MFLCs) during fibrogenesis, and exogenous FGF-2 is mitogenic for MFLCs. The aim of this study was to study the expression and role of endogenous FGF-2 in cultured human MFLCs. FGF-2 and FGF-2 receptors were studied using immunoblotting. All RNA studies used ribonuclease protection. Growth of MFLCs was studied using [³H]thymidine incorporation and direct cell counting. MFLCs expressed FGF-2 and its receptors FGF receptor 1 and FGF receptor 2. An antibody to FGF-2 blocked the mitogenic effect of transforming growth factor β1 (TGF-β1) for MFLCs but not TGF-β1-induced increase in cellular fibronectin messenger RNA (mRNA). TGF-β1 increased levels of FGF-2 and FGF receptor mRNAs in MFLCs. We have previously shown that TGF-β1 also increased platelet-derived growth factor (PDGF) A chain mRNA in these cells and that anti-PDGF antibody blunted the mitogenic effect of TGF-β1. The present results show that anti-FGF-2 and anti-PDGF-AA are not additive and that FGF-2 and PDGF-AA are not sequentially induced by TGF-β1. FGF-2 mediates the mitogenic but not the profibrogenic effect of TGF-β1 for human MFLCs, and autocrine FGF-2 and PDGF-A interact in the mediation of the mitogenic effect of TGF-β1.     

Expression of hepatocyte growth factor and transforming growth factor β1 mRNA inP. acnes and lipopolysaccharide-treated rats

Hepatocyte growth factor (HGF), a potent hepatocyte mitogen in vitro, triggers hepatocyte regeneration after partial hepatectomy and acute liver cell necrosis induced by chemicals. In contrast, tranforming growth factor ₁ inhibits hepatocyte proliferation in vitro and suppresses liver regeneration in vivo. We assessed the expression of HGF and TGF ₁ mRNA in an endotoxin-related hepatic cell necrosis model. Intravenous injection of Gram-negative lipopolysaccharide (LPS) into rats previously given heat-killedPropionibacterium acnes induced endotoxinrelated hepatic cell necrosis. In this model, serum ALT began to rise to more than 100 IU as early as 3 h after LPS injection, reaching 300 IU 12 h after injection. HGF mRNA levels in the liver did not increase significantly until 5 h after LPS injection; at 12 h, they had increased about threefold compared with controls. TGF ₁ mRNA expression increased threefold afterP. acnes treatment alone and increased further after LPS injection. In the spleen, HGF mRNA levels increased within 3 h, but in the lung no increase in HGF mRNA was observed. Early elevation of liver TGF ₁ mRNA levels and delayed elevation of HGF mRNA levels, with low expression of HGF in the lung, may play a role in the pathogenesis of endotoxin-related hepatic necrosis.This study was supported by a Grant for Scientific Research (no. 04304038) from the Japanese Ministry of Education, Science, and Culture.     

Aberrant expression of epidermal growth factor receptor and its interaction with protein kinase C δ in inflammation associated neoplastic transformation of human esophageal epithelium in high risk populations

Background and Aim: Esophageal cancer is the second most common cancer among Indian males and is mostly associated with tobacco smoking and alcohol consumption. Epidermal growth factor receptor (EGFR) is a member of Type I tyrosine kinases. Its activation causes the docking of various proteins in its cytosolic tail. In the present study we have analyzed the expression pattern of EGFR, protein kinase C δ (PKCδ), tumor necrosis factor‐α (TNF‐α), nuclear factor κB (NFκB) and the interactions between EGFR and PKCδ in various pathological conditions. Methods: Human esophageal biopsies were obtained from 93 patients with a past history of smoking and alcohol consumption: 20 showed normal mucosa, 40 with dysplasia and 33 squamous cell carcinoma (SCC). These pathological conditions were analyzed immunohistochemically for the presence of EGFR expression and then subsequently analyzed using immunoblot and immunoprecipitation. Results: A statistically significant difference of EGFR overexpression was found between low‐ and high‐grade dysplasia and carcinoma (χ² = 3.3, χ² = 3.42: P = 0.07, 0.33). A statistical significance was observed between dysplasia and SCC and in all histopathological types (χ² = 4, χ² = 4.9; P < 0.05, P = 0.18 and χ² = 26.3, 26.6; P < 0.001). EGFR tyrosine phosphorylation and its association with PKCδ was significantly higher in all histopathological types with χ² = 7.965; P < 0.05 and 4.0830; P = 0.2530. Conclusion: Altogether, our findings reveal that the activation of EGFR and its subsequent interaction with PKCδ under inflammatory conditions might positively be attributed to the transformation of normal esophageal epithelia to SCC, which could explain ongoing inflammation in normal mucosa in a population prone to smoking and alcoholism.     

An in vitro model for evaluating peripheral regulation of growth in fish: effects of 17β-estradiol and testosterone on the expression of growth hormone receptors, insulin-like growth factors, and insulin-like growth factor type 1 receptors in rainbow trout (Oncorhynchus mykiss)

A central component of growth coordination in vertebrates is the growth hormone (GH)-insulin-like growth factor-1 (IGF-1) system. To date, most studies on the control of vertebrate growth have focused on regulation of pituitary GH production and release. In this study, we used liver, muscle, and gill tissue from sexually immature rainbow trout incubated in vitro to evaluate the extrapituitary effects of 17β-estradiol (E2) and testosterone (T) on mRNA and functional expression of growth hormone receptors (GHR), insulin-like growth factors 1 and 2 (IGF-1, IGF-2), and type 1 IGF receptors (IGFR1). E2 significantly decreased steady-state levels of GHR1, GHR2, and IGF-1 mRNAs in liver as well as of GHR1 and GHR2 mRNAs in muscle and of IGF-1 and IGF-2 mRNAs in gill in a time- and concentration-dependent manner. E2 had no effect on levels of IGFR1 mRNAs in muscle or on GHR and IGFR1 mRNAs in gill. Functional expression of GHRs as assessed by ¹²⁵I-GH binding capacity was reduced by E2 in liver and muscle; however, E2 did not affect ¹²⁵I-IGF-1 binding capacity in muscle or ¹²⁵I-GH and ¹²⁵I-IGF-1 binding capacity in gill. By contrast, T increased steady-state levels of GHR1, GHR2, IGF-1, and IGF-2 mRNAs in liver, of GHR1, GHR2, IGFR1A, and IGFR1B in muscle, and of GHR1, GHR2, IGF-1, IGF-2, IGFR1A, and IGFR1B mRNAs in gill in a time- and concentration-dependent manner. Binding capacity of ¹²⁵I-GH in liver and of ¹²⁵I-GH and ¹²⁵I-IGF-1 in both muscle and gill also was increased by T. These data indicate that E2 and T directly affect peripheral aspects of the GH-IGF system, and suggest, at least in immature rainbow trout, that E2 reduces hepatic sensitivity to GH as well as reduces peripheral production of IGFs and that T increases peripheral sensitivity to GH and IGF as well as increases peripheral production of IGFs.     

Enhancement of osteopontin expression in HepG2 cells by epidermal growth factor via phosphatidylinositol 3-kinase signaling pathway

AIM:Osteopontin (OPN) is a phosphorylated glycoproteinwith diverse functions including cancer development,progression and metastasis.It is unclear how osteopontinis regulated in HepG2 cells.The aim of this study was toinvestigate the effect of epidermal growth factor on theexpression of osteopontin in HepG2 cells,and to explorethe signal transduction pathway mediated this expression.METHODS:Osteopontin expression was detected by RNAaseprotection assay and Western blot.Wortmannin,a specificinhibitor of PI3K,was used to see if PI3K signal transductionwas involved in the induction of osteopontin gene expression.RESULTS:HepG2 cells constitutively expressed low levelsof osteopontin.Treatment with epidermal growth factorincreased osteopontin mRNA and protein level in a dose-and time-dependent manner.Application of wortmannincaused a dramatic reduction of epidermal growth factor-induced osteopontin expression.CONCLUSION:Osteopontin gene expression can be inducedby treatment of HepG2 cells with epidermal growth factor.Epidermal growth factor may regulate osteopontin geneexpression through PI3K signaling pathway.Several potentialtargets in the pathway can be manipulated to block thesynthesis of osteopontin and inhibit liver cancer metastasis.     

Effects of epidermal growth factor and its signal transduction inhibitors on apoptosis in human colorectal cancer cells

AIM:The study investigated if EGF signaling inhibitors,EGFantibody and tyrphostin 51 (a tyrosine kinase inhibitor),mediated the action of EGF on apoptosis and the expressionof EGF receptors and p21 (a cyclin-dependent kinaseinhibitor) of human colorectal cancer cells.METHODS:Human colorectal adenocarcinoma cells(SW480) were incubated with 0.6mL/L dimethyl sulfoxide(DNSO,the control group),225ng/mL (37.5nmol/L) EGFin 0.6mL/L DNSO,225ng/mL EGF 2.5μg/mL (17nmol/L)EGF antibody in 0.6mL/L DMSO,or 225ng/mL EGF 215ng/mL (0.8μmol/L) tyrphostin 51 in 0.6mL/L DMSO.RESULTS:After 48h incubation,the levels of EGF in mediumsignificantly increased (P<0.05) in the EGF-treated groups.The numbers of apoptotic cells were significantly fewer(P<0.05) in the EGF EGF antibody and EGF tyrphostin51 groups than those in the control and EGF groups after12 h treatments.The expression of phosphorylated EGFreceptors in the EGF,EGF EGF antibody,and EGF tyrphostin 51 groups was 176.8%,62.4%,and 138.1% ofthe control group,respectively.The expression of p21protein in the EGF,EGF EGF antibody,and EGF tyrphostin51 groups was 115.7%,4.8%,and 61.5% of the controlgroup,respectively.CONCLUSION:The data suggest that EGF antibody andtyrphostin 51 can inhibit the action of EGF on apoptosis inhuman colorectal cancer cells through down-regulation ofEGF receptor and p21 expression.     

Expression of vascular endothelial growth factor and transforming growth factor-beta 1 on the neointimal of balloon-injured rabbit carotid arteries

Objective To make an rabbit models of restenosis and observed the expression of VEGFmRNA and TGE-β1 mRNA on the intireal proliferation in the mode. We also explored the relationship between VEGFmRNA, TGF-β1 mRNA and restenosis.     

Diverse injurious stimuli reduce protein tyrosine phosphatase-μ expression and enhance epidermal growth factor receptor signaling in human airway epithelia

In response to injury, airway epithelia utilize an epidermal growth factor (EGF) receptor (EGFR) signaling program to institute repair and restitution. Protein tyrosine phosphatases (PTPs) counterregulate EGFR autophosphorylation and downstream signaling. PTPμ is highly expressed in lung epithelia and can be localized to intercellular junctions where its ectodomain homophilically interacts with PTPμ ectodomain expressed on neighboring cells. We asked whether PTPμ expression might be altered in response to epithelial injury and whether altered PTPμ expression might influence EGFR signaling. In A549 cells, diverse injurious stimuli dramatically reduced PTPμ protein expression. Under basal conditions, small interfering RNA (siRNA)-induced silencing of PTPμ increased EGFR Y992 and Y1068 phosphorylation. In the presence of EGF, PTPμ knockdown increased EGFR Y845, Y992, Y1045, Y1068, Y1086, and Y1173 but not Y1148 phosphorylation. Reduced PTPμ expression increased EGF-stimulated phosphorylation of Y992, a docking site for phospholipase C (PLC)γ(1), activation of PLCγ(1) itself, and increased cell migration in both wounding and chemotaxis assays. In contrast, overexpression of PTPμ decreased EGF-stimulated EGFR Y992 and Y1068 phosphorylation. Therefore, airway epithelial injury profoundly reduces PTPμ expression, and PTPμ depletion selectively increases phosphorylation of specific EGFR tyrosine residues, PLCγ(1) activation, and cell migration, providing a novel mechanism through which epithelial integrity may be restored.