Stimulation of rat Schwann cell proliferation by estradiol: synergism between the estrogen and cAMP.

Schwann cells dissociated from sciatic nerves of 4- to 5-day-old rats were established in long-term primary cultures. After treatment with anti-Thy-1.1 and complement, pure Schwann cell cultures were obtained as confirmed by indirect immunofluorescence staining of the S-100 antigen. The effect of estradiol on Schwann cell proliferation was tested. The estrogen alone had no effect on cell multiplication, but in the presence of forskolin (5 microM) or dibutyryl cyclic AMP (dbcAMP, 1 microM), estradiol (100 nM) became a potent mitogen. This effect was specific since when the anti-estrogen ICI-164,384 (200 nM) was added to the cultures, it completely inhibited the mitogenic effect of estradiol. In the absence of the estrogen, ICI-164,384 did not inhibit dbcAMP-induced cell growth. The effect of progesterone was also tested in the presence of forskolin, and no influence on cell multiplication was seen. Specific and saturable binding sites for estradiol were measured in Schwann cells by whole cell assay after labeling cells at 37 degrees C with [3H]estradiol and about 3000 specific binding sites per cell were found. Treatment of the cultures with forskolin (5 microM) for 48 or 72 h, but not for shorter times, resulted in a two-fold increase in estradiol binding. Estrogen receptors were visualized inside Schwann cells with a monoclonal anti-ER antibody by indirect immunofluorescence staining. Double-label immunofluorescence of ER and S-100 showed that ER immunostaining was restricted to spindle-shaped Schwann cells expressing the S-100 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of steroid hormone receptors and steroid action in primary cultures of rat glial cells.

Primary cultures of rat glial cells were established from newborn rat forebrains. A mixed population of oligodendrocytes and astrocytes was obtained, as confirmed by indirect immunofluorescence staining with specific markers for each cell type. Receptors were measured 3 weeks after primary culture in glial cells cultured in the presence or not of 50 nM estradiol and we have identified progesterone, glucocorticoid, estrogen, and androgen receptors (PR, GR, ER and AR), but only PR was inducible by the estrogen treatment. This estrogen-induction of PR was more dramatic in glial cells derived from female offsprings than from males, as measured by binding studies and by immunohistochemical techniques with the KC 146 anti-PR monoclonal antibody. The antiestrogen tamoxifen inhibited the estrogen induction, but had no effect by itself on PR concentration. Specific binding sites for PR, GR, ER and AR were measured by whole cell assays after labeling cells with, respectively, [3H]R5020, [3H]dexamethasone, [3H]OH-tamoxifen or [3H]R1881. PR and GR were also analyzed by ultracentrifugation and after exposure of cells to agonists, both receptors were recovered from cytosol as a 9S form, and from the nuclear high-salt, tungstate ions-containing fraction as a 4-6S form. In contrast, when the antiprogestin- and antiglucocorticosteroid RU486 was used as a ligand, a non-activated 8.5S receptor complex was found for both receptors in this nuclear fraction. The 8.5S complex of the GR was further analyzed in the presence of specific antibodies and, in addition to GR, the presence of the heat shock protein hsp90 and of a 59 kDa protein was found. During primary culture, the effects of progesterone (P) and estradiol (E2) were tested on glial cell multiplication, morphology and differentiation. Cell growth was inhibited by P and stimulated by E2. Both hormones induced dramatic morphologic changes in oligodendrocytes and astrocytes and increased synthesis of the myelin basic protein in oligodendrocytes and of the glial fibrillary acidic protein in astrocytes.