CCNs, fibulin-1C and S100A4 expression in leiomyoma and myometrium: Inverse association with TGF-β and regulation by TGF-β in leiomyoma and myometrial smooth muscle cells

Abstract

Connective tissue growth factor (CTGF; CCN2) is considered to serve as downstream midiator of TGF-β action in tissue fibrosis. We tested this hypothesis in paired leiomyoma and myometrium by evaluating the expression of TGF-β1/TGF-β3 and CCN2, the other members of the CCN family, CCN3 and CCN4, as well as fibulin-1C and S100A4, calcium-binding proteins that interact with CCNs. The regulatory function of TGF-β1 on the expression of these genes was further evaluated using leiomyoma (L) and myometrial (M) smooth muscle cells (SMC). Real-time PCR, Western blotting and immunohistochemistry revealed that leiomyomas and myometrium express CCNs, fibulin-1C and S100A4, whose levels of expression with the exception of fibulin-1C were lower in leiomyomas and inversely correlated with the expression of TGF-β1 and TGF-β3 (P<0.05). The expression of these genes was menstrual cycle-independent and GnRHa therapy increased the expression of CCN2 in leiomyomas, while inhibiting CCN3, CCN4 and S100A4 in myometrium (P<0.05). TGF-β (2.5 ng/ml) in a time- and cell-dependent manner, and through MAPK and Smad pathways, differentially regulated the expression of these genes in LSMC and MSMC. We concluded that CCNs, fibulin-1C and S100A4 are expressed in leiomyomas/myometrium with relative expression levels inversely correlating with TGF-βs and influenced by GnRHa and TGF-β regulatory actions. The results suggest that unlike other fibrotic disorders, CCN2 (CTGF), at least at tissue level, may not serve as a downstream mediator of TGF-β action in leiomyomas. © 2006 Oxford University Press.