Progestin stimulates the biosynthesis of fibronectin and accumulation of fibronectin mRNA in human endometrial stromal cells

Fibronectin is a major component of decidual basement membrane. In the present study, we have investigated the effect of progestin on the synthesis and secretion of fibronectin in human endometrial stromal cells. Stromal cells were isolated during the menstrual cycle and cultured in RPMI-1640 with 2% fetal calf serum supplemented with progesterone or medroxyprogesterone acetate (MPA) in a long-term culture system. Indirect immunofluorescent staining showed that fibronectin was uniformly distributed in the intracellular and extracellular regions of stromal cells treated with MPA for 14 days. The biosynthesis and secretion of this protein and the accumulation of cellular fibronectin mRNA were studied after various culture periods. Cells were pulse-labelled with [35S]methionine to determine the amount of newly synthesized fibronectin secreted into the culture medium. A monoclonal antibody (Mab) identified human fibronectin on SDS-polyacrylamide gel electro-phoresis (SDS–PAGE), showing a predominant band (Mr 230–250 kDa) which migrated with authentic fibronectin run in parallel. In six endometrial specimens, the amount of radioactivity incorporated as [35S]fibronectin was increased by progestin. Maximal stimulation occurred after 6 days treatment with MPA. Culture beyond 16 days reduced the rate of synthesis and secretion to 40% of the maximum. The effect of progestin was dose dependent with 0.02, 0.2 and 1 μM progesterone, producing 2.0, 3.8 and 11-fold increases respectively, over the control. Medroxyprogesterone acetate was more effective than progesterone, the maximal response (10-fold increase) being achieved at 0.02 μM MPA. When MPA pretreated cells were incubated with an anti-progestin, RU 486, the synthesis and secretion of fibronectin was reduced to 20% of that in MPA treated cells. Fibronectin mRNA in stromal cells was identified by Northern blot analysis with a [32P]DNA probe prepared from cDNA of human fibronectin. A single size of mRNA was observed (7.9 kb). The amount of mRNA was increased by MPA. The maximal mRNA level was maintained between 6 and 15 days of incubation and reduced to less than 40% thereafter. Relaxin, which enhances the production of prolactin (PRL) and insulin-like growth factor binding protein (IGFBP-1), did not enhance or slightly reduced (10–20%) the synthesis of fibronectin and accumulation of fibronectin mRNA in this system, this indicates that the biosynthesis and secretion of fibronectin is specifically stimulated by progestin. The progestin-mediated induction coincides with the accumulation of intracellular mRNA in human endometrial stromal cells.