Endocrinology, Vol 133, 2545-2553, Copyright © 1993 by Endocrine Society

ARTICLES

Characterization and the regulation of inhibin/activin subunit proteins of cultured rat anterior pituitary cells

LM Bilezikjian, JM Vaughan and WW Vale
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92037.

The production of inhibin/activin by cultured rat anterior pituitary cells was evaluated using specific antisera to inhibin/activin alpha, beta A, and beta B subunit proteins (anti-alpha, anti-beta A, and anti- beta B). Cellular or secreted proteins recognized by the antisera were immunoprecipitated from metabolically labeled cells then analyzed by denaturing polyacrylamide gel electrophoresis. Immunoreactive inhibin/activin beta B proteins were visualized in both cell lysates and the media. Experiments with anti-beta B confirmed that activin-B (beta B beta B) is a local secretory product of cultured rat anterior pituitary cells. The secreted beta B-immunoreactive protein band had an apparent size of 24-25 kilodaltons (kDa) or 14-15 kDa, consistent with the size of unreduced beta B dimer or reduced monomer, respectively. Cell lysates contained two proteins that were specifically immunoprecipitated by anti-beta B. One of these had a mobility of greater than 95 kDa (unreduced) or 55-60 kDa (reduced), probably representing dimers or monomers of the beta B precursor, respectively. The second 14- to 15-kDa (reduced and unreduced) immunoreactive beta B protein band was verified to be the mature beta B monomer. Mature heterodimeric inhibin-B (alpha beta B) was not detected by either anti- alpha or anti-beta B. Multiple protein species, however, were observed to be specifically immunoprecipitated by incubation of cell lysates with anti-alpha. Mature beta A monomer was not detected in any of the samples. The regulation of cellular beta B production was monitored by evaluating its rate of synthesis in pulse-labeled cells. Treatment with either forskolin or 12-O-tetradecanoylphorbol acetate enhanced the rate of [35S]cysteine incorporation into the cellular 14- to 15-kDa beta B monomer, indicating that the activation of either protein kinase A or protein kinase C regulates its production. The rate of cellular beta B accumulation was also regulated by activin-A, inhibin-A, and follistatin; activin-A caused a 30% inhibition in contrast to the 70% stimulation by treatment with either inhibin-A or follistatin. Equimolar concentrations of activin-A and follistatin prevented the net effect produced by either factor alone. None of the immunoreactive alpha-forms was detectable under similar pulse-labeling conditions, and there was no apparent change in their level after labeling to equilibrium (up to 48 h). The observed changes in beta B accumulation may, therefore, reflect the regulated production of pituitary activin- B. Taken together, these results suggest that locally produced activin- B or gonadal activins exert an inhibitory tone on the production of pituitary activin-B and that this negative-feedback control is in turn modulated by inhibins and follistatins. The relative importance of pituitary and gonadal activins, inhibins, and follistatins in the proposed regulatory loop remains to be established.

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