Topography of amphiregulin expression in cultured human keratinocytes: Colocalization with the epidermal growth factor receptor and CD44

Summary Much of the autonomous growth of cultured keratinocytes is attributable to the signaling of amphiregulin, a heparin-binding autocrine growth factor, through the epidermal growth factor receptor. Emerging evidence suggests, moreover, that the membrane proteoglycan, CD44, is a cofactor for the interaction of heparin-binding ligands with their receptors. This model was evaluated by characterizing the patterns of the immunolabeled molecules in cultured human neonatal keratinocytes, to test the hypothesis that involvement in a common function results in coordinate segregation within or on the cell. The molecules were localized by double immunofluorescence labeling to detect amphiregulin and either the epidermal growth factor receptor or CD44, and the immunostained products were imaged by scanning laser confocal microscopy. Both amphiregulin and the epidermal growth factor receptor segregated to a perinuclear distribution and to intercellular contacts. In addition, amphiregulin localized to the outer leading edge of colonies and focally to intranuclear sites. Metabolic blockade of proteoglycan sulfation with sodium chlorate inhibited growth of the cells and concurrently enhanced the nuclear, but decreased the outer leading edge, labeling for amphiregulin. There was no nuclear or perimeter labeling for the epidermal growth factor receptor. Cultures co-immunolabeled for CD44 and amphiregulin exhibited variable perinuclear staining for both, but otherwise CD44 was distributed to intercellular contacts. The intercellular localizations of CD44 with amphiregulin and of amphiregulin with the epidermal growth factor receptor were strongly concordant. These data are consistent with a concerted function at intercellular contacts, where cytokine signaling is mediated via receptor binding and possibly regulated by the CD44 proteoglycan as cofactor. The intranuclear and perimeter labeling of amphiregulin, however, suggests that this cytokine has additional functions, both in the nucleus and as a matrix receptor.     

Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-α) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-α and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth by IGF-1 requires activation of an EGF receptor-mediated autocrine loop. © 1995 Wiley-Liss, Inc.     

Testosterone-induced growth of S115 mouse mammary tumor cells is dependent on heparan sulfate

The androgen-induced proliferation of S115 mouse mammary tumor cells has been suggested to involve autocrinic fibroblast growth factor signaling. Heparan sulfate proteoglycans are required for fibroblast growth factor signaling, presumably due to their ability to alter binding of fibroblast growth factors to their receptors. We have investigated the role of heparan sulfate proteoglycans in the testosterone-induced proliferation of S115 cells. We demonstrate that when the cells are treated with sodium chlorate, which inhibits the sulfation of endogenous heparan sulfate proteoglycans, cell growth becomes dependent on exogenous heparin. The shortest heparin oligosaccharides supporting cell growth were octasaccharides, whereas dodecasaccharides were almost as effective as native heparin. The N-, 2-O-, and 6-O-sulfate groups of heparin were all required for full testosterone response. Treatment of S115 cells with chlorate or testosterone did not alter the expression of fibroblast growth factor receptors 1 or 3, whereas the expression of fibroblast growth factor receptor 2 was down-regulated. We have previously shown that overexpression of syndecan-1 heparan sulfate proteoglycan renders S115 cells insensitive to testosterone and now demonstrate that this effect can be overcome by sodium chlorate treatment in combination with exogenous heparin. Our results suggest that heparin-like molecules are intimately involved in the androgen-mediated proliferation of S115 cells.     

A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.     

Inhibition of autonomous human keratinocyte proliferation and amphiregulin mitogenic activity by sulfated polysaccharides

Summary We previously demonstrated that human keratinocyte cultures proliferate in the absence of polypeptide growth factors (autonomous growth) and that this autonomous growth is blocked by interaction of heparin with a human keratinocyte-derived autocrine factor (KAF) which we identified as amphiregulin (AR). In the present study, we demonstrate that sulfated polysaccharides other than heparin (low and high molecular weight dextran sulfates) also inhibit the AR-mediated autonomous proliferation of human keratinocytes. Furthermore, sulfated polysaccharides such as high and low molecular weight dextran sulfates, heparan sulfate and, to a lesser extent, chondroitin sulfates B and C were also shown to be inhibitors of human keratinocyte-derived AR (k-d AR)-stimulated DNA synthesis in quiescent murine AKR-2B cell cultures. Our results demonstrate that sulfation of polysaccharides is required for AR inhibitory activity, and that several sulfated polysaccharides (other than heparin) can act as inhibitors of AR-mediated autonomous proliferation in human epidermal keratinocytes and as inhibitors of k-d AR-mediated mitogenic activity in AKR-2B cells.     

Epidermal growth factor receptor-dependent stimulation of amphiregulin expression in androgen-stimulated human prostate cancer cells.

Amphiregulin is a heparin-binding epidermal growth factor (EGF)-related peptide that binds to the EGF receptor (EGF-R) with high affinity. In this study, we report a role for amphiregulin in androgen-stimulated regulation of prostate cancer cell growth. Androgen is known to enhance EGF-R expression in the androgen-sensitive LNCaP human prostate carcinoma cell line, and it has been suggested that androgenic stimuli may regulate proliferation, in part, through autocrine mechanisms involving the EGF-R. In this study, we demonstrate that LNCaP cells express amphiregulin mRNA and peptide and that this expression is elevated by androgenic stimulation. We also show that ligand-dependent EGF-R stimulation induces amphiregulin expression and that androgenic effects on amphiregulin synthesis are mediated through this EGF-R pathway. Parallel studies using the estrogen-responsive breast carcinoma cell line, MCF-7, suggest that regulation of amphiregulin by estrogen may also be mediated via an EGF-R pathway. In addition, heparin treatment of LNCaP cells inhibits androgen-stimulated cell growth further suggesting that amphiregulin can mediate androgen-stimulated LNCaP proliferation. Together, these results implicate an androgen-regulated autocrine loop composed of amphiregulin and its receptor in prostate cancer cell growth and suggest that the mechanism of steroid hormone regulation of amphiregulin synthesis may occur through androgen upregulation of the EGF-R and subsequent receptor-dependent pathways.     

Divergent regulation of proteoglycan and glycosaminoglycan free chain expression in human keratinocytes and melanocytes

Summary Keratinocytes and melanocytes, which together form units of structure and function within human epidermis, are known to differ in expression of autocrine growth factors, particularly those with heparin binding affinity. Because such cytokines could be regulated by the endogenous heparinlike glycosaminoglycan, heparan sulfate, proteoglycan synthesis was compared between human keratinocytes and melanocytes cultured from a common donor. Following steady-state isotopic labeling under conditions of active growth (low density cultures) and growth inhibition (high density cultures), the sulfated polymers were isolated from conditioned media and cell extracts. We found that keratinocytes produced substantially more sulfated glycosaminoglycans than did the melanocytes. There was no evidence for hyaluronic acid synthesis by the melanocytes. The majority of [³⁵S]-sulfate labeling was in the heparan sulfates of the keratinocytes and in the chondroitin sulfates of the melanocytes. During the transition from active growth to growth inhibition, there was increased heparan sulfate proteoglycan and free chain synthesis by keratinocytes but not by melanocytes, and chondroitin sulfate proteoglycan production declined in both cell lineages. The differences may reflect divergent evolution as each cell type came to exploit those complex polysaccharides in different ways to regulate molecular pathways of growth and differentiation. The coupling of growth inhibition with augmented synthesis of heparan sulfates observed for the keratinocytes suggests a regulatory role in growth factor signaling in that cell type.     

Glycosaminoglycans bind granulocyte-macrophage colony-stimulating factor and modulate its mitogenic activity and signaling in human osteoblastic cells

We recently demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) is an autocrine growth factor for human osteoblastic (hOB) cells. Since GM-CSF is a member of the heparin-binding factor family, we examined the interactions between GM-CSF and glycosaminoglycans (GAGs) present in the osteoblast microenvironment. Using a bioassay in which the mitogenic activity of recombinant human (rh) GM-CSF was measured after incubation in the presence of an hOB cell layer or extracellular matrix (ECM) produced by these cells, we showed that rhGM-CSF binds to GAG components present in the ECM and that the bound rhGM-CSF retains its ability to stimulate hOB cell proliferation. Heparan sulfate compounds on the hOB cell surface were also found to sequester GM-CSF. Moreover, treatment with sodium chlorate, an inhibitor of GAG sulfation, suppressed the mitogenic activity of rhGM-CSF on hOB cells. This inhibitory effect was rescued by a low dose of heparin. Heparin was also found to promote the effect of rhGM-CSF on hOB cell proliferation, allowing nonmitogenic high doses of rhGM-CSF to stimulate hOB cell growth. Western blot analysis showed that undersulfation of cellular GAGs by chlorate inhibited the increased tyrosine phosphorylation of proteins involved in GM-CSF signaling in cloned immortalized hOB cells. The data demonstrate that GM-CSF binds to proteoglycans on the hOB cell surface and in ECM produced by these cells and that the bound GM-CSF is biologically active. Furthermore, this study shows that cellular proteoglycans play an essential role in GM-CSF signaling and biological activity in hOBs. J. Cell. Physiol. 177:187–195, 1998. © 1998 Wiley-Liss, Inc.     

Loss of HSulf-1 expression enhances autocrine signaling mediated by amphiregulin in breast cancer

Heparan sulfate (HS) glycosaminoglycans are the oligosaccharide chains of heparan sulfate proteoglycans. The sulfation of HS glycosaminoglycan residues is required for its interaction with various heparin-binding growth factors to promote their biological activities to activate their high affinity receptor tyrosine kinases. We have identified HS glycosaminoglycan-6-O-endosulfatase HSulf-1 as a down-regulated gene in ovarian, breast, and several other cancer cell lines. Here we have shown that HSulf-1 inhibits autocrine activation of the EGFR-ERK (epidermal growth factor receptor-extracellular signal-regulated kinase) pathway induced by serum withdrawal in MDA-MB-468 breast cancer cells. Short hairpin RNA-mediated down-regulation of HSulf-1 in HSulf-1 clonal lines of MDA-MB-468 led to a significant increase in autocrine activation of ERK compared with vector only control. The autocrine signaling was also inhibited with neutralization antibodies against amphiregulin and HB-EGF, the heparin-binding growth factor family of the EGF superfamily. Furthermore, HSulf-1-mediated inhibition of autocrine signaling was associated with reduced cyclin D1 levels, leading to decreased S phase fraction and increased G(2)-M fraction, as well as increased cell death. Finally, evaluation of HSulf-1 expression levels in primary invasive breast tumors by RNA in situ hybridization indicated that HSulf-1 is down-regulated in the majority (60%) of tumors, with a predominant association with lobular histology. These data suggest a potential role of HSulf-1 down-regulation in mammary carcinogenesis.     

Differential effects of a heparin antagonist (hexadimethrine) or chlorate on amphiregulin,basic fibroblast growth factor,and heparin-binding EGF-like growth factor activity

Amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF) are two recently identified members of the EGF family. Both AR and HB-EGF share with EGF the ability to interact with the type-1 EGF receptor; however, AR and HB-EGF differ from EGF in that both of these mitogens bind to heparin while EGF does not. To determine whether interactions with heparin-like molecules on the cell surface influence binding of AR and HB-EGF with EGF receptors and the subsequent mitogenic activity exerted by these growth factors, murine AKR-2B and Balb/MK-2 cells were treated with either an inhibitor of proteoglycan sulfation (chlorate) or a heparin antagonist (hexadimethrine). As expected, neither treatment significantly altered the specific binding of ¹²⁵I-EGF on AKR-2B cells. Interestingly, treatment with either chlorate or hexadimethrine inhibited the ability of AR to compete with ¹²⁵I-EGF for cell surface binding and also attenuated AR-mediated DNA synthesis. Thus, as has been suggested for other heparin-binding growth factors such as basic fibroblast growth factor (bFGF), the interaction of AR with an EGF-binding receptor appears to be facilitated by interaction with cell-associated sulfated glycosami-noglycans or proteoglycans. Unexpectedly, however, neither chlorate nor hexadimethrine treatment caused an inhibition of HB-EGF-induced mitogenic activity. Chlorate treatment did not significantly alter the ability of HB-EGF to compete with ¹²⁵I-EGF for cell surface binding sites, however, heparin and hexadimethrine reduced the ability of HB-EGF to compete for ¹²⁵I-EGF binding. These results suggest that, in AKR-2B cells, HB-EGF may mediate its mitogenic response at least in part through a receptor which appears to be selective for HB-EGF and permits HB-EGF-mediated mitogenic responses in the presence of hexadimethrine or heparin. Finally, hexadimethrine inhibited the specific binding and mitogenic activity of bFGF, suggesting that this cationic polymer can function as an antagonist of heparin-binding mitogens other than AR. © 1995 Wiley-Liss, Inc.     

Inhibition of vascular smooth muscle cell migration by heparin-like glycosaminoglycans

Previous studies have suggested that heparin-like glycosaminoglycans may be endogenous inhibitors of smooth muscle proliferation in the vessel wall. The purpose of this study was to determine the effects of exogenous glycosaminoglycans on rat vascular (aortic) smooth muscle cell migration following wounding in vitro. Our data indicate that heparin and related molecules (iota carrageenan, dextran sulfate), but not other glycosaminoglycans (hyaluronate, chondroitin, and dermatan sulfates), inhibit smooth muscle cell motility in a cell-specific, dose-dependent, and reversible fashion. The effect of heparin was maximal (60% inhibition) at 10 μg/ml; a half-maximal effect was observed at 1 μg/ml; Heparin did not significantly affect the migration of bovine aortic endothelium or Swiss 3T3 cells. These observations support the concept that heparin-like glycosaminoglycans may be important regulators of vascular smooth muscle cell function.     

Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro. I. Evidence for reversible binding and inactivation of the platelet-derived growth factor by heparin

Summary We have investigated the effects of interactions between growth factors and heparin-like glycosaminoglycans on untransformed human arterial smooth muscle cells (hASMC) in vitro. The results indicate that heparin in the presence of serum mitogens prevents the cells from entering the S phase of the cell cycle by binding and inactivating reversibly some serum mitogen(s). Our results suggest that platelet-derived growth factor (PDGF) is one of them and that it is the most potent stimulator of hASMC growth in vitro. Thymidine incorporation as well as increase in DNA content was inhibited not only by the presence of heparin in serum-containing medium but also when serum was chromatographed on Heparin-Sepharose at physiologic salt concentrations before exposure to the cells. The mitogenic activity of the unretained serum fraction was restored by the addition of PDGF AA, AB, or BB dimers or of a fraction (RF I) that dissociated from Heparin-Sepharose at 0.2 to 0.6M NaCl. Radiolabeled recombinant PDGF (c-sis) dissociated from Heparin-Sepharose within a concentration range of NaCl similar to that of RF I. Neither the unretained material nor the RF I or PDGF dimers were effective alone. The effect of RF I was significantly decreased by the addition of an anti-PDGF IgG that is known to neutralize the PDGF mitogenic activity partially. Addition of heparin abolished DNA-synthesis when the PDGF dimers or RF I were combined with the unretained fraction. A second fraction (RF II) bound strongly to Heparin-Sepharose and eluted between 1.1 and 1.6M NaCl. The RF II also induced DNA synthesis but was neither as efficient as RF I nor depending on other serum fractions for growth promotion and it was not inhibited by anti-PDGF IgG. A similar strong affinity for Heparin-Sepharose was found for labeled basic fibroblast growth factor and we cannot exclude the possibility that RF II represent fibroblast growth factor. Under these culture conditions, inhibition of hASMC proliferation was directly correlated with the expression of smooth muscle specific alpha actin isoforms in stress fibers and the suppression of a proliferating cell-specific nuclear antigen. Conversely, stimulation of hASMC proliferation was associated with the opposite phenomenon. We conclude that heparin-like glycosaminoglycans influence growth and phenotype of hASMCs in vitro by binding and inactivating PDGF. Inasmuch as heparin-like substances constitute a significant proportion of the proteoglycan-associated glycosaminoglycans of the arterial wall, such mechanisms might be important for the development of atherosclerotic lesions.     

Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction

Wingless, the Drosophila homologue of the proto-oncogene Wnt-1, encodes a secreted glycoprotein that regulates differentiation and proliferation of nearby cells. Here we report on the biochemical mechanism(s) by which the wingless signal is transmitted from cell to cell. When expressed in S2 cells, the majority (approximately 83%) of secreted wingless protein (WG) is bound to the cell surface and extracellular matrix through specific, noncovalent interactions. The tethered WG can be released by addition of exogenous heparan sulfate and chondroitin sulfate glycosaminoglycans. WG also binds directly to heparin agarose beads with high affinity. These data suggest that WG can bind to the cell surface via naturally occurring sulfated proteoglycans. Two lines of evidence indicate that extracellular glycosaminoglycans on the receiving cells also play a functional role in WG signaling. First, treatment of WG-responsive cells with glycosaminoglycan lyases reduced WG activity by 50%. Second, when WG- responsive cells were preincubated with 1 mM chlorate, which blocks sulfation, WG activity was inhibited to near-basal levels. Addition of exogenous heparin to the chlorate-treated cells was able to restore WG activity. Based on these results, we propose that WG belongs to the group of growth factor ligands whose actions are mediated by extracellular proteoglycan molecules.     

Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines

Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.     

Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins

Vascular endothelial growth factor (VEGF) is a family of glycoproteins with potent angiogenic activity. We reported previously that heparin has an affinity for VEGF165, the major isoform of VEGF, whereas 2-O-desulfated heparin and 6-O-desulfated heparin have weak but significant affinity (Ashikari-Hada, S., Habuchi, H., Kariya, Y., Itoh, N., Reddi, A. H., and Kimata, K. (2004) J. Biol. Chem. 279, 12346-12354). In this study, we first examined the effect of heparin and modified heparins (completely desulfated N-sulfated heparin, 2-O-desulfated heparin, and 6-O-desulfated heparin) on VEGF165-dependent mitogenic activity and tube formation on type I collagen gels of human umbilical vein endothelial cells. Both were enhanced by heparin, but not by modified heparins, suggesting that both the 2-O-sulfate group of hexuronic acid and the 6-O-sulfation group of N-sulfoglucosamine in heparin/heparan sulfate are necessary for VEGF165 activity. We then examined the activation of VEGF receptor (VEGFR) to understand the mechanism. We have made several new findings; 1) heparin yielded a 1.7-fold enhancement of VEGF165-induced phosphorylation of VEGFR-2; 2) depletion of cell surface heparan sulfate by heparinase/heparitinase treatment and preferential reduction of trisulfated disaccharide units of cell surface HS by sodium chlorate treatment resulted in the reduction of such phosphorylation, suggesting the involvement of a heparin-like domain in the phosphorylation of VEGFR-2; and 3) VEGF121, an isoform without the exon 7-encoded region, which has no capacity to bind to heparin, did not show these effects. It is therefore likely that a heparin-like domain of heparan sulfate/heparin forms a complex with VEGF165 and VEGFR-2 via the exon 7-encoded region, thereby enhancing VEGF165-dependent signaling.     

Autocrine signaling through Ras prevents apoptosis in vascular smooth muscle cells in vitro

Vascular smooth muscle cell (SMC) apoptosis contributes to physiological and pathological vascular remodeling. Autocrine fibroblast growth factor (FGF) signaling promotes survival in SMC in vitro. Interruption of autocrine FGF signaling results in apoptosis that can be rescued by other growth factors such as PDGF (platelet-derived growth factor) or EGF (epidermal growth factor). Such heterologous growth factor rescue is prevented by pharmacological inhibition of MAPK, implicating signaling through Ras in mediating survival. This study was designed to test the hypothesis that signaling through Ras is both necessary and sufficient to mediate SMC survival in vitro. Recombinant adenoviruses encoding dominant-negative (Ras(N17)) and constitutively active (Ras(L61)) mutants of Ras were used. Ras(N17) blocks growth factor-mediated MAPK activation and can itself induce SMC apoptosis. Ras(N17) is synergistic with inhibition of autocrine FGF signaling in triggering apoptosis and prevents heterologous growth factor rescue. Conversely, Ras(L61) prevents apoptosis resulting from inhibition of autocrine FGF signaling. Rescue by Ras(L61) can be partially prevented by pharmacological inhibition of MEK or phosphatidylinositol 3-kinase, two downstream effectors of Ras. These results suggest that Ras signaling is both necessary and sufficient to mediate survival in SMC in vitro. Further work is required to determine how these signaling events are regulated in the context of vascular remodeling in vivo.     

Autocrine extracellular signal-regulated kinase (ERK) activation in normal human keratinocytes: metalloproteinase-mediated release of amphiregulin triggers signaling from ErbB1 to ERK

ErbB signaling through extracellular signal-regulated kinase (ERK) has been implicated in regulating the expression of ErbB ligands in hyperproliferative skin disorders and wound healing. Here, we characterize the process of autocrine ERK activation in cultured normal human keratinocytes (NHKs) subjected to growth factor (GF) deprivation. Basal ERK phosphorylation was lower after 48 h than after 24 h of GF deprivation, and lowest at 30-60 min after an additional medium change. ERK phosphorylation was markedly increased by low concentrations of epidermal growth factor (EGF) (0.2-1 ng/ml) that provoked only a limited increase in ErbB1 tyrosine phosphorylation and internalization. Basal ErbB tyrosine phosphorylation and ERK phosphorylation were inhibited by two different ErbB receptor tyrosine kinase inhibitors, by the ErbB1-specific neutralizing monoclonal antibody 225 IgG, by two different metalloproteinase inhibitors, and by neutralizing antibodies against amphiregulin (AR). In contrast, these responses were unaffected by neutralizing antibodies against other ErbB1 ligands or the ErbB2 inhibitors geldanamycin and AG825. The time course of autocrine ERK phosphorylation correlated with the appearance of soluble AR, and two different metalloproteinase inhibitors blocked AR release. These results define an amphiregulin- and ErbB1-dependent mechanism by which autocrine ERK activation is maintained in NHKs, even when ErbB1 autophosphorylation and internalization are limited.     

Multiple mechanisms are responsible for transactivation of the epidermal growth factor receptor in mammary epithelial cells

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.