Novel Role for Claudin-11 in the Regulation of Osteoblasts via Modulation of ADAM10-Mediated Notch Signaling.

The claudin (Cldn) family comprises 27 members of 20 to 34 kDa transmembrane tight junction proteins. In addition to Cldns' established canonical role as barriers controlling paracellular flow of molecules, a distinct noncanonical role for them as mediators of cell signaling is now emerging. In our studies evaluating Cldn family expression levels during osteoblast differentiation, Cldn-11 showed the largest increase (60-fold). Immunohistochemistry studies revealed high Cldn-11 expression in trabecular (Tb) bone lining cells. Micro-CT analysis of femurs and vertebrae of Cldn-11 knock-out (KO) mice at 12 weeks of age exhibited a 40% (p < 0.01) reduction in Tb bone volume adjusted for tissue volume compared with control mice, a change caused by significant reductions in Tb number and thickness and increase in Tb separation. Histomorphometry and serum biomarker studies revealed that reduced bone formation, not increased resorption, is the cause for reduced Tb bone volume in the Cldn-11 KO mice. Cldn-11 KO osteoblasts expressed reduced ALP and BSP, whereas Cldn-11 overexpression in MC3T3-E1 cells increased expression of ALP and BSP. Mechanistically, Cldn-11 interacted with tetraspanin (Tspan)3 in osteoblasts, and Tspan3 knockdown reduced osteoblast differentiation. Because members of the Tspan family regulate cell functions via Notch signaling, we evaluated whether Cldn-11/Tspan3 regulates Notch signaling in osteoblasts. Accordingly, Notch targets Hey1 and Hey2 were significantly upregulated in Cldn-11 overexpressing cultures but downregulated in both Cldn-11 KO and Tspan3 knockdown osteoblasts. Because ADAM10 has been shown to interact with Tspan family members to regulate Notch signaling, we evaluated whether Cldn-11 regulates ADAM10 expression. Cldn-11 overexpressing cells express more mature ADAM10, and an ADAM10 inhibitor blocked the Cldn-11 effect on osteoblast differentiation. Based on these data, we propose Cldn-11 as a novel component of an osteoblast cell surface protein complex, comprising Tspan3 and ADAM10, which regulates Notch signaling and cell differentiation.