TRANSFORMING GROWTH-FACTOR-BETA STIMULATES ENDOMETRIAL STROMAL APOPTOSIS IN-VITRO

Active growth and differentiation of endometrial stromal cells during the decidualization of early pregnancy are followed by programmed cell death (apoptosis) in the antimesometrial region of the decidua. The location and temporal specificity of decidual cell apoptosis and the identification of transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 protein and mRNA in the decidua suggested that members of the TGF beta family control stromal apoptosis by autocrine or paracrine mechanisms. To examine this possibility, ovariectomized rats were pretreated sequentially with estradiol (2 days), no treatment (2 days), and medroxyprogesterone acetate 48 h before death. Endometrial stromal cells were selectively isolated by enzymatic digestion and cultured for 48 h before the experiments. The addition of TGF beta 1 (10(-9)-10(-12) M) stimulated a dose-dependent increase in 200-basepair nucleosomal fragmentation of DNA, indicating increased apoptosis. TGF beta 1 and TGF beta 2 had an equivalent effect in stimulating stromal apoptosis. If an autocrine or paracrine mechanism is responsible for the control of stromal apoptosis, increased apoptosis in cultures at high cell density and stromal secretion of TGF beta would be expected. Increasing the density of stromal cell cultures increased levels of nucleosomal DNA fragmentation. To examine stromal secretion of growth factor, cell cultures were incubated with defined medium, and concentrated media were subjected to immunoblot analysis using antibodies specific for TGF beta 1 and -beta 2. Only the antibody to TGF beta 2 detected secreted growth factor. The concentration of the secreted growth factor increased with increasing density of cell culture. To determine whether stromal secretion of TGF beta 2 is responsible for the increasing nucleosomal DNA fragmentation observed with increasing cell density, cells were cultured with and without the addition of neutralizing antibodies to TGF beta s. The addition of monoclonal antibodies to TGF beta 1,2,3 or TGF beta 2,3 inhibited apoptosis. These data provide evidence that TGF beta 2 controls apoptosis in endometrial stromal cells in vitro by an autocrine/paracrine mechanism and suggest that this mechanism controls apoptosis in these cells during early pregnancy.