Abstract. Type 13 transforming growth factor (TGFI3) has been shown to be both a positive and negative regulator of cellular proliferation and differentiation. The effects of TGFI3 also are cell-type specific and appear to be modulated by other growth factors. In the present study, we examined the potential of TGFI3 for control of myogenic differentiation. In mouse C-2 myoblasts, TGFfl inhibited fusion and prevented expression of the muscle-specific gene products, creatine kinase and acetylcholine receptor. Differentiation of the nonfusing muscle cell line, BC3H1, was also inhibited by TGFI3 in a dose-dependent manner (IDs0 ~0.5 ng/ml). TGFI3 was not mitogenic for either muscle cell line, indicating that its inhibitory effects do not require cell proliferation. Inhibition of differentiation required the continual presence of TGFI3 in the culture media. Removal of TGFI3 led to rapid appearance of muscle proteins, which indicates that intracellular signals generated by TGFfl are highly transient and require continuous occupancy of the TGFI~ receptor. Northern blot hybridization analysis using a muscle creatine kinase cDNA probe indicated that TGFI3 inhibited differentiation at the level of muscle-specific mRNA accumulation. These results provide the first demonstration that TGFI3 is a potent regulator of myogenic differentiation and suggest that TGFI3 may play an important role in the control of tissue-specific gene expression during development.

Proteins containing the basic-helix-loop-helix (B-HLH) domain have been shown to be important in regulating cellular differentiation. We have isolated a cDNA for a human B-HLH factor, denoted HEB, that shares nearly complete identity in the B-HLH domain with the immunoglobulin enhancer binding proteins encoded by the E2A and ITF2 genes (E proteins). Functional characterization of the protein expressed from this cDNA indicates that HEB is a third member of the E-protein class of B-HLH factors. HEB mRNA was found to be expressed in several tissues and cell types, including skeletal muscle, thymus, and a B-cell line. HEB, ITF2, and the E12 product of the E2A gene all bound to a similar spectrum of E-box sequences as homo-oligomers. All three factors also formed hetero-oligomers with myogenin, and the DNA-binding specificity and binding off-rates (dissociation rates) were modulated after hetero-oligomerization. Both homoand hetero-oligomers of these proteins were able to distinguish between very closely related E-box sequences. In addition, HEB was shown to form hetero-oligomers with the E12 and ITF2 proteins. Finally, HEB was able to activate gene expression. These data demonstrate that HEB shares characteristics with other E proteins and show that HEB can interact with members of both the myogenic regulatory class and the E-protein class of B-HLH factors. HEB is therefore likely to play an important role in regulating lineage-specific gene expression.The regulation of cell type specification in mammals is accomplished in part by interactions between sequencespecific DNA-binding proteins. One class of proteins that has been implicated in controlling these decisions binds to specific DNA sequences via a basic (B) region that lies adjacent to amino acids that are predicted to form a helixloop-helix (HLH) structure. This class of B-HLH proteins includes the myogenic regulatory factors MyoD, myogenin, myf5, and MRF4/herculin/myf6 (3,4,13,14,26,30,32,41,43). These myogenic factors can form heterodimers with a second group of B-HLH proteins, recently termed the E proteins (23), which include the E2A gene products E12 and E47 and the ITF2 protein (7,23,25,28). The heterodimers formed by these factors play a central role in regulating muscle cell differentiation, apparently via their functions as transcription factors (5,8,18,23).The HLH domain has been shown to mediate dimerization. It is required for homo-oligomerization of the E proteins and for hetero-oligomerization of the E proteins with myogenic activation factors (6, 12, 40). The B region of each of these proteins is found immediately N terminal to the HLH domain and is responsible for DNA binding (6,12,40). The identified binding sites for B-HLH proteins share a consensus sequence, NNCANNTGNN, termed an E box. E-box sequences are found to bind to tissue-specific factors or to be important for the activity of enhancers and promoters of various tissue-specific genes, such as immunoglobulin genes (21, 24), muscle-specific genes (7,17,19,22,25,31,33,34,37,42), and pan...

Several proteins of viral and cellular origin are acylated with myristic acid early during their biogenesis. To investigate the possibility that myristylation occurred cotranslationally, the BC3H1 muscle cell line, which contains a broad array of myristylated proteins, was pulse-labeled with [3H]myristic acid. Nascent polypeptide chains covalently associated with transfer RNA were isolated subsequently by ion-exchange chromatography. [3H]Myristate was attached to nascent chains through an amide linkage and was identified by thin-layer chromatography after its release from nascent chains by acid methanolysis. Inhibition of cellular protein synthesis with puromycin resulted in cessation of [3H]myristate-labeling of nascent chains, in agreement with the dependence of this modification on protein synthesis in vivo. These data represent a direct demonstration that myristylation of proteins is a cotranslational modification.

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Dietary AGEs detrimentally modulate gut microbial ecology and may partially increase colon permeability, which can adversely impact host health.