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While previous studies have indicated that exogenous TGF-alpha stimulates epithelial growth, maintenance, and repair of the gut, roles of endogenous TGF-alpha are less well-defined particularly in the small bowel. The current study examined effects of TGF-alpha knockout on adult small intestinal epithelial cell proliferation, migration, apoptosis, and damage/repair response after methotrexate treatment. Compared to normal mice, TGF-alpha gene knockout did not affect crypt cell production, mitosis position, migration, and apoptosis in non-injured intestine. RT-PCR gene expression analysis revealed presence of four out of six TGF-alpha related EGF family ligands in the normal intestine, suggesting a possible functional redundancy of the EGF family in maintenance of the intestine. Although TGF-alpha gene knockout did not significantly impair the overall mucosal repair in methotrexate-induced acute damage in the small intestine, it resulted in a higher apoptotic response in the early hours following methotrexate challenge, and a delayed and reduced crypt cell proliferation during repair. Consistently, after methotrexate challenge, intestinal TGF-alpha mRNA was found to be markedly upregulated in the early hours and during repair in the wild type, and there were similar profiles in the increased expression of all other ligands (except EGF) between the wild type and knockout intestines. Therefore, despite a possible functional redundancy among the EGF family ligands in the normal small intestine, TGF-alpha may play a role in modulating the early apoptotic events and in enhancing the subsequent reparative proliferative response in the methotrexate-damaged intestine., (Copyright 2002 Wiley-Liss, Inc.)

TGFalpha is hypothesized to be an endogenous regulator of lens fiber terminal differentiation. With immunofluorescence, TGFalpha was localized to differentiating cells in the lens epithelium and superficial fiber cell mass of the adult chicken. A similar pattern of localization was also noted when differentiating epithelial cells were cultured. Immunoneutralization of endogenous TGFalpha inhibited the accumulation of filensin, a unique intermediate filament protein subunit restricted to developing vertebrate lens fibers. ELISA assays quantified the effects of TGFalpha on filensin expression. Surprisingly, inhibition of the TGFalpha receptors' tyrosine kinase activity with nanomolar concentrations of PD153035 increased the accumulation of differentiated characteristics in the presence or absence of ligand. Morphologically, PD153035-treated cells grew as aggregated masses and spread less well onto the substrate. Accompanying these morphologic changes was a complete inhibition of cell division. Post-receptor signaling events were examined with cAMP assays and Western blotting. TGFalpha did not affect cAMP levels while isoproterenol, an additional mediator of lens cell differentiation, caused significant increases in cAMP levels. Activation of ERK2 via dual phosphorylation was noted in response to TGFalpha but not isoproterenol. PD153035 reduced, but did not eliminate, ERK2 phosphorylation in response to TGFalpha. Phosphorylation of the CREB transcription factor was also observed in response to TGFalpha or isoproterenol. These data indicate that endogenous ligands can influence the expression of differentiated characteristics in cultured chick lens cells. A focus of multiple signaling pathways affecting filensin expression is the CREB transcription factor. While increased ERK2 activation may be involved in stimulating cell division, lower levels of persistent ERK2 activation promotes differentiation thus indicating some form of signaling pathway compartmentalization. This could also mean that additional receptors for TGFalpha not inhibited by PD153035 are responsible for promoting differentiation in chick lens cells., (Copyright 2001 Wiley-Liss, Inc.)

Previously, we reported that unaggressive, growth factor-dependent FET human colon carcinoma cells downregulated their transforming growth factor alpha (TGFalpha) expression in a quiescent state (G0/G1) induced by growth factor and nutrient deprivation (Mulder, 1991, Cancer Res., 51:2256-2262). In contrast, highly aggressive, growth factor-independent HCT116 human colon carcinoma cells aberrantly upregulated this autocrine activity in the quiescent state (Mulder, 1991, Cancer Res., 51:2256-2262; Howell et al., 1998, Mol. Cell. Biol., 18:303-313). In this report, the role of autocrine TGFalpha and the mechanism of its regulation of expression during reentry into the cell cycle from a noncycling growth state were determined in FET cells. Optimal induction of DNA synthesis from a quiescent state in FET cells is dependent upon autocrine TGFalpha as well as exogenous transferrin and insulin. Reentry into the cell cycle resulting from treatment with exogenous transferrin and insulin resulted in approximately 3-fold induction of TGFalpha expression within 1 hr. TGFalpha induction was controlled at the transcription level, and the cis-controlling element was localized to the region between bp -370 - -201 relative to the translation start codon within the TGFalpha promoter. Thus neutralization of autocrine TGFalpha protein revealed that the induced TGFalpha autocrine activity was necessary for DNA synthesis and acted only in the early G1 phase of the cell cycle. Blockade of autocrine TGFalpha expression early in the cell cycle resulted in the reduction of DNA synthesis, whereas treatment with neutralization antibody at later times had no effect. This suggested that autocrine TGFalpha functions to initiate cell growth from noncycling states. This was further confirmed by the dependence of FET cells upon autocrine TGFalpha for colony formation in experiments where the plating density was sufficiently low to generate a lag phase in tissue culture. In contrast, TGFalpha autocrine activity was not required for exponential phase cells, as evidenced by the failure of TGFalpha neutralizing antibody to inhibit proliferation in this growth state. Taken together, these results suggest that autocrine TGFalpha acts primarily in the process of growth initiation by moving cells from a noncycling state back into the cell cycle, rather than supporting cell growth already initiated.

Gastrin is transcriptionally responsive to EGF stimulation (Merchant et al., 1991, Mol. Cell. Biol., 11:2686-2696). Consequently, we hypothesized that previously recognized gastrin autocrine loops (Hoosein et al., 1990, Exp. Cell. Res., 186:15-21), might be controlled by autocrine TGF alpha in human colon carcinoma cells. Therefore, we examined the interaction between these two autocrine growth factors in two colon carcinoma cell lines which utilize TGF alpha. The FET cell line requires exogenous TGF alpha/EGF for optimal growth and has a classical TGF alpha autocrine loop which is disrupted by TGF alpha or epidermal growth factor receptor (EGFr) antibodies. The HCT 116 cell line is not dependent on exogenous TGF alpha/EGF and exhibits a nonclassical TGF alpha autocrine loop which is not disrupted by neutralizing antibodies to either TGF alpha itself or the EGFr. Basal gastrin mRNA production is significantly higher in HCT 116 than FET as measured by RNase protection assay. In the FET cells, exogenous EGF stimulates gastrin mRNA production but not in HCT 116. When the TGF alpha autocrine loop in HCT 116 is disrupted by constitutive expression of antisense TGF alpha mRNA, the gastrin mRNA level is significantly repressed. In xenografts derived from these antisense clones, TGF alpha reverted to high expression, and the gastrin mRNA level was again increased. This interaction between the strong TGF alpha loop in HCT 116 and the gastrin autocrine loop may confer a growth advantage to these colon cells. Such interactions between growth factors may promote enhanced tumorigenicity to transformed cells with these strong, nonclassical autocrine loops.

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.

We have examined dependence of primary rat tracheal epithelial (RTE) on exogenous epidermal growth factor (EGF) and determined whether a TGF alpha autocrine pathway is operating in these cells. Primary RTE cells plated in serum free media (SFM) without EGF and bovine pituitary factor (BPE) show little proliferation compared to cultures propagated in media containing EGF/BPE (CSFM). Removal of EGF/BPE shortly after plating, however, results in significant proliferation, although plateau cell densities are reduced and cell morphology is significantly altered compared to cells propagated in CSFM. Addition of EGF and/or BPE to cultures propagated in SFM minus EGF/BPE restores maximum cell density. The concentration of TGF alpha peptide in media conditioned by cells propagated without EGF/BPE is lower than the concentration in the media of CSFM cultures. TGF alpha mRNA and protein levels are also significantly lower in cells late in culture compared to logarithmically growing cells regardless of the presence or absence of EGF/BPE. The proliferation of primary RTE cells propagated without EGF/BPE is inhibited by neutralizing TGF alpha antiserum and by a tyrphostin compound that blocks TGF alpha/EGF receptor tyrosine kinase activity. These results indicate that primary RTE cells utilize TGF alpha as an autocrine growth factor and that the autocrine pathway is regulated as a function of growth state of the cells. However, this pathway does not provide growth autonomy to primary RTE cells, since cultures remain dependent on exogenous EGF/BPE for sustained proliferation.

We have shown in the present study and in studies reported previously that preneoplastic and neoplastic rat tracheal epithelial (RTE) cell lines express TGF alpha and do so regardless of the mechanism by which they were transformed. In order to determine whether TGF alpha is an autocrine growth regulator of immortalized RTE cells, we have examined the function of TGF alpha/EGF receptors and the growth requirements for TGF alpha in these cells. The level of immunoprecipitated TGF alpha/EGF receptor protein in immortalized RTE cells was similar to or less than levels in primary RTE cells, indicating that chemically induced transformation of RTE cells does not involve overexpression of TGF alpha/EGF receptors. Scatchard analysis of TGF alpha/EGF receptors in the neoplastic EGV5T cell line revealed the presence of high-affinity (Kd = 0.4 nM) and low-affinity (Kd = 9.8 nM) binding sites. A tyrphostin TGF alpha/EGF receptor tyrosine kinase inhibitor decreased in a dose-dependent manner the proliferation as well as EGF-induced autophosphorylation of the TGF alpha/EGF receptor of transformed RTE cells. The inhibitory effect of tyrphostin on proliferation and receptor kinase activity was attenuated in late log and plateau phase cultures. The phosphotyrosine content of several other EGF-dependent and independent phosphoproteins was also decreased by the tyrphostin. Proliferation of transformed RTE cells was also inhibited when TGF alpha antisera was added to the media of growing cells. These data are consistent with the hypothesis that proliferation of transformed RTE cells involves autocrine regulation by TGF alpha and its receptor.

Human cellular polypeptide factors, namely interferon-alpha, interferon-gamma transforming growth factor (TGF)-alpha, and TGF-beta 1, were analyzed for their effect on motility of human sperm cells. Both interferons caused an inhibition of sperm cell motility due to direct cytotoxic effects without inducing 2'-5' oligoadenylate [2-5(A)]synthetase activity. TGF-alpha affected neither motility nor the levels of 2-5(A) synthetase in sperm cells. TGF-beta 1 had no affect on sperm motility, yet it caused an induction of 2-5(A)synthetase activity. Western immunoblot analysis of TGF-beta 1-treated sperm indicated an enhancement of a 50 kDa protein. Metabolic labeling of sperm cells revealed biosynthesis of one major protein of 50 kDa and at least five minor proteins in the range of 30-92 kDa; the level of 50 kDa protein increased after treatment with TGF-beta 1. The treatment of sperm cells with TGF-beta 1 did not affect their penetration in zona-free hamster eggs (SPA). These results indicate that TGF-beta 1 enhances expression of a 50 kDa protein related to 2-5(A) synthetase in human sperm cells along with other minor proteins, and this increase does not affect sperm motility and SPA.

The proto-oncogene c-myc and the oncogene SV40T, both of which have been implicated in the process of cellular immortalization in vitro, have been introduced via amphotropic retroviral expression vectors into the human mammary epithelial cell (HMEC) line 184A1N4 (A1N4). Two stable cell lines were established by growth in selective medium and were found to overexpress either c-myc (A1N4-myc) or SV40T antigen (A1N4-T). Neither the A1N4, A1N4-myc, or A1N4-T cells will grow in soft agar or form tumors in nude mice. However, A1N4-T or A1N4-myc cells, but not the parental A1N4 cells, form colonies in soft agar in response to either epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), or basic fibroblast growth factor (bFGF). Like EGF and TGF alpha, bFGF is moderately mitogenic for the anchorage-dependent growth (ADG) of all three cell lines. Further, co-cultivation of A1N4-T or A1N4-myc cells with primary diploid mammary fibroblasts can also induce the anchorage-independent growth (AIG) and stimulate the ADG of A1N4-T or A1N4-myc. In addition, conditioned medium obtained from these mammary fibroblasts also stimulated the AIG of the A1N4-T and A1N4-myc cells and was found to contain immunoreactive TGF alpha and bioactive FGF. The mammary fibroblasts express specific mRNA transcripts for bFGF and acidic FGF (aFGF). These results suggest that growth factors such as TFG alpha or FGF, which may be derived from the adjacent mammary stroma, might influence in a paracrine manner the phenotypic characteristics of a population of human mammary epithelial cells toward transformation.

In this study we investigated the correlation between the mitogenic effect and stimulation of Rb+ (K+) fluxes in human skin fibroblasts treated by purified growth factors. Both K+ transporters, bumetanide-sensitive and ouabain-sensitive, are stimulated 2-3-fold after addition of either fetal calf serum or purified recombinant growth factors to quiescent G0/G1 human skin fibroblasts. Three groups of mitogens were compared: i) the phorbol ester 2-O-tetradecanoyl-phorbol-13-acetate (TPA); ii) growth factors that stimulate inositol phosphate hydrolysis and subsequently activate protein kinase C--fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and alpha-thrombin; and iii) growth factors that do not activate kinase C--insulin-like growth factor-1 (IGF-1), and transforming like growth-factor-alpha (TGF-alpha). The three groups of mitogens stimulated human skin fibroblasts proliferation and Rb+ influxes in a similar dose-dependent fashion. The results indicate that both the bumetanide-sensitive and the ouabain-sensitive Rb+ fluxes are stimulated by protein kinase C-dependent and by the protein kinase C-independent pathways of the mitogenic signal.