In cell culture, type a transforming growth factor (TGF-a) stimulates epithelial cell growth, whereas TGF-p1 overrides this stimulatory effect and is growth inhibitory. Transgenic mice that overexpress TGF- In contrast to the TGF-ps, TGF-a is a mitogen for most cell types (7) and may also play a significant role in mammary gland development. TGF-a is localized in vivo in the epithelium of the advancing terminal buds and in stromal fibroblasts at the base of terminal buds (8). Furthermore, implantation of pellets containing TGF-a into regressed mammary glands of ovariectomized mice stimulates the reappearance of end buds (8). MMTV-TGF-a transgenic mice exhibit enhanced production of TGF-a in the smaller ducts of the mammary gland (9). These mice exhibit mammary epithelial hyperplasia with a marked increase in the rate of benign and malignant mammary tumor development (9,10 MATERIALS AND METHODSTransgene Detection. Transgenic mice were identified by Southern blot analysis with a probe made from either mouse TGF-31 or an EcoRI/Xho I fragment of the transgene construct containing 526 bp of rabbit P-globin exon 3 sequence as described (6). The MMTV-TGF-a transgene was detected by PCR as described (9).Transgenic Mice. The MMTV-TGF-a and MMTV-TGF-31 transgenic animals were generated in a (C57BL x DBA/2)F1 (B6D2F1) background, which has a low frequency of spontaneous mammary tumor formation (6,(9)(10)(11). Wild-type animals used in various experiments were from the same genetic background. For the crossbreeding experiments, the line 29 MMTV-TGF-a transgenic mouse line was selected (9,10

Our results demonstrate that sequential proteolytic cleavage within the ectodomain of the 50-kDa pro-AR form leads to release of a predominant N-glycosylated 43-kDa soluble AR, as well as the appearance of other cellular and soluble AR forms. Cell surface biotinylation studies using a C-terminal epitope-tagged pro-AR indicate that all cell surface forms are membrane-anchored and support that AR is released by ectodomain cleavage of pro-AR at the plasma membrane. We also show that pro-AR ectodomain cleavage is a regulated process, which can be stimulated by phorbol 12-myristate 13-acetate and inhibited by the metalloprotease inhibitor, batimastat. In addition, we provide evidence that high molecular mass AR forms may retain the full-length Nterminal pro-region, which may influence the biological activities of these forms.

EGF precursor (proEGF) is a member of the family of membrane-anchored EGF-like growth factors that bind with high affinity to the epidermal growth factor receptor (EGFR). In contrast to human transforming growth factor-α precursor (proTGFα), which is sorted basolaterally in Madin-Darby canine kidney (MDCK) cells (Dempsey, P., and R. Coffey, 1994. J. Biol. Chem. 269:16878–16889), we now demonstrate that human proEGF overexpressed in MDCK cells is found predominantly at the apical membrane domain under steady-state conditions. Nascent proEGF (185 kD) is not sorted but is delivered equally to the apical and basolateral membranes, where it is proteolytically cleaved within its ectodomain to release a soluble 170-kD EGF form into the medium. Unlike the fate of TGFα in MDCK cells, the soluble 170-kD EGF species accumulates in the medium, does not interact with the EGFR, and is not processed to the mature 6-kD peptide. We show that the rate of ectodomain cleavage of 185-kD proEGF is fourfold greater at the basolateral surface than at the apical surface and is sensitive to a metalloprotease inhibitor, batimastat. Batimastat dramatically inhibited the release of soluble 170-kD EGF into the apical and basal medium by 7 and 60%, respectively, and caused a concordant increase in the expression of 185-kD proEGF at the apical and basolateral cell surfaces of 150 and 280%, respectively. We propose that preferential ectodomain cleavage at the basolateral surface contributes to apical domain localization of 185-kD proEGF in MDCK cells, and this provides a novel mechanism to achieve a polarized distribution of cell surface membrane proteins under steady-state conditions. In addition, differences in disposition of EGF and TGFα in polarized epithelial cells offer a new conceptual framework to consider the actions of these polypeptide growth factors.

Transgenic mice expressing either the neu proto-oncogene or transforming growth factor (TGF-␣) in the mammary epithelium develop spontaneous focal mammary tumors that occur after a long latency. Since the epidermal growth factor receptor (EGFR) and Neu are capable of forming heterodimers that are responsive to EGFR ligands such as TGF-␣, we examined whether coexpression of TGF-␣ and Neu in mammary epithelium could cooperate to accelerate the onset of mammary tumors. To test this hypothesis, we interbred separate transgenic strains harboring either a mouse mammary tumor virus/TGF-␣ or a mouse mammary tumor virus/neu transgene to generate bitransgenic mice that coexpress TGF-␣ and neu in the mammary epithelium. Female mice coexpressing TGF-␣ and neu developed multifocal mammary tumors which arose after a significantly shorter latency period than either parental strain alone. The development of these mammary tumors was correlated with the tyrosine phosphorylation of Neu and the recruitment of c-Src to the Neu complex. Immunoprecipitation and immunoblot analyses with EGFR-and Neu-specific antisera, however, failed to detect physical complexes of these two receptors. Taken together, these observations suggest that Neu and TGF-␣ cooperate in mammary tumorigenesis through a mechanism involving Neu and EGFR transactivation.