Amphiregulin and hepatocyte-derived extracellular matrix regulate proliferation and autocrine growth factor expression in colon cancer cell lines of varying liver-colonizing capability

We studied the effect of two members of the epidermal growth factor (EGF) family--amphiregulin and heparin-binding EGF-like growth factor (HB-EGF)-on cell proliferation, growth factor and growth factor receptor expression, and cell differentiation in two human colon cell lines of varying liver-colonizing potential. The effect of amphiregulin and HB-EGF was assessed both in cells grown on plastic, as well as on cells grown on hepatocyte-derived extracellular matrix (ECM). We found that both colon cell lines were sensitive to HB-EGF stimulation of cell proliferation. Amphiregulin inhibited cell proliferation in KM12 cells and stimulated the strongly metastatic cell line KM12SM to a slight extent. When the cells were cultured on hepatocyte-derived ECM, amphiregulin inhibited the weakly metastatic KM12 and stimulated the growth of KM12SM. HB-EGF synergistically acted with hepatocyte-derived ECM to enhance cell proliferation in both colon cell lines. Expression of ligands of the EGF family, such as transforming growth factor-alpha (TGF-alpha) and amphiregulin, was decreased in both cell lines when cultured on ECM. Hepatocyte-derived ECM decreased expression of cripto in KM12 and increased it in KM12SM cells. Neither cripto nor TGF-alpha mRNA levels was affected by growing the cells in the presence of amphiregulin. However, amphiregulin increased expression of its own mRNA in the weakly metastatic KM12 and decreased it in the strongly metastatic KM12SM when the cells were cultured on plastic. Amphiregulin and HB-EGF stimulated expression of erb-B2 in both cell lines cultured on plastic. Surprisingly, when the cells were grown on hepatocyte-derived ECM, amphiregulin inhibited erb-B2 expression in both cell lines. We observed no effect of amphiregulin on cell differentiation as assessed by alkaline phosphatase expression. Our studies demonstrate one mechanism that could play a role in site-specific metastasis. We found an inhibitory response to an autocrine growth factor in the context of hepatocyte-derived ECM in a weakly metastatic cell and a stimulatory effect of the same growth factor when strongly metastatic cells were cultured on the same ECM.     

The heparin-binding domain of amphiregulin necessitates the precursor pro-region for growth factor secretion.

The five members of the human epidermal growth factor (EGF) family (EGF, transforming growth factor alpha [TGF-alpha], heparin-binding EGF-like growth factor [HB-EGF], betacellulin, and amphiregulin [AR]) are synthesized as transmembrane proteins whose extracellular domains are proteolytically processed to release the biologically active mature growth factors. These factors all activate the EGF receptor, but in contrast to EGF and TGF-alpha, the mature forms of HB-EGF and AR are also glycosylated, heparin-binding proteins. We have constructed a series of mutants to examine the influence of the distinct precursor domains in the biosynthesis of AR. The transmembrane and cytoplasmic domains of the precursor are not required for secretion of bioactive AR from either COS or mammary epithelium-derived cells, although proteolytic removal of the N-terminal pro-region is less efficient in the absence of the membrane anchor. Deletion of the N-terminal pro-region, however, results in rapid intracellular degradation of the molecule with no detectable secretion of active growth factor. AR secretion is preserved by replacing the native pro-region with the corresponding domain of the HB-EGF precursor but not with that of the TGF-alpha precursor. In the absence of any N-terminal pro-region, secretion of the molecule is restored by deleting the N-terminal heparin-binding domain of mature AR. Both EGF and TGF-alpha, in contrast, can be secreted without their pro-regions. However, if the protein is fused with the AR heparin-binding domain, TGF-alpha secretion is inhibited unless the AR pro-region is also present. We propose that the heparin-binding domain of mature AR necessitates the presence of a specific structural motif in an N-terminal pro-region to permit proper folding, and thus secretion, of a bioactive molecule.     

Epiregulin is Up-regulated in pancreatic cancer and stimulates pancreatic cancer cell growth

Epiregulin belongs to the epidermal growth factor (EGF) family of polypeptides. Previous studies have underscored the important role of the EGF family of ligands and receptors in the pathology of pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP). It is not known, however, whether epiregulin may also have a role in these diseases. Therefore, in the present study we investigated the expression and function of epiregulin in five pancreatic cancer cell lines and in PDAC and CP tissue samples. Epiregulin mRNA was present at high (MIA-PaCa-2 cells) or moderate levels (ASPC-1, CAPAN-1, and T3M4) in most cells, but was below detection levels in PANC-1 cells. All the cell lines exhibited a dose-dependent increase in growth in response to recombinant human epiregulin. Epiregulin mRNA levels were increased 2.1-fold in PDAC samples (P < 0.01) and 1.7-fold in CP samples (P < 0.01), when compared with the normal controls. There was no correlation between epiregulin mRNA levels and tumor stage or grade. By in situ hybridization, a moderate to intense epiregulin mRNA signal was present in most pancreatic cancer cells in PDAC. In contrast, only a weak (normal pancreas) to moderate (CP) signals were present in the ductal and acinar cells in CP. These findings suggest that epiregulin may contribute to the pathobiology of PDAC, and may also have a role in CP.     

HaCaT keratinocyte migration is dependent on epidermal growth factor receptor signaling and glycogen synthase kinase-3alpha

After epithelial disruption by tissue injury, keratinocytes migrate from the wound edge into a provisional matrix. This process is stimulated by growth factors that signal through epidermal growth factor (EGF) receptor, including EGF, heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha), and by for example keratinocyte growth factor (KGF) and TGF-beta1 that function through different receptors. We have previously shown that keratinocyte migration induced by EGF or staurosporine is dependent on the activity of glycogen synthase kinase-3 (GSK-3). In the present study, we show that keratinocyte migration induced by TGF-beta1, KGF, EGF, TGF-alpha and staurosporine depends on EGFR signaling, involves autocrine HB-EGF expression and is potently blocked by GSK-3 inhibitors SB-415286 and LiCl. Inhibition of GSK-3 also retards wound reepithelialization in vivo in mice. Moreover, inhibition of GSK-3 activity prevented cell rounding that is an early event in EGFR-mediated keratinocyte migration. Isoform-specific GSK-3alpha and GSK-3beta knockdown and overexpression experiments with siRNAs and adenoviral constructs, respectively, revealed that GSK-3alpha is required for keratinocyte migration, whereas excessive activity of GSK-3beta is inhibitory. Thus, induction of keratinocyte migration is conveyed through EGFR, promoted by endogenous HB-EGF and requires GSK-3alpha activity.     

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.     

Structure of heparin-binding EGF-like growth factor. Multiple forms, primary structure, and glycosylation of the mature protein.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a newly described member of the epidermal growth factor (EGF) family that is mitogenic for BALB/c 3T3 cells, inhibits the binding of 125I-EGF to its receptor, and triggers autophosphorylation of the EGF receptor. HB-EGF was purified from the conditioned medium of U-937 cells using cation exchange, copper affinity, heparin affinity, and two rounds of C4 reversed phase liquid chromatography. The elution profile of the first round of C4 column chromatography contained four growth factor activity peaks with similar specific biological activities. N-terminal and tryptic fragment microsequencing demonstrated that these peaks contained different structural forms of the HB-EGF protein. Some of the differences in the various forms of HB-EGF were found to be due to N-terminal heterogeneity. Microsequencing of tryptic fragments indicated that the mature HB-EGF polypeptide can contain at least 86 of the 208 amino acids predicted by nucleotide sequence to be the HB-EGF precursor molecule. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the various forms of HB-EGF have apparent molecular masses of 19-23 kDa. Further analysis of the most predominant form of HB-EGF found in U-937 cell conditioned medium indicated that it has a pI of 7.2-7.8 and is O-glycosylated.     

Herstatin, an autoinhibitor of the human epidermal growth factor receptor 2 tyrosine kinase, modulates epidermal growth factor signaling pathways resulting in growth arrest

Herstatin is an autoinhibitor of the ErbB family consisting of subdomains I and II of the human epidermal growth factor receptor 2 (ErbB-2) extracellular domain and a novel C-terminal domain encoded by an intron. Herstatin binds to human epidermal growth factor receptor 2 and to the epidermal growth factor receptor (EGFR), blocking receptor oligomerization and tyrosine phosphorylation. In this study, we characterized several early steps in EGFR activation and investigated downstream signaling events induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-alpha) in NIH3T3 cell lines expressing EGFR with and without herstatin. Herstatin expression decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation despite receptor occupancy by ligand with normal binding affinity. Akt stimulation by EGF and TGF-alpha, but not by fibroblast growth factor 2, was almost completely blocked in the presence of herstatin. Surprisingly, EGF and TGF-alpha induced full activation of MAPK in duration and intensity and stimulated association of the EGFR with Shc and Grb2. Although MAPK was fully stimulated, herstatin expression prevented TGF-alpha-induced DNA synthesis and EGF-induced proliferation. The herstatin-mediated uncoupling of MAPK from Akt activation was also observed in Chinese hamster ovary cells co-transfected with EGFR and herstatin. These findings show that herstatin expression alters EGF and TGF-alpha signaling profiles, culminating in inhibition of proliferation.     

Epidermal growth factor and transforming growth factor-alpha decrease gamma interferon receptors and induction of intercellular adhesion molecule (ICAM-1) on cultured keratinocytes.

The link between the epidermal keratinocytes of the skin and the activated T lymphocytes of the immune system is mediated by a variety of cytokines, including gamma interferon (IFN-gamma). We studied the influence of keratinocyte mitogens such as transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and somatomedin-C (SM-C) on the ligand binding of 32P-labeled IFN-gamma to cultured keratinocytes derived from normal appearing adult human skin. Keratinocytes placed in a medium devoid of mitogens become growth arrested, and these quiescent cells expressed 2.4 times (28,900 versus 12,200 sites/cell) as many high affinity IFN-gamma receptors (Kd = 0.22 nM) compared to keratinocytes which were actively growing in medium containing TGF-alpha (25 ng/ml) or EGF (10 ng/ml). The reduction in IFN-gamma receptor sites by TGF-alpha/EGF was mitogen specific, as adding SM-C (500 ng/ml) did not have any effect on ligand binding, although it similarly stimulated keratinocyte growth. The reduction in IFN-gamma receptors was time dependent, occurring primarily after 24-48 hours of change in tissue culture conditions. The reduction in the number of high affinity IFN-gamma receptors by TGF-alpha/EGF had immunobiological consequences, because quiescent keratinocytes in basal medium had an increased expression of HLA-DR and intercellular adhesion molecule-1 (ICAM-1) induced by IFN-gamma, compared to actively growing TGF-alpha/EGF treated keratinocytes. These results suggest that rapidly proliferating keratinocytes exposed to TGF-alpha/EGF but not SM-C are capable of altering their response to IFN-gamma by decreasing their number of cell surface high affinity receptors for IFN-gamma.     

Induction of human microvascular endothelial tubular morphogenesis by human keratinocytes: involvement of transforming growth factor-alpha.

Transforming growth factor-alpha(TGF-alpha), homologous to epidermal growth factor(EGF), is closely involved in hyperproliferation of human keratinocytes. Psoriasis is a common hyperproliferative skin disease characterized by hyperproliferation of keratinocytes and abnormal development of dermal capillary networks. In this study, we have examined whether keratinocytes could enhance angiogenesis. TGF-alpha or EGF efficiently stimulated formation of tubular-like structures of human omental microvascular endothelial(HOME) cells in type I collagen gels. Human keratinocytes produced TGF-alpha. To examine whether co-cultured keratinocytes could induce tubulogenesis of HOME cells in collagen gel, we have developed a co-culture system with human keratinocytes. Surprisingly, there appeared new development of many tubular-like structures of HOME cells in collagen gels when co-cultured with keratinocytes. This keratinocytes-dependent tubulogenesis was almost completely blocked when anti-TGF-alpha-antibody was present. The TGF-alpha molecules derived from keratinocytes appeared to enhance tubulogenesis of human microvascular endothelial cells. We propose the hypothesis that secretory TGF-alpha from human keratinocytes may promote an autocrine loop to proliferate the skin keratinocytes and also a paracrine loop to induce the skin angiogenesis.     

Amphiregulin-dependent proliferation of cultured human keratinocytes: Autocrine growth,the effects of exogenous recombinant cytokine,and apparent requirement for heparin-like glycosaminoglycans

Amphiregulin, a member of the epidermal growth factor family with heparin binding affinity, functions as a natural regulator of keratinocyte growth. Autocrine signaling by amphiregulin and the effects of exogenous recombinant cytokine were studied in serum-free cultures of human neonatal keratinocytes. A metabolic inhibitor of proteoglycan sulfation was used to assess the role of cellular heparin-like glycosaminoglycans in amphiregulin-dependent growth. Keratinocytes plated at >10³ cells/cm² grew in an autocrine manner in the absence of exogenous epidermal growth factor or amphiregulin. Incubation of keratinocytes with an amphiregulin-blocking antibody indicated that ～70% of autocrine growth is mediated by endogenous amphiregulin. Proliferation potential in the presence of recombinant human amphiregulin was dose dependent and saturable and above ～1 ng/ml was comparable to that achieved with similar concentrations of epidermal growth factor. Sodium chlorate, which blocks glycosaminoglycan sulfation, reversibly inhibited epidermal growth factor-dependent proliferation by 42%, exogenous amphiregulin-dependent proliferation by 75%, and autocrine growth by 95%; concurrent incubation with 1-100 μg/ml heparin partially reversed this inhibition. Exogenous heparin in the absence of chlorate, however, nearly completely inhibited growth under autocrine conditions and in the presence of recombinant amphiregulin. Structure-function studies indicate that the polymerization level, high sulfate group density, and possibly iduronic acid content of heparin-like moieties correlate with their inhibitory activity. Collectively, these observations indicate that amphiregulin is the major autocrine factor for keratinocytes and demonstrate that exogenous amphiregulin is an effective growth promoting factor with molar potency similar to that of epidermal growth factor. Autocrine and paracrine signaling by amphiregulin may require cellular heparin-like glycosaminoglycans, presumably as matrix or membrane proteoglycans, whereas soluble glycosaminoglycans inhibit signaling, possibly by competing for cytokine binding. © 1994 wiley-Liss, Inc.     

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.     

Epiregulin is not essential for development of intestinal tumors but is required for protection from intestinal damage

Epiregulin, an epidermal growth factor family member, acts as a local signal mediator and shows dual biological activity, stimulating the proliferation of fibroblasts, hepatocytes, smooth muscle cells, and keratinocytes while inhibiting the growth of several tumor-derived epithelial cell lines. The epiregulin gene (Ereg) is located on mouse chromosome 5 adjacent to three other epidermal growth factor family members, epigen, amphiregulin, and betacellulin. Gene targeting was used to insert a lacZ reporter into the mouse Ereg locus and to ablate its function. Although epiregulin is broadly expressed and regulated both spatially and temporally, Ereg null mice show no overt developmental defects, reproductive abnormalities, or altered liver regeneration. Additionally, in contrast to previous hypotheses, Ereg deficiency does not alter intestinal cancer susceptibility, as assayed in the ApcMin model, despite showing robust expression in developing tumors. However, Ereg null mice are highly susceptible to cancer-predisposing intestinal damage caused by oral administration of dextran sulfate sodium.     

Regulation of TGF-alpha expression in human keratinocytes: PKC-dependent and -independent pathways.

Transforming growth factor-alpha (TGF-alpha) is an autocrine growth factor for epidermal keratinocytes that can induce its own expression (autoinduction). Because the regulation of this process may be important for the control of epidermal growth, we examined the roles of EGF receptor tyrosine kinase and protein kinase C (PKC) in TGF-alpha autoinduction in cultured human keratinocytes. Antiphosphotyrosine immunoblot analysis demonstrated that EGF and TGF-alpha rapidly and markedly stimulated tyrosine phosphorylation of a 170 kDa protein in growth factor-deprived keratinocytes. This protein was identified as the EGF receptor by immuno-precipitation using anti-EGF receptor mAbs. Tyrosine phosphorylation and TGF-alpha mRNA accumulation in response to EGF and TGF-alpha were both inhibited by a monoclonal antibody against the EGF receptor and by the EGF receptor tyrosine kinase inhibitor RG50864, demonstrating the involvement of the tyrosine kinase activity of the receptor in TGF-alpha autoinduction. The monoclonal antibody inhibited keratinocyte growth and TGF-alpha autoinduction with similar potency (IC50 approximately 0.1 microgram/ml). TGF-alpha and the PKC activator tetradecanoyl phorbol 12-myristyl, 13-acetate (TPA) had similar effects on TGF-alpha steady-state mRNA levels, suggesting that PKC activation might be a downstream mediator of TGF-alpha autoinduction. However, down-regulation of more than 90% of keratinocyte PKC activity by bryostatin pretreatment abrogated the induction of TGF-alpha mRNA in response to TPA without affecting the autoinductive response or EGF-stimulated tyrosine phosphorylation. These results indicate that EGF receptor and PKC stimulate TGF-alpha gene expression by different pathways, and suggest that PKC is not required for TGF-alpha autoinduction in this system. Moreover, the fact that EGF-stimulated tyrosine phosphorylation and TGF-alpha autoinduction were not potentiated after PKC down-regulation suggests that PKC does not exert a tonic inhibitory influence on EGF receptor tyrosine kinase activity in normal human keratinocytes.     

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.     

Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability

We previously implicated tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming growth factor-alpha (TGF-alpha), pro-TGF-alpha. Here we examined TGF-alpha processing in a physiologically relevant cell model, primary keratinocytes, showing that cells lacking TACE activity shed dramatically less TGF-alpha as compared with wild-type cultures and that TGF-alpha cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-alpha and TACE cDNAs increased shedding of mature TGF-alpha with concomitant conversion of cell-associated pro-TGF-alpha to a processed form. Purified TACE accurately cleaved pro-TGF-alpha in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-alpha containing only the ectodomain at the C-terminal site. In vitro, TACE accurately cleaved peptides corresponding to cleavage sites of several epidermal growth factor (EGF) family members, and transfection of TACE into TACE-deficient cells increased the shedding of amphiregulin and heparin-binding EGF (HB-EGF) proteins. Consistent with the hypothesis that TACE regulates EGF receptor (EGFR) ligand availability in vivo, mice heterozygous for Tace and homozygous for an impaired EGFR allele (wa-2) were born with open eyes significantly more often than Tace(+/+)Egfr(wa-2)(/)(wa-2) counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.     

Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation.     

Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells.

Normal human mammary epithelial cells (HMECs) proliferate in a serum-free defined growth medium in the absence of epidermal growth factor (Li and Shipley, 1991). Amphiregulin (AR) is a heparin-regulated, EGF-like growth factor. Our observation that one strain of HMECs produce AR mRNA (Cook et al., 1991 a) stimulated us to determine whether AR expression was a common phenomenon in HMECs and whether AR could act as an autocrine growth factor to support the EGF-independent growth of these cells. In this study, we detected high levels of AR expression in four separate HMEC strains while one immortal mammary cell line (HBL-100) and six mammary tumor-derived cell lines had low to undetectable levels of AR. The EGF-independent growth of HMECs was blocked by the addition of heparin or a monoclonal anti-EGF receptor antibody to the culture medium, implicating AR as an autocrine growth mediator. This hypothesis is further supported by the fact that medium conditioned by HMECs contains secreted AR protein. A mammary tumor-derived cell line, Hs578T, which proliferates in an EGF-independent manner, does not express detectable levels of AR and is not growth inhibited by heparin. Examination of the same cell types for expression of transforming growth factor type-alpha (TGF-alpha) mRNA revealed coordinate expression of AR and TGF-alpha in these cells. These data suggest that both AR and TGF-alpha mRNA are produced in much greater abundance by normal HMECs than in tumor-derived cells in culture, and that AR is an important autostimulatory factor for the growth of normal HMECs.