Role of activin-A and myostatin and their signaling pathway in human myometrial and leiomyoma cell function.

CONTEXT: Uterine leiomyomas are highly prevalent benign tumors of premenopausal women and the most common indication for hysterectomy. However, the exact etiology of this tumor is not fully understood. OBJECTIVE: The objective of the study was to evaluate the role of activin-A and myostatin and their signaling pathways in human myometrial and leiomyoma cells. DESIGN: This was a laboratory study. SETTING: Myometrial and leiomyoma cells (primary and cell lines) were cultured in vitro. PATIENTS: The study included premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. INTERVENTIONS: Primary myometrial and leiomyoma cells and/or cell lines were treated with activin-A (4 nM) and myostatin (4 nM) for different days of interval (to measure proliferation rate) or 30 minutes (to measure signaling molecules) or 48 hours to measure proliferating markers, extracellular matrix mRNA, and/or protein expression by real-time PCR, Western blot, and/or immunocytochemistry. RESULTS: We found that activin-A and myostatin significantly reduce cell proliferation in primary myometrial cells but not in leiomyoma cells as measured by a CyQUANT cell proliferation assay kit. Reduced expression of proliferating cell nuclear antigen and Ki-67 were also observed in myometrial cells in response to activin-A and myostatin treatment. Activin-A also significantly increased mRNA expression of fibronectin, collagen1A1, and versican in primary leiomyoma cells. Finally, we found that activin-A and myostatin activate Smad-2/3 signaling but do not affect ERK or p38 signaling in both myometrial and leiomyoma cells. CONCLUSIONS: This study results suggest that activin-A and myostatin can exert antiproliferative and/or fibrotic effects on these cell types via Smad-2/3 signaling.

Possible role of RKIP in cytotrophoblast migration: immunohistochemical and in vitro studies.

Raf kinase inhibitor protein (RKIP) regulates growth and differentiation signaling of mitogen-activated protein kinases (MAPK), GRK2 and NF-kappaB pathways each of which regulates cytotrophoblast differentiation and normal placental development. We show here that RKIP is expressed in human normal and preeclampic placentas as detected by immunostaining. RKIP was detected in villous cytotrophoblast in normal placenta and switched to syncytiotrophoblast in pre-eclampsia (PE)-complicated pregnancies. RKIP was also localized in extravillous cytotrophoblast of cell islands and cell columns both in normal and in PE placentas, although staining was less uniform in the latter specimens. In order to test RKIP involvement in cytotrophoblast function, we performed in vitro studies on HTR-8/SVneo cells, a first trimester cytotrophoblast cell line. We show that the RKIP inhibitor locostatin reduces ERK phosphorylation and impairs HTR-8/SV neo cells motility in wound closure experiments. We also document the presence of GRK2 mRNA, the reduction of phosphorylated RKIP expression by locostatin and the induction of PAI mRNA expression in HTR-8/SV neo cells, suggesting the involvement of GRK2 and NF-kappaB pathways in these cells. In conclusion, our work provides evidence that RKIP is a novel factor expressed in cytotrophoblast cells where it likely regulates cell migration.