Regulation of collagenase gene expression by serotonin and progesterone in rat uterine smooth muscle cells.

B.D Wilcox,L Rydelek-Fitzgerald,J.J Jeffrey

doi:10.1016/s0021-9258(19)36750-x

B.D Wilcox, L Rydelek-Fitzgerald + Show 1 more

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https://doi.org/10.1016/s0021-9258(19)36750-x

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Abstract

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.

Highlights

Studies utilizing rat uterine explants (Jeffrey et al, 1971a, 1971b) as well asrat myometrial smooth muscle cells in culture (Jeffreyet al., 1990) indicate a strong repressive effect of progestational steroids on collagenase synthesis in uitro. These studies, did not identify the existence of an inducer of collagenase synthesis in the uterusQ. uite recently onstrate that serotonin- and PMA-inducedincreases in it hasbeen found in this laboratory that rat uterine collagenase mRNA are dependent on protein synthesis
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 influenceof progesterone
Northern blot analysisdemonstrated a 6-7-fold decrease in hybridizable collagenase mRNA over the 5 days in which cells were maintained inmedium containing charcoal-stripped FBSS.ecreted collagenase decreased to undetectable levels

Summary

SerRoetognuilnation of CollagenasEe xGperneession

Reagents and Chemicals-DMEM’ was obtainedfromGIBCOBRL, Gathersburg, MD. FBS was from Hyclone, Logan, UT. Collagenase Actiuity-In all experiments, collagenolytic activity of conditioned medium harvested from cell cultures was assayed on reconstituted [“Clglycine-labeledguineapig skintype I collagen fibrils as previously described (Jeffrey et al, 1990; Nagai et al, 1966). Cells were lysed by adding 2-ml lysis solution directly to 75-cmZ culture flasks,homogenized in a dounce homogenizer, and incubated for 1 h a t 45 “C. Poly(A’) mRNA was allowed to adsorb for 45 min a t room temperature with gentle shaking, thenwas centrifuged for 10 min a t 4500 X g to pellet oligo(dT)-cellulose. Nuclei were isolated by centrifugation a t 500 X g for 10 min at 4 “C, washed once with PBS, resuspended in 100 pl 2 X labeling buffer (100 mM Tris-HC1, pH 8.3, 10 mM MgCl,, 600 mM KC1, 60% (v/v) glycerol) and frozen at -80 “C. Labeled RNA was hybridized to purified cDNAs exactly asdescribed for Northern hybridizations but were allowed to incubate for 4 days a t 42 “C

RESULTS

Serotonin Regulation of Collagenase GeneExpression

DISCUSSION

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