The expression of transforming growth factor-βs and TGF-β receptor mRNA and protein and the effect of TGF-βs on human myometrial smooth muscle cells in vitro

Abstract

In this study we investigated the expression of transforming growth factor-β (TGF-β) isoform and TGF-β receptor mRNA and protein, and the effect of TGF-β1-3 on the rate of DNA synthesis and proliferation of human myometrial smooth muscle cells in vitro. To determine these, we utilized primary cultures of myometrial smooth muscle cells, standard and competitive quantitative reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, enzyme-linked immunoassay, radioreceptor assay, [3H] thymidine incorporation and cell proliferation assay. Standard RT-PCR and immunocytochemistry revealed that myometrial smooth muscle cells express TGFβ1-3 and TGF-β type I-III receptor (TGF-βR) mRNA and protein. Quantitative RT-PCR, using an external synthetic RNA standard, indicated that the cells express 10 copies/cell of TGF-β1 and TGF-β2, less than one copy/cell of TGF-β3 and TGF-β type IR, three copies/cell of type IIIR, and >200 copies/cell of TGF-β type IIR mRNA. The cells also synthesized and released TGF-β1 at the rate of 7.8 ± 0.7 ng/106 cells, of which 1.4 ± 0.2 ng/106 cells was in an active form. The rate of [3H] thymidine incorporation or proliferation of subconfluent quiescent smooth muscle cells was not altered by TGF-βs (0.1-10 ng/ml) under serum-free conditions, nor in the presence of 10% fetal bovine serum (FBS). TGF-β1-3 at 0.25-0.5 ng/ml in the presence of 2% FBS, which induces half maximal stimulation of these cells, stimulated the rate (P <0.05), whereas at higher doses it reduced the rate of [3H]-thymidine incorporation compared to the controls. The effect of TGF-β was partially reversible using neutralizing antibodies specific to TGF-β1, TGF-β2 (10 μg/ml) or TGF-β3 (3-6 μg/ml). TGF-βs had no significant effect on cell proliferation determined by cell counting. The data indicate that human myometrial smooth muscle cells express the necessary components of the TGF-β system, suggesting an autocrine/paracrine role for TGF-βs in myometrium.