Effects of estrogen, raloxifene, and levormeloxifene on the expression of Rho-kinase signaling molecules in urethral smooth muscle cells

Abstract

Objectives: To investigate the effects of estrogen, raloxifene, and levormeloxifene on the expression of Rho-kinase signaling molecules in urethral smooth muscle cells (USMCs).

Methods: USMCs were isolated from female rats. Expression of calponin and estrogen receptors α (ERα) was detected by immunofluorescence staining. Cells were treated with estrogen, raloxifene, or levormeloxifene at 0, 1, 10, and 100 nmol/L for 48 h and then processed for Western blotting with antibodies against RhoA, Rho kinase I and II (Rock-I and Rock-II), myosin light chain (MLC), phosphorylated MLC, and β-actin. Protein expression was quantitated by densitometry, followed by statistical analysis with β-actin as control.

Results: USMCs expressed calponin and ERα. Treatment of USMCs with estrogen, raloxifene or levormeloxifene resulted in decreased expression of RhoA, Rock-I, Rock-II, and p-MLC in a dosage-dependent manner.

Conclusions: Estrogen, raloxifene, and levormeloxifene may affect urinary continence by inhibiting the expression of Rho-kinase signaling molecules.

FIG. 1

Localization of estrogen receptor and calponin in rat urethra smooth muscle cells (USMCs). UMSC at passage 4 were processed for immunofluorescence staining as described in Materials and Methods. Red fluorescence indicates expression of calponin or ERα. Blue fluorescence indicates cell nuclei (DAPI).

FIG. 2

Effects of estrogen on the expression of RhoA, Rock-I, Rock-II, MLC, and p-MLC. USMCs were treated with estrogen at 0, 1, 10, and 100 nM and analyzed by western blotting for RhoA, Rock-I, Rock-II, MLC, and p-MLC. Three independent experiments were conducted for each drug. One representative graph of the three experiments is shown in panel A for each drug. The expression levels of RhoA, Rock-I, and Rock-II are displayed as relative expression against β-Actin expression (panel B). MLC phosphorylation level is the ratio (in percentage) between p-MLC and MLC expression levels (panel C). Asterisks denote significant difference in comparison with control (0 nM). *, P<0.05; **, P<0.01.

FIG. 3

Effects of raloxifene on the expression of RhoA, Rock-I, Rock-II, MLC, and p-MLC. USMCs were treated with raloxifene at 0, 1, 10, and 100 nM and analyzed by western blotting for RhoA, Rock-I, Rock-II, MLC, and p-MLC. Three independent experiments were conducted for each drug. One representative graph of the three experiments is shown in panel A for each drug. The expression levels of RhoA, Rock-I, and Rock-II are displayed as relative expression against β-Actin expression (panel B). MLC phosphorylation level is the ratio (in percentage) between p-MLC and MLC expression levels (panel C). Asterisks denote significant difference in comparison with control (0 nM). *, P<0.05; **, P<0.01.

FIG. 4

Effects of levormeloxifene on the expression of RhoA, Rock-I, Rock-II, MLC, and p-MLC. USMCs were treated with levormeloxifene at 0, 1, 10, and 100 nM and analyzed by western blotting for RhoA, Rock-I, Rock-II, MLC, and p-MLC. Three independent experiments were conducted for each drug. One representative graph of the three experiments is shown in panel A for each drug. The expression levels of RhoA, Rock-I, and Rock-II are displayed as relative expression against β-Actin expression (panel B). MLC phosphorylation level is the ratio (in percentage) between p-MLC and MLC expression levels (panel C). Asterisks denote significant difference in comparison with control (0 nM). *, P<0.05; **, P<0.01.