Activin responsive PAI-1 promoter activity in granulosa cells is dependent on attachment to extracellular matrix

Abstract

Objective: We have previously shown in a rodent organ culture model that preantral follicle development may be regulated by an interaction between extracellular matrix (ECM) and activin-a (1). The objectives of this study were 1) to characterize the activin receptors in a spontaneously immortalized rodent granulosa cell line (SIGC), 2) to show that these receptors in SIGC are functional, 3) to demonstrate that activin responsiveness is dependent on the presence of ECM. Design: An in vitro study of SIGC. SIGC carry the characteristics of preantral/undifferentiated granulosa cells (2) and express integrins that bind collagen (C), and laminin (L)(3). Materials/Methods: Expression of activin receptor type IB, IIA, and IIB were determined by Western blotting. To determine whether these receptors are functional, SIGC were transfected by electroporation with 40 mg of p3TP-lux and 10 mg of GFP plasmid in a serum-free medium. The p3TP-lux reporter gene contains the Activin-responsive PAI-1 promoter. As determined by the percentage of GFP positive cells the transfection efficiency was approximately 20–30%. After serum starvation SIGC were plated on laminin (L), collagen (C), or polylysine (P, negative control) coated coverslips and treated with 0–20 ng/mL of activin-a for 48 hours. The promoter activity was measured by a luminometer and corrected for protein concentration. Results: SIGC expressed activin type IB, IIA, and IIB receptors. The results of transfection experiments are summarized in the figure. The p3TP-lux promoter activity was increased with activin-a treatment in a dose dependent fashion on ECM but not on polylysine. There was no significant difference in promoter activity between cells plated on L and C. The promoter activity was negligible in cells plated on P. Conclusions: 1) SIGC express functional activin receptors and thus are suitable for studying the effects of activin treatment on granulosa cells, 2) In the SIGC model, promoter response to activin-a is dependent on the presence of ECM. Given the known interaction between tyrosine kinase-type receptor signaling (growth factor signaling) and ECM-induced integrin signaling on cell growth and differentiation (4), a similar interaction may exist between the serine/threonine kinase type receptor (i.e activin) signaling and integrin signaling pathways. 1. Oktay et al. Biol Reprod. 2000;63:457–61. 2. Stein et al. Cancer Res. 1991;51:696–706. 3. Oktay et al. J Soc Gynecol Investig. 2002;9:338A. 4. Oktay et al. J Cell Biol. 1999;145:1461–9. Supported by: ASRM-Serono Research grant to K.O., and by the Center for Reproductive Medicine & Infertility.