Involvement of transforming growth factor alpha in the release of luteinizing hormone-releasing hormone from the developing female hypothalamus.

Little is known about the presence of trophic factors in the hypothalamus and the role they may play in regulating the functional development of hypothalamic neurons. We have investigated the ability of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) to affect the release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide that controls reproductive development. We have also determined whether the genes encoding EGF and TGF-alpha are expressed in the prepubertal female hypothalamus. Northern blot analysis of poly(A)+ RNA utilizing a single-stranded EGF cDNA probe failed to reveal the presence of EGF mRNA in either the hypothalamus or the cerebral cortex at any age studied (fetal day 18 to postnatal day 36). In contrast, both a complementary RNA probe and a double-stranded TGF-alpha cDNA recognized in these regions a 4.5-kilobase (kb) mRNA species identical to TGF-alpha mRNA. The abundance of TGF-alpha mRNA was 3-4 times greater in the hypothalamus than in the cerebral cortex. Both EGF and TGF-alpha (2-100 ng/ml) elicited a dose-related increase in LHRH release from the median eminence of juvenile rats in vitro. They also enhanced prostaglandin E2 (PGE2) release. The transforming growth factors TGF-beta 1 and -beta 2 were ineffective. Only a high dose of basic fibroblast growth factor was able to increase LHRH and PGE2 release. Blockade of the EGF receptor transduction mechanism with RG 50864, a selective inhibitor of EGF receptor tyrosine kinase activity, prevented the effect of both EGF and TGF-alpha on LHRH and PGE2 release but failed to inhibit the stimulatory effect of PGE2 on LHRH release. Inhibition of prostaglandin synthesis abolished the effect of TGF-alpha on LHRH, indicating that PGE2 mediates TGF-alpha-induced LHRH release. The results indicate that the effect of EGF and TGF-alpha on LHRH release is mediated by the EGF/TGF-alpha receptor and suggest that TGF-alpha rather than EGF may be the physiological ligand for this interaction. Since in the central nervous system most EGF/TGF-alpha receptors are located on glial cells, the results also raise the possibility that--at the median eminence--TGF-alpha action may involve a glial-neuronal interaction, a mechanism by which the trophic factor first stimulates PGE2 release from glial cells, and then PGE2 elicits LHRH from the neuronal terminals.     

Sarcoma growth factor from conditioned medium of virally transformed cells is composed of both type alpha and type beta transforming growth factors.

Sarcoma growth factor (SGF) derived from conditioned medium of Moloney sarcoma virus-transformed cells and partially purified by gel filtration (crude SGF) has been characterized by its ability both to compete with epidermal growth factor (EGF) for binding to membrane receptors and to induce anchorage-independent growth of untransformed cells. We now show that further purification of crude SGF by reverse-phase HPLC on muBondapak C18 and CN columns at pH 2 resolves it into two distinctly different polypeptides, which we call types alpha and beta transforming growth factors (TGFs). Type alpha TGF (TGF-alpha), but not type beta TGF (TGF-beta), competes for binding to the EGF receptor and induces the formation of small colonies (1,000-2,000 micron2) of normal rat kidney cells in soft agar. Both TGF-beta and EGF or TGF-alpha must be present in order to induce the formation of large colonies (7,000-15,000 micron2). Based on EGF competing equivalents as determined from a radioreceptor assay with 125I-labeled EGF in normal rat kidney cells, the relative ability of EGF and TGF-alpha to potentiate TGF-beta-dependent colony formation is in the order conditioned-medium TGF-alpha greater than EGF greater than intracellular TGF-alpha. Suboptimal concentrations of the same polypeptides give additive potentiation of the TGF-beta-dependent colony-forming response; saturating levels potentiate a similar maximum response whether used alone or in various combinations. The data indicate that the EGF-competing activity of crude SGF is due to its TGF-alpha component alone, whereas the soft-agar colony-forming activity is due to the combined action of two distinct polypeptides, TGF-alpha and TGF-beta.     

Potential role of the epidermal growth factor receptor in human pancreatic cancer

The epidermal growth factor (EGF) receptor is a transmembrane protein that has tyrosine kinase activity. It is activated by both EGF and transforming growth factor-alpha (TGF-alpha). Human pancreatic cancer cells overexpress the EGF receptor and exhibit a parallel increase in EGF receptor mRNA without a detectable increase in the number of gene copies coding for the receptor. These cells also produce TGF-alpha and are capable of binding exogenous TGF-alpha. They often recycle EGF, but markedly and rapidly degrade TGF-alpha. However, TGF-alpha is 10-100-fold more potent than EGF in enhancing their anchorage-independent growth. Both growth factors induce EGF receptor down-regulation, but EGF is more efficient than TGF-alpha in this regard. The concomitant overexpression of the EGF receptor and production of TGF-alpha, the recycling of EGF, and the attenuated ability of TGF-alpha to down-regulate the EGF receptor may combine to provide a distinct growth advantage to human pancreatic cancer cells.     

Transforming growth factor-alpha stimulates proliferation of mammotrophs and corticotrophs in the mouse pituitary

Oestrogen stimulates the proliferation of pituitary cells. The present study was designed to clarify the involvement of transforming growth factor-alpha (TGF-alpha) in the oestrogen-induced growth of mouse pituitary cells in vitro. Anterior pituitary cells obtained from ICR male mice were cultured in a primary serum-free culture system. Proliferation of pituitary cells was detected by monitoring the cellular uptake of bromodeoxyuridine. Secretory cell types were immunocytochemically determined. Treatment with TGF-alpha (0.1 and 1 ng/ml) for 5 days stimulated cell proliferation. Since TGF-alpha binds to the epidermal growth factor (EGF) receptor, this action may be exerted through the EGF receptor. Oestradiol-17beta (OE(2), 10(-)(9) M) stimulated mammotrophic and corticotrophic cell proliferation. RG-13022, an EGF receptor inhibitor, inhibited the cell proliferation induced by EGF or OE(2), showing that the EGF receptor was involved in the growth response in mammotrophs and corticotrophs. Treatment with antisense TGF-alpha oligodeoxynucleotide (ODN) inhibited the cell proliferation induced by OE(2), but treatment with antisense EGF ODN did not. RT-PCR analysis revealed that OE(2) stimulated TGF-alpha mRNA and EGF receptor mRNA expression. These results indicate that TGF-alpha mediates the stimulatory effect of oestrogen on the pituitary cell proliferation in a paracrine or autocrine manner, and that EGF receptor expression is stimulated by oestrogen.     

Morphologic and mitogenic responses of rabbit synovial fibroblasts to transforming growth factor beta require transforming growth factor alpha or epidermal growth factor

We tested the hypothesis that normal synovial fibroblasts might proliferate in response to transforming growth factors (TGFs), peptides that are extracted with acid-ethanol from bovine kidney or salivary gland and that cause anchorage-independent growth of normal cells. A 72-hour exposure of confluent monolayers of rabbit synovial fibroblasts in 10% fetal calf serum to partially purified TGF-beta in the presence of TGF-alpha gave a 2- to 5-fold increase in incorporation of 3H-thymidine, protein content, and cell number. Similar results were obtained with high pressure liquid chromatography-purified TGF-beta in the presence of epidermal growth factor (EGF) (a type of TGF-alpha). By itself, purified TGF-beta was not mitogenic; it depended absolutely on EGF. However, only TGF-beta along with EGF, and not EGF alone, induced a marked morphologic change: a piling up of cells into foci resembling those commonly seen in primary cultures of rheumatoid synovial cells. Mitogenic responses induced by the TGF-beta-EGF combination were prevented by all-trans-retinoic acid but not by indomethacin or dexamethasone. The data indicate that TGF-beta, a peptide extracted from normal cells, can act in concert with EGF to cause proliferation and piling up of synovial cells and raise the possibility that these factors may play a role in rheumatoid arthritis and other proliferative but nonmalignant diseases as well.     

A variant epidermal growth factor receptor exhibits altered type alpha transforming growth factor binding and transmembrane signaling.

Epidermal growth factor (EGF) and type alpha transforming growth factor (TGF-alpha) bind to a specific region in subdomain III of the extracellular portion of the EGF receptor (EGFR). Binding leads to receptor dimerization, auto-and transphosphorylation on intracellular tyrosine residues, and activation of signal transduction pathways. We compared the binding and biological actions of EGF and TGF-alpha in Chinese hamster ovary (CHO) cells expressing either wild-type human EGFR (HER497R) or a variant EGFR that has an arginine-to-lysine substitution in the extracellular domain at codon 497 (HER497K) within subdomain IV of EGFR. Both receptors exhibited two orders of binding sites with radioiodinated EGF (125I-EGF). Similar results were obtained with 125I-TGF-alpha in cells expressing HER497R. In contrast, only one order of low-affinity binding sites was seen with 125I-TGF-alpha in the case of HER497K. Although EGF and TGF-alpha enhanced tyrosine phosphorylation of both receptors, CHO cells expressing HER497K exhibited an attenuated growth response to EGF and TGF-alpha and a reduced induction of the protooncogenes FOS, JUN, and MYC. Moreover, high concentrations of TGF-alpha (5 nM) inhibited growth in these cells but not in cells expressing HER497R. These findings indicate that a region in subdomain IV of EGFR regulates signal transduction across the cell membrane and selectively modulates that binding characteristics of TGF-alpha.     

The human transforming growth factor type alpha coding sequence is not a direct-acting oncogene when overexpressed in NIH 3T3 cells.

A peptide secreted by some tumor cells in vitro imparts anchorage-independent growth to normal rat kidney (NRK) cells and has been termed transforming growth factor type alpha (TGF-alpha). To directly investigate the transforming properties of this factor, the human sequence coding for TGF-alpha was placed under the control of either a metallothionein promoter or a retroviral long terminal repeat. These constructs failed to induce morphological transformation upon transfection of NIH 3T3 cells, whereas viral oncogenes encoding a truncated form of its cognate receptor, the EGF receptor, or another growth factor, sis/platelet-derived growth factor 2, efficiently induced transformed foci. When NIH 3T3 clonal sublines were selected by transfection of TGF-alpha expression vectors in the presence of a dominant selectable marker, they were shown to secrete large amounts of TGF-alpha into the medium, to have downregulated EGF receptors, and to be inhibited in growth by TGF-alpha monoclonal antibody. These results indicated that secreted TGF-alpha interacts with its receptor at a cell surface location. Single cell-derived TGF-alpha-expressing sublines grew to high saturation density in culture. However, when plated as single cells on contact-inhibited monolayers of NIH 3T3 cells, they failed to form colonies, whereas v-sis- and v-erbB-transfected cells formed transformed colonies under the same conditions. Moreover, TGF-alpha-expressing sublines were not tumorigenic in nude mice. These and other results imply that TGF-alpha exerts a growth-promoting effect on the entire NIH 3T3 cell population after secretion into the medium but little, if any, effect on the individual cell synthesizing this factor. It is concluded that the normal coding sequence for TGF-alpha is not a direct-acting oncogene when overexpressed in NIH 3T3 cells.     

Decreased expression of epidermal growth factor receptor and mRNA of its ligands in Helicobacter pylori-infected gastric mucosa

BACKGROUND: Epidermal growth factor (EGF) and TGF-alpha play a central role in maintaining gastric mucosal integrity. Little is known about the regulative role of the four other widely expressed epidermal growth factor receptor ligands, heparin-binding EGF, amphiregulin, betacellulin and cripto in the gastric mucosa. METHODS: Nineteen patients with Helicobacter pylori-positive gastritis and 32 healthy controls were investigated. Mucosal mRNA expression of EGF receptor ligands was determined by quantitative PCR before and after H. pylori eradication. PCR products were analyzed by soft laser scanning densitometry. Moreover, the effect of chronic active gastritis on EGF receptor expression was assessed by [125I] EGF receptor autoradiography. Immunohistochemistry was performed for TGF-alpha to localize growth factor expression. RESULTS: Antral and oxyntic biopsies showed strong mRNA expressions for TGF-alpha, amphiregulin and heparin binding EGF, but not for EGF, cripto and betacellulin. mRNA expression was significantly reduced down to 50% in H. pylori infection, significantly lower compared to normal gastric mucosa, and increased after eradication therapy. Moreover, chronic gastritis was associated with decreased antral EGF receptor binding compared to healthy controls, possibly reflecting reduced autoinduction. Immunohistochemical analyses localized TGF-alpha in the cytoplasma of gastric epithelial cells and revealed its increased expression after H. pylori eradication. CONCLUSIONS: The data presented suggest that amphiregulin, heparin binding EGF and TGF-alpha are important EGF receptor ligands in the gastric mucosa. H. pylori infection apparently suppresses their mRNA as well as receptor expression that is reversed by H. pylori eradication. This deficiency of the gastroprotective EGF system may contribute to the gastric pathogenicity of H. pylori infection.     

Transforming growth factor alpha (TGF-alpha) increases cell number in a human pancreatic cancer cell line but not in normal mouse pancreas.

BACKGROUND: The pancreas harbors growth factors such as the epidermal growth factor (EGF) family. The physiological and pathophysiological roles of growth factors in normal pancreas remain unsettled. Human pancreatic cancer overexpresses the EGF receptor, and the ligands EGF and transforming growth factor alpha (TGF-alpha). The aim of the present experiments was to study the effect of TGF-alpha in a pancreatic cancer cell line and in normal mouse pancreas. METHOD: The LN-36 cell line, established from a pancreatic duct cell adenocarcinoma, was incubated with TGF-alpha or EGF. The effect of an EGF receptor-specific, tyrosine kinase inhibitor (tyrphostin B56) with or without growth factors was also studied. The cell number was measured with the XTT-colorimetric method. TGF-alpha, the tyrphostins A25, B48, and B56, were in separate experiments infused during 1 wk to normal female mice by subcutaneous (sc) minipumps. RESULTS: The LN-36 cell line responded to TGF-alpha and EGF with increased cell number; +61% with 10(-10) M TGF-alpha and +34% with 10(-9) M EGF. Tyrphostin B56 at a concentration of 10(-5) M reduced the cell number by 76%, but when incubated together with growth factors the reduction was only 44% with TGF-alpha, and 39% with EGF. Infusion of TGF-alpha increased mouse pancreatic wet weight and protein content but was without effect on DNA synthesis, measured as incorporation of tritiated thymidine. Infusion of three different tyrphostins did not influence mice pancreas. CONCLUSION: The results support the role of TGF-alpha to maintain growth of pancreatic cancer cells by the EGF receptor. Infusion of TGF-alpha induced hypertrophy in normal mouse pancreas.     

Production of transforming growth factor alpha in human pancreatic cancer cells: evidence for a superagonist autocrine cycle.

Previous work showed that cultured human pancreatic cancer cells overexpress the epidermal growth factor (EGF) receptor. In the present study, we sought to determine whether some of these cell lines produce transforming growth factor alpha (TGF-alpha). Utilizing a radiolabeled TGF-alpha cDNA in hybridization experiments, we determined that ASPC-1, T3M4, PANC-1, COLO-357, and MIA PaCa-2 cell lines expressed TGF-alpha mRNA. Serum-free medium conditioned by T3M4 and ASPC-1 cells contained significant amounts of TGF-alpha protein. Although unlabeled TGF-alpha readily competed with 125I-labeled EGF for binding, each cell line exhibited lower surface binding and internalization of 125I-labeled TGF-alpha as compared to 125I-labeled EGF. Both TGF-alpha and EGF significantly enhanced the anchorage-independent growth of PANC-1, T3M4, and ASPC-1 cells. However, TGF-alpha was 10- to 100-fold more potent than EGF. These findings suggest that the concomitant overexpression of EGF receptors and production of TGF-alpha may represent an efficient mechanism for certain cancer cells to obtain a growth advantage.     

The central role of human chorionic gonadotropin in the formation of human placental syncytium

A number of trophoblast products, including human chorionic gonadotropin (hCG), can increase the formation of human placental syncytium through the differentiation of mononuclear cytotrophoblasts. The present study investigated the central role of hCG in this process by using antisense receptor phosphorothioate oligodeoxynucleotides (ODNs). Culturing cytotrophoblasts with the hCG/LH receptor antisense, but not sense, ODN resulted in a significant decrease in receptor protein levels and inhibited spontaneous as well as exogenous hCG induced increase in differentiation. The hCG/LH receptor antisense ODN also inhibited epidermal growth factor (EGF), TGF-alpha, leukemia inhibitory factor (LIF), but not 8-bromo-cAMP, induced increases in differentiation, suggesting that hCG is required for EGF, TGF-alpha and LIF, but not for the cAMP actions. Although antisense EGF receptor and LIF receptor ODNs inhibited EGF and LIF induced increase in differentiation, respectively, they were ineffective against hCG, suggesting that they use separate pathways, but they both converge on a common pathway requiring the hCG actions. Mechanism of action studies revealed that EGF treatment activates its receptors and MAPK, both of which are required for EGF to increase the differentiation, cAMP levels and activate protein kinase A. In summary, our results demonstrate that hCG is an autocrine and paracrine regulator that is required for EGF, TGF-alpha, and LIF, but not for cAMP to increase human placental syncytium formation. Direct activation of protein kinase A seems to bypass the hCG pathway, perhaps by targeting genes associated with the differentiation.     

Transforming growth factor alpha contributes to the mechanism by which hypothalamic injury induces precocious puberty.

It has long been known that lesions of the hypothalamus lead to female sexual precocity. While an increased production of luteinizing hormone-releasing hormone (LHRH), the neurohormone that controls sexual development, appears to mediate the advancement of puberty induced by these lesions, little is known about the mechanism(s) by which hypothalamic injury activates LHRH secretion. Since brain lesions result in accumulation of neurotrophic/mitogenic activities in the injured area, we tested the hypothesis that transforming growth factor alpha (TGF-alpha), a mitogenic polypeptide recently shown to stimulate LHRH release, is produced in response to hypothalamic injury and mediates the effect of the lesion on puberty. Radiofrequency lesions of the preoptic area-anterior hypothalamic area (POA-AHA) of 22-day-old female rats resulted in precocious puberty within 7 days after the operation. RNA blot hybridization revealed that lesion-induced puberty was preceded by an increase in TGF-alpha mRNA levels in the POA-AHA. Epidermal growth factor (EGF) mRNA was undetectable in both intact and lesioned hypothalami. TGF-alpha mRNA levels, quantitated by RNase protection assays, were 3.5-fold greater in lesioned animals approaching puberty than in age-matched controls. Immunohistochemical studies, utilizing single- and double-staining procedures, demonstrated the presence of TGF-alpha precursor-like immunoreactivity in reactive astrocytes surrounding the lesion site. Hybridization histochemistry showed increased TGF-alpha mRNA expression in cells of the same area, further implicating reactive astrocytes as a site of TGF-alpha synthesis. The actions of TGF-alpha are mediated by its interaction with EGF receptors. Continuous infusion of RG-50864, an inhibitor of EGF receptor kinase activity, at the site of injury prevented the advancement of puberty induced by the lesion. These results suggest that TGF-alpha acting via EGF-like receptors contributes to the acceleration of puberty induced by anterior hypothalamic lesions. They also indicate that activation of TGF-alpha gene expression in glial cells is a component of the hypothalamic response to injury.     

Stimulatory actions of insulin-like growth factor-I and transforming growth factor-alpha on intestinal neurotensin and peptide YY.

Proliferation of the gastrointestinal mucosa is stimulated by the growth factors, insulin-like growth factor-I (IGF-I) and transforming growth factor-alpha (TGF-alpha), or the closely related epidermal growth factor (EGF), as well as the gastrointestinal hormones, gastrin, neurotensin (NT), and peptide YY (PYY). The stimulatory actions of these growth factors or gastrointestinal hormones on the gastrointestinal mucosa may be direct or mediated in part by gastrointestinal peptides or the growth factors, respectively. The purpose of these studies therefore was to examine the effects of IGF-I and TGF-alpha on stomach gastrin and intestinal NT and PYY gene expression [i.e. messenger RNA (mRNA), peptide levels] and secretion. Mice were given recombinant human IGF-I (3, 6 mg/kg BW/day x 14 days). Transgenic mice with the rat TGF-alpha gene linked to a metallothionein promoter were used as a model of chronic TGF-alpha excess. IGF-I and TGF-alpha did not affect gastrin gene expression. Steady-state intestinal NT and PYY mRNA and peptide levels were elevated in a dose-related manner by IGF. TGF-alpha also increased intestinal expression of NT and PYY peptide, but not mRNA levels. Basal serum levels of PYY were elevated by IGF-I and TGF-alpha. IGF-I and TGF-alpha did not increase intestinal chromogranin A (CGA) gene expression, a marker of endocrine cells, or the density of PYY-containing cells in the colon, indicating that the elevations in intestinal gut peptide gene expression by IGF-I and TGF-alpha are not due simply to an increased number of enteroendocrine cells. IV infusion of EGF also stimulated release of PYY in the dog. Together, these findings indicate that IGF-I and TGF-alpha may cause secretion of gut hormones and exert a major upregulatory influence on the regulation of intestinal peptide hormone homeostasis.     

Effects of transforming growth factor-alpha on chicken adipocyte precursor cells in vitro.

The hyperplastic capacity of adipose tissue resides in a group of fibroblast-like adipocyte precursor cells. There is evidence to suggest that their proliferation and differentiation is regulated by insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) but there is less information about other growth factors which may also participate in adipocyte precursor cell hyperplasia. Transforming growth factor-alpha (TGF-alpha) is a 50 amino acid polypeptide which has been shown to stimulate proliferation in both neoplastic and normal cell types acting through the epidermal growth factor (EGF) receptor. We have studied the regulation of DNA synthesis and the activity of lipoprotein lipase by TGF-alpha in chicken adipocyte precursor cells in vitro. Both TGF-alpha and EGF stimulated incorporation of [3H]thymidine into DNA in a dose-dependent manner. TGF-alpha was approximately 180-fold more potent than EGF. Addition of TGF-alpha in combination with IGF-I, TGF-beta 1 or platelet-derived growth factor produced a synergistic increase in DNA synthesis. Short-term incubation with TGF-alpha reduced lipoprotein lipase activity by 23%. These results show that TGF-alpha is a potent mitogen in these adipocyte precursor cells and can inhibit their differentiation in vitro and may participate in the regulation of adipose tissue development in vivo.     

Tumor secretion of growth factors.

An increasing number of polypeptide growth factors have been identified that regulate not only cell proliferation but also an extraordinary range of cell activities, including matrix protein deposition and resolution, the maintenance of cell viability, cell differentiation, inflammation, and tissue repair. Normal cells appear to require growth factors for proliferation and for maintenance of viability. Cells that secrete a polypeptide growth factor have an advantage in growth. These factors can act either externally through cell surface receptors or internally during the transport of receptors and growth factors through the endoplasmic reticulum and Golgi apparatus, causing autocrine stimulation of cell growth. Depending on the cell type, growth factors can also be potent inhibitors of cell growth rather than stimulators of growth, and the effect can depend on the presence or absence of growth factors. Among the growth factors considered, IGFs are unusual in that they function both as endocrine and as autocrine/paracrine agents. IGF-II, which is associated with fetal growth, is the IGF most frequently expressed by tumors. There is now convincing evidence that some tumors secrete sufficient IGF-II to have systemic endocrine effects as recognized as nonislet cell tumor hypoglycemia. PDGF is normally highly concentrated in platelets and has major significance in stimulation of cellular proliferation in inflammation and wound repair. Normally, this proliferation is self-limited, but the secretion of PDGF by tumors and its effects on cell proliferation of tumors persist. The fact that PDGF B monomer has an identical structure with that of the proto-oncogene C-cis further strengthens the connection between PDGF and tumor growth. EGF has a restricted role in normal physiology, but its close relative, TGF-alpha, is widely distributed in normal and neoplastic tissues. The common receptor for EGF and TGF-alpha is present in many normal and neoplastic cell types. The EGF receptor is the product of the C-erb gene. The oncogene V-cis is a truncated form of the EGF receptor whose tyrosine kinase activity is not dependent on ligand binding. TGF-beta exists in multiple forms. Although it can transform the morphology of certain cell lines in culture, it probably does not act generally as a mitogenic agent. Its major physiologic role in the body appears to be the stimulation of mesenchymal matrix formation. It is of special importance in the regulation of bone matrix formation. Its expression is increased in many tumors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential tubulogenic and branching morphogenetic activities of growth factors: implications for epithelial tissue development.

At least two kidney epithelial cell lines, the Madin-Darby canine kidney (MDCK) and the murine inner medullary collecting duct line mIMCD-3, can be induced to form branching tubular structures when cultured with hepatocyte growth factor (HGF) plus serum in collagen I gels. In our studies, whereas MDCK cells remained unable to form tubules in the presence of serum alone, mIMCD-3 cells formed impressive branching tubular structures with apparent lumens, suggesting the existence of specific factors in serum that are tubulogenic for mIMCD-3 cells but not for MDCK cells. Since normal serum does not contain enough HGF to induce tubulogenesis, these factors appeared to be substances other than HGF. This was also suggested by another observation: when MDCK cells or mIMCD-3 cells were cocultured under serum-free conditions with the embryonic kidney, both cell types formed branching tubular structures similar to those induced by HGF; however, only in the case of MDCK cells could this be inhibited by neutralizing antibodies against HGF. Thus, the embryonic kidney produces growth factors other than HGF capable of inducing tubule formation in the mIMCD-3 cells. Of a number of growth factors examined, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) were found to be tubulogenic for mIMCD-3 cells. Whereas only HGF was a potent tubulogenic factor for MDCK cells, HGF, TGF-alpha, and EGF were potent tubulogenic factors for mIMCD-3 cells. Nevertheless, there were marked differences in the capacity of these tubulogenic factors to induce tubulation as well as branching events in those tubules that did form (HGF >> TGF-alpha > EGF). Thus, at least three different growth factors can induce tubulogenesis and branching in a specific epithelial cell in vitro (though to different degrees), and different epithelial cells that are capable of forming branching tubular structures demonstrate vastly different responses to tubulogenic growth factors. The results are discussed in the context of branching morphogenesis during epithelial tissue development.