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We have investigated the effect of type (3 transforming growth factor (TGF-() on the differentiation of skeletal muscle myoblasts. TGF-(3 potently (IDso 10 pM) prevents established cell lines and primary cultures of rat and chicken embryo myoblasts from fusing into multinucleated myotubes. Inhibition of morphological differentiation by TGF-,( correlates with inhibition of the expression of muscle-specific fiiRNAs and proteins, strong induction of extracellular matrix type I collagen and fibronectin, and a marked tendency of the treated myoblasts to aggregate into densely multilayered arrays or clusters. Myogenic differentiation can resume after removal of TGF-j3 from the medium. Examination of the time of action of TGF-(3 shows that myoblasts stochastically reach a point beyond which they become insensitive to the inhibitory action of TGF-(8. This resistance of committed myoblasts to the inhibitory action of TGF-.3 is not associated with any measurable change in the number or affinity of TGF-(3 receptors in those cells. The results indicate that TGF-(3 is a potent inhibitor of myogenesis and may regulate muscle development in vivo.Transforming growth factor ,3 (TGF-/3), a hormonally active polypeptide found in normal and transformed tissues, is a potent regulator of cell development (for review, see ref. 1). At picomolar concentrations TGF-P induces anchorageindependent growth of fibroblasts but inhibits the growth of certain tumor-derived as well as normal cells (2-6). In addition to its effects on cell proliferation, TGF-,B inhibits adipogenic differentiation without altering the growth rate of preadipocytes (7). Many cells, the growth or differentiation of which is regulated by TGF-,B, respond to this factor with a marked increase in the production and accumulation of the exetracellular matrix proteins fibronectin (8) and collagen (8,9). 'Available evidence suggests that the induction of an abundant extracellular matrix by TGF-,B mediates cellular responses to this factor such as the stimulation of anchorageindependent proliferation (8). These actions of TGF-P are presumably mediated by specific cell surface receptors: three structurally distinct cell surface glycoproteins that exhibit the properties of high-affinity receptors for TGF-P have been identified in mammalian and avian cells (10-12). It is not known whether all three receptor forms are involved in the mediation of TGF-,B actions or whether one receptor form is a signaling receptor, whereas the others have some other function(s).The widespread distribution of TGF-,3 and its receptors in different cell types and tissues suggests that this factor is involved in an ample spectrum of developmental processes in vivo. To obtain further information on the range of cellular targets for TGF-,B, we have investigated the effects of this factor on the differentiation of skeletal muscle myoblasts.These studies reveal a strong inhibitory action of TGF-,3 on myogenesis. METHODSL6Eq rat skeletal muscle myoblasts (13) were grown in Dulbecco's modified Eagle ...

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Mutants of Chinese hamster ovary cells have been found that no longer produce heparan sulfate. Characterization of one of the mutants, pgsD-677, showed that it lacks both N-acetylglucosaminyl-and glucuronosyltransferase, enzymes required for the polymerization of heparan sulfate chains. pgsD-677 also accumulates 3-to 4-fold more chondroitin sulfate than the wild type. Cell hybrids derived from pgsD-677 and wild type regained both transferase activities and the capacity to synthesize heparan sulfate. Two segregants from one of the hybrids reexpressed the dual enzyme deficiency, the lack of heparan sulfate synthesis, and the enhanced accumulation of chondroitin sulfate, suggesting that all of the traits were genetically linked. These fin gs indicate that the pgsD locus may represent a gene involved in the coordinate control of glycosaminoglycan formation.Proteoglycans consist of a core protein and one or more covalently attached glycosaminoglycan chains. Typical animal cells produce proteoglycans bearing chondroitin (dermatan) sulfate or heparan sulfate chains, but the composition varies considerably among different cells (1, 2). To study the regulation of proteoglycan composition, we have isolated Chinese hamster ovary (CHO) cell mutants defective in glycosaminoglycan biosynthesis (3-6). Many of these mutants bear mutations in genes involved in the formation of both heparan sulfate and chondroitin sulfate chains (3, 5). Here we describe a CHO cell mutant, pgsD-677, that specifically lacks heparan sulfate. The mutation in pgsD-677 affects both N-acetylglucosaminyl (GlcNAc)-and glucuronosyl (GlcA)-transferase activities required for heparan sulfate polymerization, suggesting that some form of coordinate regulation of these enzymes exists.EXPERIMENTAL PROCEDURES Cell Cultures. CHO cells (CHO-Ki) were obtained from the American Type Culture Collection (CCL-61). All mutants were identified by colony autoradiography (7), and the purity of each strain was ensured by its isolation from cultures containing only mutant colonies. Cells were maintained in Ham's F12 (8) medium (Mediatech, Washington) supplemented with 10% (vol/vol) fetal bovine serum (HyClone) or in sulfate-deficient medium (4).Cell fusion studies required the isolation of a CHO-K1 subline resistant to thioguanine and ouabain (OT-1). Wildtype cells were treated with 10 ,uM 6-thioguanine in hypoxanthine-free F12 medium supplemented with dialyzed fetal bovine serum. A resistant mutant was isolated and then treated with mutagen (7), and a ouabain-resistant clone was selected in growth medium containing 1 mM ouabain. The introduction of these markers did not alter the proteoglycan composition of the cells.Cell hybrids were generated by co-plating 2 x 105 cells of pgsD-677 and OT-1 in individual wells of a 24-well plate. After overnight incubation, the mixed monolayers were treated for 1 min with 50% (wt/wt) poly(ethylene glycol) (PEG 3320) prepared in F12 medium without serum (9). After 1 day the cells were harvested with trypsin, and multiple 100-mm-diam...

Endoglin is an homodimeric membrane antigen with capacity to bind transforming growth factor-beta (TGF-beta) and whose expression is up-regulated on myeloid cells upon differentiation to macrophages. We have isolated full-length cDNA clones from a lambda gt 10 library, prepared from phorbol 12-myristate 13-acetate-differentiated HL60 cells by screening with an endoglin-specific cDNA probe from endothelial cells. Sequencing of the largest clone (3073 bp), revealed that the leader sequence contains 25 residues and that the 586 amino acids of the extracellular and transmembrane domains were identical to those described for endothelial endoglin. However, the cytoplasmic tail encoded by this cDNA clone contains only 14 amino acids as opposed to the 47 residues previously reported, suggesting the existence of two alternative endoglin variants. The expression of these isoforms was demonstrated by polymerase chain reaction analyses on endothelial cells, myelomonocytic cell lines HL-60 and U-937, and placenta. Independent cDNA constructs corresponding to both forms were transfected into mouse fibroblasts leading to the expression of two distinct endoglin molecules. Both forms were shown to bind TGF-beta 1 and, when overexpressed in transfected mouse fibroblasts, to form disulfide-linked homodimers, indicating that the cysteine residues present in the extracellular domain are responsible for the dimerization.

Abstract. Transforming growth factors/~1 and/~2 bind with high affinity to the core protein of a 250-350-kD cell surface proteoglycan. This proteoglycan (formerly referred to as the type III TGF-/3 receptor) coexists in many cells with the receptor implicated in TGF-/~ signal transduction (type I TGF-/3 receptor), but its function is not known. We report here that soluble TGF-/3-binding proteoglycans are released by several cell types into the culture media, and can be found in serum and extracellular matrices. As has been shown for the membrane-bound form, the soluble proteoglycans have a heterogeneous core protein of 100-120 kD that carries chondroitin sulfate and/or heparan sulfate glycosaminoglycan chains and a small amount of N-linked carbohydrate. The membrane-bound form of this proteoglycan is hydrophobic and associates with liposomes, whereas the soluble forms lack a membrane anchor and do not associate with liposomes. Differences in the electrophoretic migration of the soluble and membrane forms of this proteoglycan suggest additional structural differences in their core proteins and glycosaminoglycan chains. These soluble and membrane-bound proteoglycans, for which we propose the name "betaglycans," might play distinct roles in periceUular retention, delivery, or clearance of activated TGF-B.

Ki-1 (CD30)+ cute T-cell