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Primary cultures of uterine smooth muscle cells from post-partum rats express interstitial collagenase in response to serotonin and the serotonin-dependent production of interleukin-1 (IL-1) [Wilcox, B.D., Dumin, J.A. and Jeffrey, J.J. Serotonin regulation of interleukin-1 messenger RNA in rat uterine smooth muscle cells. Relationship to the production of interstitial collagenase J. Biol. Chem., 269, (1994a), 29658]. Transient transfections of these cells indicate that rat collagenase transcription is regulated via a proximal consensus AP-1 site within an extended palindrome. Mutation of either the AP-1 site or extended palindrome (EP) decreases promoter activity to approximately 30% of the wild-type. Electrophoretic mobility shift assays reveal the binding of smooth muscle cell nuclear proteins to the AP-1 EP. This binding is barely detectable after mutation of the EP and is completely eliminated by mutation of the AP-1 heptamer. Competition experiments demonstrate that binding to the AP-1 EP is specific and of higher affinity than binding to oligonucleotides containing a mutated EP. Binding to the AP-1 EP is higher when smooth muscle cells are cultured in the presence of serotonin than in its absence. Although IL-1 is required for collagenase transcription, binding to the AP-1 EP appears to be IL-1-independent. FosB, Fra-2, c-Jun, JunB and, most abundantly, JunD bind the AP-1 EP in the absence and presence of serotonin. In contrast, Fra-1 expression and binding are serotonin-dependent suggesting that the activation of Fra-1 may be a key component of collagenase transcriptional activation.

The activation of the gene for interstitial collagenase in myometrial smooth muscle cells is absolutely dependent upon the presence of serotonin. Our previous studies investigating the mechanisms of this induction demonstrated that the mRNAs of both interleukin-1 (IL-1) isoforms, IL-1alpha and IL-1beta, are induced by serotonin and that the induction of IL-1 is required for the subsequent induction of collagenase. These data provided compelling evidence that serotonin-induced IL-1 acts via an autocrine loop in activating the collagenase gene. The experiments described here were designed to examine the potential role of each IL-1 isoform in collagenase production by using neutralizing antisera specific to each isoform of the cytokine. The antisera were examined for their ability to inhibit the serotonin-dependent production of the mRNA for collagenase and of the cytokines themselves. Neutralizing antiserum against IL-1alpha, but not against IL-1beta, inhibited the induction of the mRNA for collagenase and of the mRNAs for both IL-1alpha and IL-1beta. Western analysis indicated that detectable levels of IL-1alpha protein, but not that of IL-1beta, are produced at the time of serotonin-dependent collagenase induction. In contrast, significant levels of IL-1beta protein are detected only when bacterial lipopolysaccharide is added to the cells. Taken together, the results of our study indicate that IL-1alpha, but not IL-1beta, plays an obligatory role in multiple serotonin-mediated gene regulations in the myometrial smooth muscle cell. In addition, the data suggest that IL-1beta production has the potential for modifying myometrial function in pathological settings, particularly that of uterine infection.

The regulation of collagen gene expression by serotonin was investigated in rat uterine smooth muscle cells. Serotonin treatment of myometrial cells caused decreases of up to 10-fold in levels of type I collagen mRNA. Decreases in secreted type 1 collagen protein paralleled decreases in collagen mRNA. The effective half-life of collagen mRNA in serotonin-treated cells was approximately 1.7 days. Selective 5-HT2 receptor agonists mimicked the effects of serotonin, while the effects of serotonin were blocked by 5-HT2 antagonists. Nuclear run-on analysis showed that serotonin-dependent decreases in collagen mRNA are accompanied by decreased transcription. Progesterone analogs, which inhibit the serotonin-dependent activation of the gene for interstitial collagenase, had no effect on the ability of serotonin to decrease collagen mRNA. Conversely, the cell-permeable cAMP analog, 8-bromo-cAMP, mimicked the effects of serotonin on type I collagen mRNA and protein. Serotonin also decreased levels of the mRNAs for type III collagen and fibronectin, but had no effect on the mRNAs for type IV collagen. These results indicate that serotonin, previously shown to upregulate the interstitial collagenase gene, downregulates the gene for type I collagen and other extracellular matrix proteins, possibly by a novel mechanism of action downstream of 5-HT2 receptor binding.

Previous studies have shown that the production of interstitial collagenase by rat myometrial smooth muscle cells is dependent on serotonin. These cells fail to produce collagenase early in culture, however, and produce the enzyme only 8-12 days after confluence. During the early quiescent period, collagenase production can be induced by low concentrations of bacterial endotoxin. Under these conditions, interleukin (IL)-1 alpha and IL-1 beta mRNAs increase coincident with collagenase and collagenase mRNA. Serotonin removal decreases IL-1 alpha and IL-1 beta mRNAs, and effect that is rapidly reversed upon readdition of serotonin. Conversely, serotonin-dependent increases in IL-1 mRNAs are blocked by progesterone. Experiments with 5-HT2 receptor agonists and antagonists indicate that induction is mediated by the 5-HT2 receptor subtype. In serotonin-treated cells late in culture, IL-1 mRNAs increase coincident with the production of collagenase. Similarly, exogenous IL-1 fully substitutes for lipopolysaccharide in stimulating myometrial cells to produce collagenase early in culture. Cells treated with IL-1 receptor antagonist fail to make IL-1 mRNAs or collagenase but produce collagenase and IL-1 mRNAs following antagonist removal. These results indicate that serotonin-dependent IL-1 production by the myometrial cell is required for collagenase production and that IL-1 participates in its own production via an autocrine mechanism.

This report seeks to further define the requirements for the previously established induction of collagenase gene expression by serotonin and inhibition by progesterone in primary cultures of rat uterine smooth muscle cells. Detectable increases in collagenase production were observed after as little as 3 h exposure of cells to 5 microM serotonin, with maximal induction occurring after approximately 8 h of exposure. The apparent half-life of collagenase mRNA upon removal of serotonin was estimated to be approximately 12 h, and was not dependent on the duration of induction. Inhibition by either cycloheximide or progesterone showed similar half lives for collagenase mRNA, however a much shorter half-life (6 h) was obtained in the presence of actinomycin D. These experiments suggest that neither serotonin induction nor progesterone inhibition of collagenase synthesis represents a primary effect on collagenase gene transcription. Rather they appear to be secondary to changes that occur at one or more primary intermediate genes whose induction or decay must occur prior to changes in collagenase transcription. The progesterone receptor antagonist, RU-486, abrogates the ability of progesterone to inhibit serotonin-induced collagenase gene expression, indicating that the effects of progestins in this system likely are receptor-mediated. Finally, the present studies demonstrate that pretreatment of cells for times as long as 5 days with medroxyprogesterone in the absence of serotonin is unable to prevent subsequent serotonin-induced collagenase mRNA increases. These data suggest the possibility of a unique interaction between the molecular pathways of inducer and inhibitor, one in which serotonin may help mediate the progesterone-dependent repression of the levels of collagenase mRNA.

Recent studies have shown that serotonin (5-hydroxytryptamine; 5-HT) is required for the induction of interstitial collagenase in cultured rat and human myometrial smooth muscle cells. The present study was performed to determine which serotonin receptor subtype mediates the induction of collagenase in these cells. [125I]DOI ((+/- )-1-(2,5-dimethoxy-4-[125I]iodophenyl)-2-aminopropane), a 5-HT2 receptor agonist radioligand, bound specifically to sites in myometrial cell membranes, and exhibited binding characteristics essentially identical to those observed with brain 5-HT2 receptors. Radioligands selective for other serotonin receptor subtypes (5-HT1 and 5-HT3) failed to yield detectable binding. Northern blot analysis demonstrated the presence of 5-HT2 mRNA in the uterine smooth muscle cell cultures, whereas transcripts for 5-HT1A and 5-HT1C receptors were not detectable. Moreover, RT-PCR indicated that 5-HT2 receptor mRNA is present in freshly isolated uterine tissue as well. Selective antagonists of the 5-HT2 receptor, ketanserin and spiperone, displayed concentration-dependent inhibition of serotonin-mediated collagenase induction in the myometrial cultures. These antagonists yielded IC50 values of 4.7 nM and 2.7 nM respectively, characteristic of values expected from a 5-HT2 receptor-mediated response. In addition, a number of selective 5-HT2 receptor agonists (quipazine, alpha-methyl-serotonin, DOI) mimicked the ability of serotonin to induce collagenase production, whereas compounds selective for 5-HT1 and 5-HT3 receptor subtypes had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of collagenase gene expression by serotonin and progesterone was investigated in primary cultures of rat uterine smooth muscle cells. Northern blot analysis demonstrates that serotonin (5-hydroxytryptamine (5-HT)), when administered to cells in serotonin-depleted medium, causes 6-8-fold increases in levels of collagenase mRNA. Selective serotonin 5-HT2 receptor agonists were able to mimic the effect of the natural hormone, while the induction by serotonin could be blocked by 5-HT2 receptor antagonists. Addition of phorbol ester (PMA) to 5-HT-depleted cultures fully mimicked the effect of 5-HT on collagenase mRNA induction. Treatment with progesterone analogs caused a decrease in collagenase mRNA, even in the presence of saturating levels of serotonin or PMA. In all experiments, levels of secreted collagenase were observed to correspond to levels of collagenase mRNA. Experiments with cycloheximide demonstrate that serotonin- and PMA-induced increases in collagenase mRNA are dependent on protein synthesis. Furthermore, nuclear run-on analysis shows that mRNA increases are accompanied by increases in initiation of transcripts. These data indicate that transcription of collagenase mRNA in myometrial smooth muscle cells is stimulated by serotonin, possibly via activation of protein kinase C, but is in some way prevented by the negative influence of progesterone.