A.F. Purchio

γ‐Interferon‐Induced Activation of Latent Transforming Growth Factor‐β by Human Monocytes

Transforming growth factor-ps (TGFps), a family of at least four different homologous disulfide-linked homodimeric polypeptides, are potent modulators of cell growth and differentiation.’-3 Analysis of cDNA clones encoding mammalian TGFPs and appropriate cell culture supernatants indicates that the mature molecule is cleaved from the carboxy terminus of a larger glycosylated precurso+6 and is secreted in a latent, inactive form?-9 Transient acidification activates E F P ; however, the physiological mechanism(s) of TGFB secretion and activation, which initiate its potent autocrine and paracrine effects in vivo, are poorly understood. In this study, we asked whether cell association was involved in activation of ‘EFp. The ’’latentrecombinant complex used contains one dimeric TGFpl molecule associated with a disulfide-bonded dimeric remnant of the precursor. Fresh human monocytes stimulated by y-interferon (yIFN), activates the “latent” recombinant E F P l complex ( L m F P l ) in a dose-dependent manner. The activated X F p l released into the media is neutralized by a TGFPl monoclonal antibody and has a mass (24 kD) identical to native TGFB1. Thus, TGFSl activation by monocytes may require y IFN-mediated gene expression as well as a cell-associated processing event. We have recently reported the expression of simian ‘EFP type 1 cDNAIO in Chinese hamster ovary (CHO) cells using dehydrofolate reductase (dhfr) gene amplification.” One CHO cell transfectant, designated clone 17, secretes mg amounts of latent

Ovarian transforming growth factor-β (TGFβ): cellular site(s), and mechanism(s) of action☆

It is the objective of the experiments reported herein to examine the possible relevance of transforming growth factor-beta (TGF beta) to theca-interstitial cell function, and to further characterize the established interaction of TGF beta with the granulosa cell. In examining the interaction of TGF beta (10 ng/ml) with murine theca-interstitial cells, no significant effect was observed on either basal or human chorionic gonadotropin (hCG)-stimulated androsterone accumulation. In contrast, given murine granulosa cells, TGF beta (10 ng/ml) produced dose- and time-dependent augmentation of follicle-stimulating hormone (FSH)-supported aromatase activity with a minimal and median effective doses of 20 +/- 3 and 123 +/- 24 pg/ml, respectively and a minimal time requirement of less than or equal to 48 h. The ability of TGF beta to augment FSH hormonal action could not be accounted for by alteration(s) of specific FSH binding (13965 +/- 298 and 12614 +/- 694 cpm/4 X 10(5) cells for FSH and FSH + TGF beta). However, TGF beta proved capable of exerting a direct upregulatory effect on stimulatable adenylate cyclase activity, further enhancement occurring at site(s) distal to cAMP generation (dibutyryl cyclic AMP (Bt2cAMP) = 1.4 +/- 0.2 ng/culture; Bt2cAMP + TGF beta = 4.1 +/- 0.6 ng/culture). Taken together, our findings are in keeping with the notion that TGF beta, possibly of intraovarian origin, comprises the central signal of autocrine or paracrine loop(s) capable of amplifying gonadotropin action at the level of the granulosa, but not theca-interstitial cell.

Bone-derived and recombinant transforming growth factor β′S are potent inhibitors of tumor cell growth

Two naturally occurring chrondogenesis inducing peptides have been purified to homogeneity from demineralized bovine bone. Cartilage-inducing factors A and B are the bone-derived equivalents of transforming growth factor-beta types I and II. Both peptides exhibit identical biological activities in chondrogenesis assays and stimulate anchorage independent cell growth. In this study we show that both bone-derived factors are potent (ng/ml) inhibitors of both DNA synthesis and the anchorage independent growth of a variety of human and non-human tumor cells. Unique in this study is also a comparison of the activities of these polypeptide growth factors with recombinant transforming growth factor type I expressed in mammalian cells.

High-Level Expression of TGF-β2 and the TGF-β2(414) Precursor in Chinese Hamster Ovary Cells

AbstractChinese hamster ovary (CHO) clones secreting high levels of transforming growth factor-β2 (TGF-β2) were obtained after transfection with a cDNA clone coding for the 414-amino acid TGF-β2 precursor and subsequent amplification with methotrexate. The TGF-β2 was secreted in a latent form since acidification was necessary for detection of maximal levels of bioactivity. Amino- and carboxy-terminal sequencing of purified recombinant TGF-β2 indicated that correct processing of mature TGF-β2 had occurred. In addition to mature TGF-β2, the recombinant CHO clones secreted larger proteins having molecular weights of 85, 105, and 130 kD, which consisted of both mature and pro-region sequences when analyzed by immunoblotting using site-specific anti-peptide antibodies. Analysis of serum-and cell-free media from recombinant CHO cells metabolically labeled with [3Hglucosamine and [32Porthophosphate indicated that pro-TGF-β2 was glycosylated and phosphorylated. Two-dimensional electrophoretic analysis of acid hyd...

Structural requirements of diacylglycerols for binding and activating phospholipid-dependent, Ca++-sensitive protein kinase

Phospholipid-dependent, Ca++-sensitive protein kinase (protein kinase C) is activated by phorbol esters and diacylglycerols. A series of diacylglycerols was synthesized with different substituents at positions 1 and 2 in order to expand known structure-activity relationships for these compounds with respect to binding and activating purified protein kinase C. Compounds were synthesized with saturated and unsaturated long chain fatty acyl groups at position 1 and acetyl, butyryl, or hexanoyl groups at position 2. Binding to protein kinase C correlated well with in-vitro activation of the enzyme. These diacylglycerols activated protein kinase C in an intact cellular system causing the phosphorylation of pp60c-src. This indicates that the length of the fatty acyl group at C2 is critical and that the existence of unsaturation in the fatty acyl group at C1 is not essential.

Synthesis of an active hybrid growth factor (GF) in bacteria: transforming GF-α/vaccinia GF fusion protein

A hybrid gene encoding for a polypeptide consisting of the first 33 N-terminal amino acid (aa) residues of transforming growth factor-alpha (TGF-alpha) and a C terminus consisting of 20 aa residues of vaccinia growth factor (VGF) was chemically synthesized and expressed as a fusion protein in Escherichia coli. The primary structure of the hybrid gene product maintained the same positioning of the three disulfide bonds found in each parent molecule thus conserving the first two loop regions of TGF-alpha and the third loop region of VGF. After cleavage with CNBr its renatured biological activity was found to be comparable to TGF-alpha and VGF with respect to binding to the epidermal growth factor receptor, stimulation of DNA synthesis and induction of anchorage-independent growth of NRK cells in the presence of TGF-beta. Thus, we suggest that similar domains can be interchanged within the same family of molecules and equivalent functionality maintained.