Differential Expression of Claudin Family Members during Osteoblast and Osteoclast Differentiation: Cldn-1 Is a Novel Positive Regulator of Osteoblastogenesis

摘要

Claudins (Cldns), a family of 27 transmembrane proteins, represent major components of tight junctions. Aside from functioning as tight junctions, Cldns have emerging roles as regulators of cell proliferation and differentiation. While Cldns are known to be expressed and have important functions in various tissues, their expression and function in bone cells is ill-defined. In this study, the expression of Cldns was examined during osteoblast and osteoclast differentiation. The expression of Cldn-1, -7, -11, and -15 was downregulated during early stages of osteoclast differentiation, whereas Cldn-6 was upregulated. Moreover, the expression of several Cldns increased 3–7 fold in fully differentiated osteoclasts. As for osteoblasts, the expression of several Cldns was found to increase more than 10-fold during differentiation, with some peaking at early, and others at late stages. By contrast, only expression of Cldn-12, and -15 decreased during osteoblast differentiation. In subsequent studies, we focused on the role of Cldn-1 in osteoblasts as its expression was increased by more than 10 fold during osteoblast differentiation and was found to be regulated by multiple osteoregulatory agents including IGF-1 and Wnt3a. We evaluated the consequence of lentiviral shRNA-mediated knockdown of Cldn-1 on osteoblast proliferation and differentiation using MC3T3-E1 mouse osteoblasts. Cldn-1 knockdown caused a significant reduction in MC3T3-E1 cell proliferation and ALP activity. Accordingly, expression levels of cyclinD1 and ALP mRNA levels were reduced in Cldn-1 shRNA knockdown cells. We next determined if Cldn-1 regulates the expression of Runx-2 and osterix, master transcription factors of osteoblast differentiation, and found that their levels were reduced significantly as a consequence of Cldn-1 knockdown. Moreover, knocking down Cldn-1 reduced β-catenin level. In conclusion, the expression of Cldn family members during bone cell differentiation is complex and involves cell type and differentiation stage-dependent regulation. In addition, Cldn-1 is a positive regulator of osteoblast proliferation and differentiation.