Metalloproteinases and transforming growth factor-alpha mediate substance P-induced mitogen-activated protein kinase activation and proliferation in human colonocytes

Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of proinflammatory cytokines from colonic epithelial cells. SP also stimulates cell proliferation, a critical event in tissue healing during chronic colitis, via transactivation of the epidermal growth factor (EGF) receptor (EGFR) and activation of mitogen-activated protein kinase (MAPK). Here we examined the mechanism by which SP induces EGFR and MAPK activation. We used non-transformed human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) as well as untransfected U373 MG cells expressing high levels of endogenous NK-1R. Exposure of both cell lines to SP (10(-7) m) stimulated EGFR activation (1 min) followed by extracellular signal-regulated protein kinase (ERK1/2) activation (2-5 min). SP-induced ERK1/2 activation was blocked by pretreatment with the metalloproteinase inhibitor Batimastat/GM6001, the EGFR phosphorylation inhibitor AG1478, and the tumor necrosis factor-alpha-converting enzyme (TACE) inhibitor TAPI-1. Pretreatment with antibodies against potential EGFR ligands suggested that transforming growth factor-alpha (TGFalpha), but not the other EGFR ligands EGF, heparin-binding EGF, or amphiregulin, mediates SP-induced EGFR transactivation. SP stimulated TGFalpha release into the extracellular space that was measurable within 2 min, and this release was inhibited by metalloproteinase inhibitors and the TACE inhibitor TAPI-1. SP also induced MAPK-mediated cell proliferation that was inhibited by TACE, matrix metalloproteinase (MMP), EGFR, and MEK1 inhibitors. Thus, in human colonocytes, NK-1R-induced EGFR and MAPK activation and cell proliferation involve matrix metalloproteinases (most likely TACE) and the release of TGFalpha. These signaling mechanisms may be involved in the protective effects of NK-1R in chronic colitis.     

Heparin binding epidermal growth factor-like growth factor is a transforming growth factor beta-regulated gene in intestinal epithelial cells.

Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an EGF-related peptide with prominent effects on cell growth and migration. We explored potentially unique characteristics of HB-EGF in the intestinal epithelial cell line RIE-1. HB-EGF stimulated [(3)H]thymidine incorporation to a level equivalent to transforming growth factor alpha (TGFalpha). HB-EGF also rapidly activated MAPK and induced cyclin D1 in mid-G1 with kinetics similar to TGFalpha. Unlike TGFalpha, HB-EGF mRNA was induced within 1 h by a variety of stimuli, including TGFbeta1. Maximal induction by TGFbeta (7-fold) occurred within 2 h of treatment. Actinomycin D decay curves showed that TGFbeta1 had no effect on HB-EGF mRNA half-life (T(1/2) 20 min). Induction of HB-EGF by TGFbeta1 was not affected by pretreatment with the MEK inhibitor PD-98059 while inhibition of protein kinase C either partially (calphostin C) or completely (staurosporin) blocked induction. Our results suggest that major differences exist in the regulation of the closely related EGF family members TGFalpha and HB-EGF. TGFbeta and HB-EGF, structurally unrelated peptides with potent effects on wound healing, may function coordinately to mediate responses to wounding or cell injury in the intestinal epithelium.     

Signal transduction of betacellulin in growth and migration of vascular smooth muscle cells

Epidermal growth factor (EGF) family ligands have been implicated in cardiovascular diseases because of their enhanced expression in vascular lesions and their promoting effects on growth and migration of vascular smooth muscle cells (VSMCs). Betacellulin (BTC), a novel EGF family ligand, has been shown to be expressed in atherosclerotic lesions and to be a potent growth factor of VSMCs. However, the molecular mechanisms downstream of BTC involved in mediating vascular remodeling remain largely unknown. Therefore, the aim of this study was to examine the effects of BTC on signal transduction, growth, and migration in VSMCs. We found that BTC stimulated phosphorylation of EGF receptor (EGFR) at Tyr1068, which was completely blocked by an EGFR kinase inhibitor, AG-1478. BTC also phosphorylated ErbB2 at Tyr877, Tyr1112, and Tyr1248 and induced association of ErbB2 with EGFR, suggesting their heterodimerization in VSMCs. In postreceptor signal transduction, BTC stimulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2, Akt, and p38 mitogen-activated protein kinase (MAPK). Moreover, BTC stimulated proliferation and migration of VSMCs. ERK and Akt inhibitors suppressed migration markedly and proliferation partially, whereas the p38 inhibitor suppressed migration partially but not proliferation. In addition, we found the presence of endogenous BTC in conditioned medium of VSMCs and an increase of BTC on angiotensin II stimulation. In summary, BTC promotes growth and migration of VSMCs through activation of EGFR, ErbB2, and downstream serine/threonine kinases. Together with the expression and processing of endogenous BTC in VSMCs, our results suggest a critical involvement of BTC in vascular remodeling. epidermal growth factor receptors; ErbB2; migration; signal transduction     

Extracellular regulated kinase5 is expressed in fetal mouse submandibular glands and is phosphorylated in response to epidermal growth factor and other ligands of the ErbB family of receptors

Growth factors and their receptors regulate development of many organs through activation of multiple intracellular signaling cascades including a mitogen‐activated protein kinase (MAPK). Extracellular regulated kinases (ERK)1/2, classic MAPK family members, are expressed in fetal mouse submandibular glands (SMG), and stimulate branching morphogenesis. ERK5, also called big mitogen‐activated protein kinase 1, was recently found as a new member of MAPK super family, and its biological roles are still largely unknown. In this study, we investigated the expression and function of ERK5 in developing fetal mouse SMGs. Western blotting analysis showed that the expression pattern of ERK5 was different from the pattern of ERK1/2 in developing fetal SMGs. Both ERK1/2 and ERK5 were phosphorylated after exposure to ligands of the ErbB family of receptor tyrosine kinases (RTKs). Phosphorylation of ERK1/2 was strongly induced by epidermal growth factor (EGF) in SMG rudiments at embryonic day 14 (E14), E16 and E18. However, ERK5 phosphorylation induced by EGF was clearly observed at E14 and E16, but not at E18. Branching morphogenesis of cultured E13 SMG rudiments was strongly suppressed by administration of U0126, an inhibitor for ERK1/2 activation, whereas the phosphorylation of ERK5 was not inhibited by U0126. BIX02188, a specific inhibitor for ERK5 activation, also inhibited branching morphogenesis in cultured SMG rudiments. These results show that EGF‐responsive ERK5 is expressed in developing fetal mouse SMG, and suggest that both ERK1/2 and ERK5 signaling cascades might play an important role in the regulation of branching morphogenesis.     

Influences of dopaminergic lesion on epidermal growth factor-ErbB signals in Parkinson's disease and its model: neurotrophic implication in nigrostriatal neurons

Epidermal growth factor (EGF) is a member of a structurally related family containing heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor alpha (TGFalpha) that exerts neurotrophic activity on midbrain dopaminergic neurons. To examine neurotrophic abnormality in Parkinson's disease (PD), we measured the protein content of EGF, TGFalpha, and HB-EGF in post-mortem brains of patients with Parkinson's disease and age-matched control subjects. Protein levels of EGF and tyrosine hydroxylase were decreased in the prefrontal cortex and the striatum of patients. In contrast, HB-EGF and TGFalpha levels were not significantly altered in either region. The expression of EGF receptors (ErbB1 and ErbB2, but not ErbB3 or ErbB4) was down-regulated significantly in the same forebrain regions. The same phenomenon was mimicked in rats by dopaminergic lesions induced by nigral 6-hydroxydopamine infusion. EGF and ErbB1 levels in the striatum of the PD model were markedly reduced on the lesioned side, compared with the control hemisphere. Subchronic supplement of EGF in the striatum of the PD model locally prevented the dopaminergic neurodegeration as measured by tyrosine hydroxylase immunoreactivity. These findings suggest that the neurotrophic activity of EGF is maintained by afferent signals of midbrain dopaminergic neurons and is impaired in patients with Parkinson's disease.     

Transforming growth factor-alpha short-circuits downregulation of the epidermal growth factor receptor

Transforming growth factor-alpha (TGFalpha) is an epidermal growth factor receptor (EGFR) ligand which is distinguished from EGF by its acid-labile structure and potent transforming function. We recently reported that TGFalpha induces less efficient EGFR heterodimerization and downregulation than does EGF (Gulliford et al., 1997, Oncogene, 15:2219-2223). Here we use isoform-specific EGFR and ErbB2 antibodies to show that the duration of EGFR signalling induced by a single TGFalpha exposure is less than that induced by equimolar EGF. The protein trafficking inhibitor brefeldin A (BFA) reduces the duration of EGF signalling to an extent similar to that seen with TGFalpha alone; the effects of TGFalpha and BFA on EGFR degradation are opposite, however, with TGFalpha sparing EGFR from downregulation but BFA accelerating EGF-dependent receptor loss. This suggests that BFA blocks EGFR recycling and thus shortens EGF-dependent receptor signalling, whereas TGFalpha shortens receptor signalling and thus blocks EGFR downregulation. Consistent with this, repeated application of TGFalpha is accompanied by prolonged EGFR expression and signalling, whereas similar application of EGF causes receptor downregulation and signal termination. These findings indicate that constitutive secretion of pH-labile TGFalpha may perpetuate EGFR signalling by permitting early oligomer dissociation and dephosphorylation within acidic endosomes, thereby extinguishing a phosphotyrosine-based downregulation signal and creating an irreversible autocrine growth loop.     

ErbB2 and ErbB3 receptors mediate inhibition of calcium-dependent chloride secretion in colonic epithelial cells

We have previously demonstrated that epidermal growth factor (EGF) inhibits calcium-dependent chloride secretion via a mechanism involving stimulation of phosphatidylinositol 3-kinase (PI3-K). The muscarinic agonist of chloride secretion, carbachol (CCh), also stimulates an antisecretory pathway that involves transactivation of the EGF receptor (EGFR) but does not involve PI3-K. Here, we have examined if ErbB receptors, other than the EGFR, have a role in regulation of colonic secretion and if differential effects on ErbB receptor activation may explain the ability of the EGFR to propagate diverse signaling pathways in response to EGF versus CCh. Basolateral, but not apical, addition of the ErbB3/ErbB4 ligand alpha-heregulin (HRG; 1-100 ng/ml) inhibited secretory responses to CCh (100 microM) across voltage-clamped T(84) epithelial cells. Immunoprecipitation/Western blot studies revealed that HRG (100 ng/ml) stimulated tyrosine phosphorylation and dimerization of ErbB3 and ErbB2, but had no effect on phosphorylation of the EGFR. HRG also stimulated recruitment of the p85 subunit of PI3-K to ErbB3/ErbB2 receptor dimers, while the PI3-K inhibitor, wortmannin (50 nM), completely reversed the inhibitory effect of HRG on CCh-stimulated secretion. Further studies revealed that, while both EGF (100 ng/ml) and CCh (100 microM) stimulated phosphorylation of the EGFR, only EGF stimulated phosphorylation of ErbB2, and neither stimulated ErbB3 phosphorylation. EGF, but not CCh, stimulated the formation of EGFR/ErbB2 receptor dimers and the recruitment of p85 to ErbB2. We conclude that ErbB2 and ErbB3 are expressed in T(84) cells and are functionally coupled to inhibition of calcium-dependent chloride secretion. Differential dimerization with other ErbB family members may underlie the ability of the EGFR to propagate diverse inhibitory signals in response to activation by EGF or transactivation by CCh.     

Role of mitochondrial Bax, caspases, and MAPKs for ceramide-induced apoptosis in renal proximal tubular cells

It remains elusive whether crosstalk exists among mitochondrial Bax, caspases, and mitogen-activated protein kinases (MAPKs), and whether epidermal growth factor (EGF), which may activate MAPKs, affects ceramide-induced apoptosis through the crosstalk in renal proximal tubular cells (RPTCs). Effect of ceramide on expression of mitochondrial Bax and phosphorylated (p)-ERK, p38MAPK and JNK, that of MAPKs inhibition, and of EGF in the presence or absence of MAPKs inhibition on ceramide-induced apoptosis were examined in HK-2 cells. Apoptosis and expression of mitochondrial Bax and p-MAPKs were measured by Hoechst 33258 staining and Western blotting. C2-ceramide, but not dihydroC2-ceramide, inactive C2-ceramide, induced apoptosis at 24 h. C2-ceramide enhanced the mitochondrial Bax expression at 1 h, which was peaked at 3–6 h and decreased at 24 h, but remained increased, compared to control. An inhibitor of caspases, zVAD-fmk, ameliorated ceramide-induced apoptosis, suggesting a role of caspases for ceramide-induced apoptosis. C2-ceramide enhanced the expression of p-ERK and p-p38MAPK, but not p-JNK, at 1 h, which was increased till 24 h. An inhibitor of ERK, PD98059, or of p38MAPK, SB202190, failed to affect C2-ceramide-induced apoptosis. EGF, which enhanced the expression of p-ERK and p-p38MAPK but not p-JNK, ameliorated C2-ceramide-induced apoptosis without affecting mitochondrial Bax. Inhibition of ERK or p38MAPK failed to abolish the protective effect of EGF on C2-ceramide-induced apoptosis. Mitochondrial Bax and caspases, but not MAPKs, play a role for ceramide-induced apoptosis in RPTCs. EGF ameliorates ceramide-induced apoptosis in Bax- and MAPKs-independent pathways. The mechanism of ceramide-induced apoptosis and anti-apoptotic effect of EGF deserves further investigations.     

The MAPK(ERK-1,2) pathway integrates distinct and antagonistic signals from TGFalpha and FGF7 in morphogenesis of mouse mammary epithelium

Transforming growth factor-alpha (TGFalpha) and fibroblast growth factor-7 (FGF7) exhibit distinct expression patterns in the mammary gland. Both factors signal through mitogen-activated kinase/extracellular regulated kinase-1,2 (MAPK(ERK1,2)); however, their unique and/or combined contributions to mammary morphogenesis have not been examined. In ex vivo mammary explants, we show that a sustained activation of MAPK(ERK1,2) for 1 h, induced by TGFalpha, was necessary and sufficient to initiate branching morphogenesis, whereas a transient activation (15 min) of MAPK(ERK1,2), induced by FGF7, led to growth without branching. Unlike TGFalpha, FGF7 promoted sustained proliferation as well as ectopic localization of, and increase in, keratin-6 expressing cells. The response of the explants to FGF10 was similar to that to FGF7. Simultaneous stimulation by FGF7 and TGFalpha indicated that the FGF7-induced MAPK(ERK1,2) signaling and associated phenotypes were dominant: FGF7 may prevent branching by suppression of two necessary TGFalpha-induced morphogenetic effectors, matrix metalloproteinase-3 (MMP-3/stromelysin-1), and fibronectin. Our findings indicate that expression of morphogenetic effectors, proliferation, and cell-type decisions during mammary organoid morphogenesis are intimately dependent on the duration of activation of MAPK(ERK1,2) activation.     

HB-EGF promotes epithelial cell migration in eyelid development

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR) and ERBB4. Here, we show that HB-EGF-EGFR signaling is involved in eyelid development. HB-EGF expression is restricted to the tip of the leading edge of the migrating epithelium during eyelid closure in late gestation mouse embryos. Both HB-EGF null (HB(del/del)) and secretion-deficient (HB(uc/uc)) mutant embryos exhibited delayed eyelid closure, owing to slower leading edge extension and reduced actin bundle formation in migrating epithelial cells. No changes in cell proliferation were observed in these embryos. In addition, activation of EGFR and ERK was decreased in HB(del/del) eyelids. Crosses between HB(del/del) mice and waved 2 mice, a hypomorphic EGFR mutant strain, indicate that HB-EGF and EGFR interact genetically in eyelid closure. Together with our data showing that embryos treated with an EGFR-specific kinase inhibitor phenocopy HB(del/del) embryos, these data indicate that EGFR mediates HB-EGF-dependent eyelid closure. Finally, analysis of eyelid closure in TGFalpha-null mice and in HB-EGF and TGFalpha double null mice revealed that HB-EGF and TGFalpha contribute equally to and function synergistically in this process. These results indicate that soluble HB-EGF secreted from the tip of the leading edge activates the EGFR and ERK pathway, and that synergy with TGFalpha is required for leading edge extension in epithelial sheet migration during eyelid closure.     

Specific signaling cascades involved in cell spreading during healing of micro-wounded gastric epithelial monolayers

Mechanisms that specifically modulate cell spreading and/or cell migration following epithelial wounding are poorly understood. Using micro-wounded human gastric epithelial monolayers, we show herein that EGF and TGFalpha maximally increase spreading of epithelial sheets under a cell proliferation-independent mechanism. Treatment of confluent HGE-17 cells with the phosphatidylinositol 3-kinase inhibitor, LY294002, and the epidermal growth factor receptor inhibitor, PD153035, strongly reduced basal and TGFalpha-stimulated cell spreading. While pharmacological inhibition of pp60src-kinase activity also attenuated basal epithelial spreading, addition of the mTOR/p70S6K inhibitor rapamycin or a specific siRNA targeting ILK sequence did not affect the kinetic rates of wound closure. Epithelial wound healing was initiated by actin purse-string contraction followed by lamellae formation. Conversely, disruption of actin and tubulin stability with cytochalasin D and nocodazole, respectively, inhibited epithelial sheet spreading. Finally, antibodies directed against the alpha3 integrin subunit, but not against the alpha6 or alpha2 subunits, attenuated epithelial sheet spreading as well as lamellae formation. In conclusion, the current investigation establishes that EGF/TGFalpha and the alpha3beta1 integrin, pp60c-src, EGFR and PI3K pathways are mainly associated with the cell spreading of the restitution process during healing of human gastric epithelial wounds.     

Selective cleavage of ErbB4 by G-protein-coupled gonadotropin-releasing hormone receptor in cultured hypothalamic neurons

Gonadotropin‐releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons). GnRH neurons have a GnRH receptor belonging to the G‐protein‐coupled receptors. The stimulation of this receptor activates extracellular signal‐regulated kinase (ERK). In the present study, we found that epidermal growth factor receptor (EGFR) and ErbB4 were expressed in immortalized GnRH neurons (GT1‐7 cells). AG1478, a relatively specific inhibitor of the ErbB family, and small interfering RNA (siRNA) for ErbB4 inhibited the GnRH‐induced activation of ERK in GT1‐7 cells, suggesting that EGFR and ErbB4 were necessary for the activation. In addition, GnRH induced the cleavage of ErbB4 and accumulation of an 80‐kDa fragment. After treatment of the cells with 50 nM GnRH for 5 min, about 80% of ErbB4 was cleaved. Biotinylation of cell surface proteins revealed that more than 70% of the cell surface ErbB4 was cleaved by GnRH treatment. A higher concentration and longer treatment were necessary for GnRH to induce ErbB4 cleavage than ERK activation. TAPI‐2, an inhibitor of tumor necrosis factor‐α‐converting enzyme (TACE), and siRNA for TACE inhibited the cleavage of ErbB4, suggesting that TACE was involved. After ErbB4 cleavage, the activation of ERK by neuregulin 1 was almost completely inhibited. These results suggest that the down‐regulation of ErbB4 expression is induced by G‐protein‐coupled receptor stimulation. J. Cell. Physiol. 227: 2492–2501, 2012. © 2011 Wiley Periodicals, Inc.     

Activation of extracellular signal-regulated kinase by ultraviolet is mediated through Src-dependent epidermal growth factor receptor phosphorylation. Its implication in an anti-apoptotic function.

Ultraviolet (UV) irradiation stimulates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), which is a member of the mitogen-activated protein kinase (MAPK) superfamily and implicated in stress-induced apoptosis. UV also induces the activation of another MAPK member, extracellular signal-regulated kinase (ERK), which is typically involved in a growth-signaling cascade. However, the UV-induced signaling pathway leading to ERK activation, together with the physiological role, has remained unknown. Here we examined the molecular mechanism and physiological function of UV-induced ERK activation in human epidermoid carcinoma A431 cells that retain a high number of epidermal growth factor (EGF) receptors. UV-induced ERK activation was accompanied with the Tyr phosphorylation of EGF receptors, and both responses were completely abolished in the presence of a selective EGF receptor inhibitor (AG1478) or the Src inhibitor PP2 and by the expression of a kinase-dead Src mutant. On the other hand, SAPK/JNK activation by UV was partially inhibited by these inhibitors. UV stimulated Src activity in a manner similar to the ERK activation, but the Src activation was insensitive to AG1478. UV-induced cell apoptosis measured by DNA fragmentation and caspase 3 activation was enhanced by AG1478 and an ERK kinase inhibitor (U0126) but inhibited by EGF receptor stimulation by the agonist. These results indicate that UV-induced ERK activation, which provides a survival signal against stress-induced apoptosis, is mediated through Src-dependent Tyr phosphorylation of EGF receptors.     

The emergence of ErbB2 expression in cultured rat hepatocytes correlates with enhanced and diversified EGF-mediated signaling

The proliferative effects of EGF in liver have been extensively investigated in cultured hepatocytes. We studied the effects of EGF, insulin, and other growth regulators on the expression, interaction, and signaling of ErbB receptors in primary cultures of adult rat hepatocytes. Using immunological methods and ErbB tyrosine kinase inhibitors, we analyzed the expression and signaling patterns of the ErbB kinases over 120 h of culture. Basal and EGF-stimulated protein tyrosine phosphorylation increased as cells adapted in vitro. EGF receptor (EGFr) expression declined in the first 24 h, whereas ErbB3 expression rose. Although ErbB2 was not present in freshly isolated hepatocytes, EGF and insulin independently induced ErbB2 while suppressing ErbB3 expression. Low concentrations of EGF and insulin synergistically stimulated ErbB2 expression and DNA synthesis. The greatest increase in ErbB2, which is normally expressed by fetal and neonatal hepatocytes, occurred shortly before the onset of DNA synthesis (> 40 h). EGF promoted EGFr and ErbB2 coassociation, stimulating tyrosine phosphorylation of both proteins. In contrast, heregulin beta1 (HRG-beta1) did not promote ErbB2 and ErbB3 coassociation. A selective tyrphostin inhibitor of ErbB2 suppressed EGF-stimulated DNA synthesis, but maximum suppression required the blockade of the EGFr kinase as well. Maximal EGF stimulation of DNA synthesis in vitro depends on the induction of ErbB2 and involves an EGFr-ErbB2 heterodimer. The ability of insulin to induce ErbB2 suggests both a mechanism for the synergy between insulin and EGF and a possible metabolic control of ErbB2 in vivo.     

Cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis: role in decreased responsiveness to inflammatory cytokines

Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) are major signaling molecules activated in human neutrophils stimulated by cytokines. Both molecules were cleaved at the N-terminal portion in neutrophils undergoing apoptosis induced by in vitro culture alone or treatment with TNF and/or cycloheximide. The cleavage of both molecules was inhibited by G-CSF and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a caspase inhibitor, both of which can inhibit neutrophil apoptosis. In a cell-free system, ERK and p38 MAPK were not cleaved by recombinant caspase-3 or caspase-8 while gelsolin was cleaved by caspase-3 under the same condition. The cleavage of both molecules appears to be specific to mature neutrophils, since it was not detected in immature cells (HL-60 and Jurkat) undergoing apoptosis, indicating that proteases responsible for the cleavage of both molecules may develop during differentiation into mature neutrophils. Concomitant with the cleavage of ERK and p38 MAPK, GM-CSF- and TNF-induced superoxide release, adherence, and phosphorylation of ERK and p38 MAPK were decreased in neutrophils undergoing apoptosis. In addition, GM-CSF- and TNF-induced superoxide release and adherence were inhibited by PD98059 MAPK/ERK kinase inhibitor) as well as SB203580 (p38 MAPK inhibitor), suggesting possible involvement of ERK and p38 MAPK in superoxide release and adherence induced by these cytokines. These findings indicate that ERK and p38 MAPK are cleaved and degraded in neutrophils undergoing apoptosis in a caspase-dependent manner and the cleavage of both molecules may be partly responsible for decreased functional responsiveness to inflammatory cytokines.     

Regulation of the EGFR/ErbB signalling by clathrin in response to various ligands in hepatocellular carcinoma cell lines

Membrane receptor intracellular trafficking and signalling are frequently altered in cancers. Our aim was to investigate whether clathrin‐dependent trafficking modulates signalling of the ErbB receptor family in response to amphiregulin (AR), EGF, heparin‐binding EGF‐like growth factor (HB‐EGF) and heregulin‐1β (HRG). Experiments were performed using three hepatocellular carcinoma (HCC) cell lines, Hep3B, HepG2 and PLC/PRF/5, expressing various levels of EGFR, ErbB2 and ErbB3. Inhibition of clathrin‐mediated endocytosis (CME), by down‐regulating clathrin heavy chain expression, resulted in a cell‐ and ligand‐specific pattern of phosphorylation of the ErbB receptors and their downstream effectors. Clathrin down‐regulation significantly decreased the ratio between phosphorylated EGFR (pEGFR) and total EGFR in all cell lines when stimulated with AR, EGF, HB‐EGF or HRG, except in HRG‐stimulated Hep3B cells in which pEGFR was not detectable. The ratio between phosphorylated ErbB2 and total ErbB2 was significantly decreased in clathrin down‐regulated Hep3B cells stimulated with any of the ligands, and in HRG‐stimulated PLC/PRF/5 cells. The ratio between phosphorylated ErbB3 and total ErbB3 significantly decreased in clathrin down‐regulated cell lines upon stimulation with EGF or HB‐EGF. STAT3 phosphorylation levels significantly increased in all cell lines irrespective of stimulation, while that of AKT remained unchanged, except in AR‐stimulated Hep3B and HepG2 cells in which pAKT was significantly decreased. Finally, ERK phosphorylation was insensitive to clathrin inhibition. Altogether, our observations indicate that clathrin regulation of ErbB signalling in HCC is a complex process that likely depends on the expression of ErbB family members and on the autocrine/paracrine secretion of their ligands in the tumour environment.     

Role of epidermal growth factor and ErbB2 receptors in 3T3-L1 adipogenesis

Objective: Epidermal growth factor (EGF) stimulates proliferation in 3T3‐L1 preadipocytes, but EGF action in differentiation is less clear. EGF promotes differentiation at concentrations <1 nM but inhibits differentiation at higher concentrations, suggesting a dual role in adipogenesis. We hypothesized that differences in EGF receptor activation and downstream signaling mediate distinct biological effects of EGF at low vs. high abundance. Research Methods and Procedures: We compared the effects of low (0.1 nM) vs. high (10 nM) EGF on the activation of EGF receptors, proximal signaling molecules Src and Shc, and the downstream mitogen‐activated protein kinase (MAPK) pathways extracellular regulated kinase (ERK) and p38 in proliferating and differentiated 3T3‐L1 cells. Results: Both low and high EGF activated ERK and p38 in preadipocytes. Src inhibitors PP1 and PP2 blocked ERK and p38 activation by low but not high EGF, and only high EGF increased Shc phosphorylation. Selective inhibition of the EGF receptor (EGFR) with AG1478 blocked ERK and p38 activation at both concentrations; however, selective inhibition of the ErbB2 receptor (EB2R) with AG825 or small interfering RNA (siRNA) blocked low but not high EGF activation of ERK and p38. Coimmunoprecipitation of EGFR with EB2R and Src was observed with low EGF in preadipocytes but at both concentrations in adipocytes. EB2R inhibition during differentiation decreased p38 activity and peroxisome proliferator‐activated receptor γ (PPARγ) abundance. Discussion: Our results show that EGFR homodimers mediate action of EGF at high abundance, but at low abundance, EGF promotes differentiation through EGFR/EB2R heterodimer activation of Src and p38. These results may partially explain the observations that high EGF concentrations inhibit, whereas low concentrations support, preadipocyte differentiation.     

Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes

Fibroblast growth factors (FGFs) regulate long bone development by affecting the proliferation and differentiation of chondrocytes. FGF treatment inhibits the proliferation of chondrocytes both in vitro and in vivo, but the signaling pathways involved have not been clearly identified. In this report we show that both the MEK-ERK1/2 and p38 MAPK pathways, but not phospholipase C gamma or phosphatidylinositol 3-kinase, play a role in FGF-mediated growth arrest of chondrocytes. Chemical inhibitors of the MEK1/2 or the p38 MAPK pathways applied to rat chondrosarcoma (RCS) chondrocytes significantly prevented FGF-induced growth arrest. The retinoblastoma family members p107 and p130 were previously shown to be essential effectors of FGF-induced growth arrest in chondrocytes. The dephosphorylation of p107, one of the earliest events in RCS growth arrest, was significantly blocked by MEK1/2 inhibitors but not by the p38 MAPK inhibitors, whereas that of p130, which occurs later, was partially prevented both by the MEK and p38 inhibitors. Furthermore, by expressing the nerve growth factor (NGF) receptor, TrkA, and the epidermal growth factor (EGF) receptor, ErbB1, in RCS cells we show that NGF treatment of the transfected cells caused growth inhibition, whereas EGF did not. FGF- and NGF-induced growth inhibition is accompanied by a strong and sustained activation of ERK1/2 and p38 MAPK and a decrease of AKT phosphorylation, whereas EGF induces a much more transient activation of p38 and ERK1/2 and increases AKT phosphorylation. These results indicate that inhibition of chondrocyte proliferation by FGF requires both ERK1/2 and p38 MAPK signaling and also suggest that sustained activation of these pathways is required to achieve growth inhibition.     

Activation of MAPKs by angiotensin II in vascular smooth muscle cells. Metalloprotease-dependent EGF receptor activation is required for activation of ERK and p38 MAPK but not for JNK

In cultured vascular smooth muscle cells (VSMC), the vasculotrophic factor, angiotensin II (AngII) activates three major MAPKs via the G(q)-coupled AT1 receptor. Extracellular signal-regulated kinase (ERK) activation by AngII requires Ca(2+)-dependent "transactivation" of the EGF receptor that may involve a metalloprotease to stimulate processing of an EGF receptor ligand from its precursor. Whether EGF receptor transactivation also contributes to activation of other members of MAPKs such as p38MAPK and c-Jun N-terminal kinase (JNK) by AngII remains unclear. In the present study, we have examined the effects of a synthetic metalloprotease inhibitor BB2116, and the EGF receptor kinase inhibitor AG1478 on AngII-induced activation of MAPKs in cultured VSMC. BB2116 markedly inhibited ERK activation induced by AngII or the Ca(2+) ionophore without affecting the activation by EGF or PDGF. BB2116 as well as HB-EGF neutralizing antibody inhibited the EGF receptor transactivation by AngII, suggesting a critical role of HB-EGF in the metalloprotease-dependent EGF receptor transactivation. In addition to the ERK activation, activation of p38MAPK and JNK by AngII was inhibited by an AT1 receptor antagonist, RNH6270. and EGF markedly activate p38MAPK, whereas but not EGF markedly activates JNK, indicating the possible contribution of the EGF receptor transactivation to the p38MAPK activation. The findings that both BB2116 and AG1478 specifically inhibited activation of p38MAPK but not JNK by AngII support this hypothesis. From these data, we conclude that ERK and p38MAPK activation by AngII requires the metalloprotease-dependent EGF receptor transactivation, whereas the JNK activation is regulated without involvement of EGF receptor transactivation.