Abstract. Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF/31 cDNA contains high levels of latent TGF/31. The amino-terminal region of the TGF/~I precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical crosslinking of CHO-conditioned medium using bis-(sulfosuccinimidyl)-suberate (BS 3) followed by immunoblot analyses indicates that latent recombinant TGF/31 contains both the cleaved amino-terminal glycopeptide and mature TGF/31 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF/3 binding protein(s) in latent recombinant TGF/31. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF/3 competing activity. In addition, immunoblot analysis of plasmintreated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF/31 is released. Thus, activation of latent TGFB1 may occur by proteolytic nicking within the amino-terminal glycopeptide thereby causing a disruption of tertiary structure and noncovalent bonds, which results in the release of active, mature TGF/~I. Acid activation of latent TGF/~, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide.

The transforming growth factors 13 (TGFs-[3) are potent inhibitors of cell proliferation and are usually secreted in a latent form. TGF-IM, TGF-B2, and TGF-I33 are expressed in distinct but overlapping patterns in the developing mouse mammary gland. To study the role of transforming growth factor-B1 (TGF-I31) in normal mammary development and in mammary neoplasia, we have constructed three transgenic mouse lines that express a simian TGF-B1 s223/22s mutated to produce a constitutively active product under the control of the MMTV enhancer/promoter. Expression of the transgene, as confirmed by in situ hybridization, immunohistochemistry, and Northern blot analysis, was associated with marked suppression of the normal pattern of mammary ductal tree development in female transgenics. Reduction in total ductal tree volume was observed at 7 weeks, soon after estrous begins, and was most apparent at 13 weeks, as ductal growth in the normal mammary gland declines. This effect was seen in all three lines. However, during pregnancy, alveolar outgrowths developed from the hypoplastic ductal tree, and lactation occurred, therefore, all transgenic females could feed full litters. Unlike many other transgenic mouse models in which expression of growth factors or oncogenes under control of the MMTV promoter leads to mammary epithelial hyperplasia and increased tumor formation, the MMTV-TGF-~I s223/22s transgene causes conditional hypoplasia of the mammary ductal tree and no spontaneous tumors have been detected in the MMTV-TGF-[~I s223/22s transgenic animals.

Regulation of transforming growth factor Il1 (TGFII1), TGF(2, and TGF0i3 mRNAs in murine fibroblasts and keratinocytes by TGFIII and TGFI2 was studied. In quiescent AKR-2B fibroblasts, in which TGFOi induces delayed stimulation of DNA synthesis, TGFII1 autoregulation of TGFI1 expression was observed as early as 1 h, with maximal induction (25-fold) after 6 to 12 h. Increased expression of TGF(II mRNA was accompanied by increased TGFO protein production into conditioned medium of AKR-2B cells. Neither TGFI32 nor TGFI3 mRNA, however, was significantly induced, but both were apparently down regulated at later times by TGFI31. Protein synthesis was not required for autoinduction of TGFfII mRNA in AKR-2B cells. Nuclear run-on analyses and dactinomycin experiments indicated that autoregulation of TGF(l expression is complex, involving both increased transcription and message stabilization. In contrast to TGF(11, TGFj32 treatment of quiescent AKR-2B cells increased expression of TGFI1, TGFI82, and TGF,3 mRNAs, but with different kinetics. Autoinduction of TGF,I2 mRNA occurred rapidly with maximal induction at 1 to 3 h, enhanced TGFI83 mRNA levels were observed after 3 h, and increased expression of TGFI1 occurred later, with maximal mRNA levels obtained after 12 to 24 h. Nuclear run-on analyses indicated that TGFI2 regulation of TGF,I2 and TGF,3 mRNA levels is transcriptional, while TGFP2 induction of TGFI{1 expression most likely involves both transcriptional and posttranscriptional controls. In BALB/MK mouse keratinocytes, minimal autoinduction of TGF(H occurred at only the 12-and 24-h time points and protein synthesis was required for this autoinduction. The results of this study provide an example in which TGFI1 and TGF,2 elicit different responses and demonstrate that expressions of TGFII1, TGF,I2, and TGFj13 are regulated differently. The physiological relevance of TGFIV1 autoinduction in the context of wound healing is discussed.

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