Localization and regulation of IL-1alpha in rat myometrium during late pregnancy and the postpartum period.

Interleukin-1 (IL-1) has been implicated as a participant in preterm labor that is induced by bacterial infection. Previously, we showed that serotonin-induced production of IL-1alpha by myometrial smooth muscle cells in vitro is also essential for the synthesis of interstitial collagenase. It is therefore likely that IL-1alpha production in uterine tissues has implications for both the normal physiology of involution and for the pathophysiological mechanisms of preterm labor. The objective of this study was to characterize the serotonin-induced production of IL-1alpha by myometrial cultures in vitro and to assess the production of IL-1alpha and its relationship to collagenase production in vivo during pregnancy and the postpartum period. Immunohistochemistry demonstrated IL-1alpha protein in the nuclei and cytoplasm of serotonin-treated myometrial cells. IL-1alpha levels were decreased by treatment with progesterone or IL-1-receptor antagonist but were unaffected by lipopolysaccharide. Western analysis of myometrium from pregnant rats showed low levels of IL-1alpha during midpregnancy with increased concentrations at days 21 and 22 and postpartum. IL-1alpha mRNA levels also increased from days 15 to 22. Levels of mRNA for IL-1beta also increased, although to a lesser degree than IL-1alpha. Both mRNAs decreased postpartum. Conversely, mRNA for interstitial collagenase was barely detectable at term but increased postpartum. Together, these data show that serotonin stimulates IL-1alpha production in vitro and indicate that normal myometrium from pregnant rats is an identifiable source of IL-1 during late pregnancy. The findings are consistent with the possibility that myometrial IL-1alpha participates in normal labor as well as the postpartum production of interstitial collagenase.

Progesterone mediates decreases in uterine smooth muscle cell interleukin-1alpha by a mechanism involving decreased stability of IL-1alpha mRNA.

The regulation, by progesterone, of serotonin-induced interleukin-1alpha production was studied in primary cultures of rat uterine smooth muscle cells. Prior reports from this laboratory have demonstrated that these cells produce IL-1alpha and IL-1beta mRNAs in response to the hormonal action of serotonin. Results of the present study indicate that treatment of myometrial smooth muscle cells with medroxyprogesterone acetate (MPA) results in a marked decrease in IL-1alpha protein as measured by western blot analysis. These decreases occur even in the presence of maximally-inducing concentrations of serotonin. MPA-mediated changes in IL-1alpha protein are characterized by a rapid decline in IL-1alpha mRNA levels. This inhibition by medroxyprogesterone also occurs when cells are stimulated to produce IL-1alpha by PMA rather than serotonin. Thus, when cells are cultured in the presence of both inducer and inhibitor, the inhibitor, progesterone, clearly dominates in the control of IL-1alpha expression. This effect is concentration-dependent, can be mimicked by native progesterone or glucocorticoids, but is unaffected by estradiol. The ability of progestins to decrease IL-1alpha mRNA is blocked by both inhibitors of transcription and translation and by treatment with RU-486. Progesterone had no effect on chloramphenicol acetyl transferase (CAT) transcription from two different IL-1alpha promoter constructs, indicating that progesterone's action appears to be dependent on post-transcriptional rather than transcriptional regulation. Conversely, progesterone accelerated the normal rate of decay of IL-1alpha mRNA that occurs following the removal of serotonin from the cultures. These results suggest that progesterone decreases IL-1alpha levels by stimulating the production of an intracellular intermediate that decreases the stability of IL-1alpha mRNA.

Decreased tyrosine phosphorylation of focal adhesion kinase after estradiol treatment of MCF-7 human breast carcinoma cells.

MCF-7 human breast carcinoma cultures grown in the presence of 17-beta estradiol form solid, multicellular nodules, a process that reflects changes in cell-substrate adhesions and loss of growth inhibition. We examined the effects of estradiol on the status of tyrosine phosphorylation in focal adhesion kinase (FAK) and the association of FAK with paxillin using immunoprecipitations and then probing western blots for FAK, phosphotyrosine, and paxillin. Culture of MCF-7 cells for seven days in the presence of 1 nM E2 resulted in decreased tyrosine phosphorylation of FAK compared to controls. The estradiol-induced effect was blocked by 100 nM of the estrogen receptor antagonist 4-hydroxytamoxifen, indicating dephosphorylation of FAK is an estrogen receptor-mediated event. FAK immunoprecipitated from either estradiol or DMSO-treated cells phosphorylated the exogenous substrate poly(Glu,Tyr), suggesting that the potential kinase activity of FAK was not changed by estradiol. Estradiol treatment also resulted in a reduced association between FAK and paxillin. The decreased phosphorylation levels and reduced association between FAK and paxillin may be important steps leading to the loss of stable focal contacts and loss of growth inhibition during MCF-7 nodulation.

Expression of messenger RNAs for complement inhibitors in human tissues and tumors.

The mRNAs coding for three complement inhibitors produced by human cells, complement cytolysis inhibitor (CLI), decay-accelerating factor (DAF), and CD59, are characteristically distributed among normal tissues. High levels of CLI mRNA are expressed in tissues that express low levels of DAF mRNA and vice versa. Therefore, the expression of these mRNAs shows a mutually exclusive relationship, with the possible exception of the lung, where all these mRNAs are expressed. In contrast, CD59 mRNA is rather uniformly expressed in all tumor cell lines examined, whereas the mRNA for either of the two other complement inhibitors is overexpressed in some specific tumor cells, e.g., HeLa cells overexpress DAF mRNA, while A172 cells overexpress CLI mRNA. These two cell lines were resistant to antibody-dependent complement cytotoxicity. Expression of CLI and DAF mRNA was induced in cells treated with the antitumor drug N-(chloroetyl)-N'-cyclohexyl-N-nitrosourea; these cells became resistant to complement cytotoxicity. A similar pattern of expression was detected in tumor samples obtained during surgery, with a relatively uniform expression of CD59 mRNA and occasional overexpression of CLI or DAF mRNA. These findings suggest that overexpression of complement inhibitors mRNA and of the corresponding proteins may contribute to tumor cell resistance to complement-mediated cytotoxicity.