The production, survival, and function of monocytes and macrophages is regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor Fms. Binding of M-CSF to Fms induces the tyrosine phosphorylation and association of a 150-kD protein with the phosphotyrosine-binding (PTB) domain of Shc. We have cloned p150 using a modified yeast two-hybrid screen.

A polypeptide termed oncostatin M, which inhibits the replication of A375 melanoma and other human tumor cells, but not normal human fibroblasts, has been isolated from serum-free supernatants of U-937 histiocytic lymphoma cells that have been induced to differentiate into macrophage-like cells following treatment with the phorbol ester phorbol 12-myristate 13-acetate. No such growth inhibitory activity is detected in the supernatant of untreated U-937 cells, indicating that the protein is induced or increased in expression in the phorbol ester-induced differentiated cells. Oncostatin M is stable between pH 2 and 11 and after heating for 1 hr at 560C but is not stable at 90TC. Purification of oncostatin M has been achieved by gel chromatography and reversed-phase HPLC, using sequentially acetonitrile and n-propanol in the presence of aqueous trifluoroacetic acid. The apparent molecular weight of oncostatin M is -18,000, as determined by gel chromatography, and 28,000, as determined by polyacrylamide gel electrophoresis. (vol/vol) fetal bovine serum, L-glutamine, and penicillin-streptomycin. All of the following cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum, L-glutamine, and penicillin-streptomycin. The A375 cell line was derived from a human melanoma (26); A549, from a lung carcinoma (26); HTB10, from a neuroblastoma (27); SK-MEL-28, from a melanoma (28); and WI-26 and WI-38 cells were derived from human embryonic lung (29,30).Cell Growth Inhibition Assay. The assay was performed in flat 96-well plates (3596; Costar, Cambridge, MA). Human A375 melanoma cells were used as a sensitive indicator cell line. Cells (3 x 103 cells) in 0.1 ml of DMEM supplemented with 10% (vol/vol) fetal bovine serum and penicillinstreptomycin were placed in each well. Three hours later, 0. 1 ml of test samples was added to each well. Plates were incubated at 37°C for 3 days. Then 0.025 ml (0.5 ,uCi Abbreviations: PMA, phorbol 12-myristate 13-acetate; GIA, growth inhibitory activity. 9739The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Recently, the simian type 1 transforming growth factor beta (TGF-I1) cDNA was expressed at high levels in Chinese hamster ovary (CHO) cells by dihydrofolate reductase-induced gene ampliffication (L. E. Gentry, N. R. Webb, G. J. Lim, A. M. Brunner, J. E. Ranchalis, D. R. Twardzik, M. N. Lioubin, H. Marquardt, and A. F. Purchio, Mol. Cell. Biol. 7:3418-3427, 1987). We have now purified and characterized the recombinant proteins released by these cells. Analyses of the precursor proteins by amino acid sequencing identified potentially important proteolytic processing sites. Signal peptide cleavage occurs at the Gly-29-Leu-30 peptide bond of pre-pro-TGF-I1, yielding pro-TGF-j11 (30 to 390). In addition, proteolytic processing of the precursor to yield mature TGF-II1 occurs at the dibasic cleavage site immediately preceding Ala-279, indicating that CHO cells possess the appropriate processing enzyme. Greater than 95% of the biological activity detected in the conditioned medium of the CHO transfectant was due to mature, properly processed growth factor. Highly purified recombinant TGF-,1 had the same specific biological activity as natural TGF-111. The concentration of TGF-j1 required for half-maximal inhibition of MvlLu mink lung epithelial cell growth was approximately 1 to 2 pM. Purified precursor inhibited mink lung cell proliferation at 50 to 60 pM concentrations. The purified precursor preparation was shown to consist of pro-TGF-I1 (30 to 390), the pro region of the precursor (30 to 278), and mature TGF-01 (279 to 390) interlinked by at least one disulfide bond with the pro portion of the precursor. These recombinant forms of TGF-j1l should prove useful for further structural and functional studies.Type 1 transforming growth factor beta (TGF-B1) belongs to a closely related family of polypeptides with potent cellular modulating activities (for reviews, see references 12 and 19). This growth factor molecule appears to be intimately associated with cell growth and differentiation and may play pivotal roles in the autocrine or paracrine regulation of these processes. Although numerous studies have addressed important cellular and physiological properties of TGF-pl, very little information is available concerning structural features and posttranslational processing events necessary for its expression and function.The molecular cloning of TGF-p1 cDNA from several species (4, 5, 18) and analysis of the predicted protein sequences has provided some insight into the structure and processing of this polypeptide growth factor. Sequence analysis has predicted that the mature 112-amino-acid chain of TGF-pl is derived from the C terminus of a 390-aminoacid pre-pro-TGF-,1 by proteolytic cleavage. Proteolytic cleavage is predicted to occur following a dibasic Arg-Arg sequence and immediately preceding the N-terminal Ala residue of the mature growth factor. Structural studies of mature TGF-13l have revealed that TGF-,1 is homodimeric and interlinked by disulfide bonds (1,6,16). The precursor for TGF-,1l contains a typical hyd...

Recombinant type 1 transforming growth factor beta (TGF-,) was expressed to high levels in CHO cells by using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-I and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the simian virus 40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines that expressed amplified TGF-, nucleic acid sequences. The expression plasmid DNA was amplified greater than 35-fold in one of the methotrexate-selected transfectants.The major proteins secreted by these cells consisted of latent as judged by immunoblots by using site-specific anti-peptide antibodies derived from various regions of the TGF-, precursor. Levels of recombinant TGF-0 protein secreted by these cells approached 30 ,ug/24 h per 107 cells and required prior acidification for optimal activity; nonacidified supernatants were approximately 1% as active as acidified material. Antibodies directed toward sequences present in the mature growth factor readily identified a proteolytically processed recombinant TGF-, which, on sodium dodecyl sulfate-polyacrylamide gels, comigrated with highly purified natural TGF-4. In addition to mature recombinant TGF-jA, site-specific antibodies demonstrated the existence of larger TGF-3 precursor polypeptides. The availability of biologically active recombinant type 1 TGF-I8 and precursor forms should provide a means to examine the structure, function, and potential in vivo therapeutic use of this growth factor.Results of recent studies have suggested that transforming growth factor beta (TGF-P) consists of a family of closely related biologically active polypeptides with potent cellularmodulating activities. Three molecular forms of TGF-P (types 1, 2 and 1.2) have been identified and characterized.Type 1 TGF-P was the first growth factor isolated and, as a result, the best characterized (for reviews, see references 23 and 47). sources have been obtained. The predicted protein sequences derived from the cDNAs reveal a remarkable homology. The mature forms of the human and simian growth factor differ from the murine factor by one amino acid. Analysis of the various TGF-,B cDNA clones suggests that the mature 112 amino acid chain of type 1 TGF-P is derived from a much larger precursor polypeptide by proteolytic cleavage. The full-length precursor of TGF-,1 shows a high degree of structural conservation and constitutes 391 amino acids for human and 390 amino acids for both murine and simian precursors. A typical leader sequence is observed in the precursor, as well as three potential N-linked glycosylation sites. A dibasic sequence immediately precedes the amino-terminal alanine residue of the mature growth factor, indicating the involvement of a dibasic protease in processing of the growth factor.To understand in more detail the structure and processing of type 1 TGF-P, as well as to provide a source for large...

Activation of erbB-1 receptors by glial TGFalpha has been shown to be a component of the developmental program by which the neuroendocrine brain controls mammalian sexual development. The participation of other members of the erbB family may be required, however, for full signaling capacity. Here, we show that activation of astrocytic erbB-2/erbB-4 receptors plays a significant role in the process by which the hypothalamus controls the advent of mammalian sexual maturation. Hypothalamic astrocytes express both the erbB-2 and erbB-4 genes, but no erbB-3, and respond to neuregulins (NRGs) by releasing prostaglandin E(2) (PGE(2)), which acts on neurosecretory neurons to stimulate secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual development. The actions of TGFalpha and NRGs in glia are synergistic and involve recruitment of erbB-2 as a coreceptor, via erbB-1 and erbB-4, respectively. Hypothalamic expression of both erbB-2 and erbB-4 increases first in a gonad-independent manner before the onset of puberty, and then, at the time of puberty, in a sex steroid-dependent manner. Disruption of erbB-2 synthesis in hypothalamic astrocytes by treatment with an antisense oligodeoxynucleotide inhibited the astrocytic response to NRGs and, to a lesser extent, that to TGFalpha and blocked the erbB-dependent, glia-mediated, stimulation of LHRH release. Intracerebral administration of the oligodeoxynucleotide to developing animals delayed the initiation of puberty. Thus, activation of the erbB-2-erbB-4 receptor complex appears to be a critical component of the signaling process by which astrocytes facilitate the acquisition of female reproductive capacity in mammals.