Rat uterine stromal cells: thrombin receptor and growth stimulation by thrombin

The estrogen-stimulated maturation of the immature rat uterus is mediated by peptide growth factors whose expression is regulated by estradiol. We present evidence that thrombin is a uterine growth factor. When an immature rat is given a single injection of estradiol, the uterus increases 50% in wet weight within 3 h through the imbibition of water and plasma proteins, including prothrombin. Tissue factor, the initiator of coagulation, is induced 3- to 4-fold over the same time period. Thrombin is generated in situ from prothrombin through the coagulation cascade. It acts as a growth factor through the proteolytically activated thrombin receptor. Thrombin's role as a growth factor in uterine stromal cells is proven by two lines of evidence: demonstrations that the proteolytically activated thrombin receptor is present and that cultured cells are stimulated to grow by thrombin. Thrombin receptor in the uterus is demonstrated by reverse transcription-PCR for receptor messenger RNA by specific [125I]peptide labeling of a membrane-bound binding protein of about 60 kDa and by Western blot with a thrombin receptor antipeptide antibody. Thrombin's effectiveness as a growth factor is shown by thrombin-stimulated growth of primary stromal cell cultures, with maximum stimulation at 100 nM. That the effect is mediated by the proteolytically activated thrombin receptor is shown by the inhibition of growth by hirudin, a highly specific inhibitor of thrombin; the absence of enhanced growth with Pro-Phe-Arg-chloromethyl ketone-thrombin, an active site-inhibited thrombin derivative; and the stimulation of growth by the thrombin receptor-activating peptide.