Abstract
Sialomucin complex (SMC, rat Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ASGP-1 (for ascites sialoglycoprotein-1) and a transmembrane subunit ASGP-2, produced from a single gene and precursor. SMC expression is tightly regulated in mammary gland; the level in lactating mammary gland is about 100-fold that in virgin gland. In rat mammary epithelial cells, SMC is post-transcriptionally regulated by Matrigel by inhibition of SMC precursor synthesis. SMC is also post-transcriptionally regulated by transforming growth factor-beta (TGFbeta). The repression of SMC expression by TGFbeta is rapid, is independent of TGFbeta-induced cell cycle arrest, and does not require new protein synthesis. Unlike Matrigel, TGFbeta does not reduce SMC protein synthesis, as SMC precursor accumulation is equivalent in TGFbeta-treated and untreated cells. Instead, SMC precursor in TGFbeta-treated cells is more persistent and does not become processed as rapidly into mature ASGP-1 and ASGP-2, indicating that TGFbeta disrupts processing of SMC precursor. These results indicate that SMC, a product of normal mammary gland and milk, is regulated by TGFbeta by a novel post-translational mechanism. Thus, SMC is regulated by multiple post-transcriptional mechanisms, which serve to repress potential deleterious effects of overexpression.
Highlights
TGFb1 is a member of a family of growth factors that have been shown to have extensive effects on the maturation and function of normal mammary gland
SMC (ASGP-2) Expression in Cultured MEC in the Presence or Absence of transforming growth factor-b (TGFb)—We have shown previously that SMC/ Muc4 protein is induced rapidly when isolated mammary epithelial cells are cultured as a monolayer on plastic tissue culture dishes
In all tissues studied to date, including mammary gland [8, 17], ASGP-1 and ASGP-2 recognizes the remaining SMC (ASGP-2) are present as a complex, allowing us to use immunoblotting of ASGP-2 for the analysis of SMC
Summary
Materials—The MAT-B1 ascites subline of the 13762 rat mammary adenocarcinoma was maintained by weekly passage [28]. After an additional 24 h cells were washed twice with PBS, starved for 30 min in Cys/Metfree Dulbecco’s minimal essential medium supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, 2 mM glutamine, and 10 mM Hepes, and incubated in 1 ml of labeling medium (starvation medium 1 550 mCi/ml [35S]Cys 1 [35S]Met) (EXPRE35S35S Protein Labeling Mix, NEN Life Science Products) for times ranging from 0 to 6 h. For pulse-chase studies, labeled cells were washed twice with prelabeling medium and incubated in Ham’s F-12 supplemented with 10% FCS and 200 pM TGFb, where indicated, for times ranging from 0 to 8 h. Cell lysates (equivalent counts used for samples for each time point) were immunoprecipitated with polyclonal anti-ASGP-2 antiserum and protein A-agarose beads (Sigma) overnight at 4 °C with rotation. Diluted samples (equivalent total counts per time point) were analyzed by SDS-PAGE and fluorography with Fluoro-Hance autoradiography enhancer (Research Products International Corp., Mount Prospect, IL)
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