Disruption of claudin-18 diminishes ovariectomy-induced bone loss in mice

Abstract

Claudin-18 (Cldn-18), a member of the tight junction family of proteins, is a negative regulator of RANKL-induced osteoclast differentiation and bone resorption (BR) in vivo. Since estrogen deficiency decreases bone mass in part by a RANKL-mediated increase in BR, we evaluated whether estrogen regulates Cldn-18 expression in bone. We found that Cldn-18 expression was reduced in the bones of estrogen deficient mice, whereas it was increased by estrogen treatment in osteoblasts and osteoclasts in vitro. We next evaluated the role of Cldn-18 in mediating estrogen-induced bone loss. Cldn-18 knockout (KO) and littermate wild-type (WT) mice were ovariectomized (OVX) or sham operated at 6 wk of age, and the skeletal phenotype was evaluated at 14 wk of age. PIXImus revealed that total body, femur, and lumbar BMD were reduced 8-13% (P < 0.05) after 8 wk of OVX compared with sham in WT mice. As expected, total body, femur, and lumbar BMD were reduced 14-21% (P < 0.05) in Cldn-18 KO sham mice compared with sham WT mice. However, ovariectomy failed to induce significant changes in BMD of total body, femur, or vertebra in the Cldn-18 KO mice. μCT analysis of the distal femur revealed that trabecular (Tb) bone volume was decreased 50% in the OVX WT mice compared with sham that was caused by a 26% decrease in Tb number and a 30% increase in Tb separation (all P < 0.05). By contrast, none of the Tb parameters were significantly different in OVX Cldn-18 KO mice compared with sham KO mice. Histomorphometric analyses at the Tb site revealed that neither osteoclast surface nor osteoclast perimeter was increased significantly as a consequence of OVX in either genotype at the time point examined. Based on our findings, we conclude that the estrogen effects on osteoclasts may in part be mediated via regulation of Cldn-18 signaling.

Fig. 1.

Estrogen regulates claudin-18 (Cldn-18) mRNA levels. A: real-time PCR analysis of Cldn-18 mRNA levels in the bones of Sham and overiectomized (OVX) mice (n = 6). B: real-time PCR analysis of Cldn-18 mRNA levels in bone marrow stromal cells (BMSCs), primary osteoblasts (OB), MC3T3-E1 cells, and RAW264.7 cells treated with estrogen (10 nM) or vehicle for 24 h (n = 3–6). †P < 0.05, Sham vs. OVX; *P < 0.05; **P < 0.001, estrogen vs. vehicle. Error bars represent SE.

Fig. 2.

Longitudinal in vivo assessment of bone mineral density (BMD) in female wild-type (WT) and Cldn-18 knockout (KO) mice (n = 8–10/group). Left: total body BMD. Middle: femur BMD. Right: lumbar BMD. *P < 0.05; **P < 0.001, Sham vs. OVX. Error bars represent SE.

Fig. 3.

Difference (in %) in ratio of trabecular bone volume to total volume (BV/TV), trabecular number (Tb.N), and connectivity density (Conn.D) between OVX and Sham at the vertebrae and distal femur in WT and Cldn-18 KO mice (n = 8–10/group). *P < 0.05; **P < 0.001, Sham vs. OVX. Error bars represent SE.

Fig. 4.

Serum alkaline phosphatase (ALP; A) and tartrate-resistant acid phosphatase (TRAP; B) activity in WT and Cldn-18 KO mice at 14 wk of age (n = 12–13/group). *P < 0.05 Sham vs. OVX. Error bars represent SE.