Differential expression of claudin family members during osteoblast and osteoclast differentiation: Cldn-1 is a novel positive regulator of osteoblastogenesis.

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

The expression level of “non-classic” Cldns during RANKL induced osteoclast differentiation.

<p>The expression level of “non-classic” Cldns (Cldn 11-13, -20, -22, and -23) during primary OC differentiation was evaluated by real time RT-PCR, at different time points (0, 1, and 6 days). Values (means ± SEM; n = 4) are presented as fold change from day 0 (untreated cells), data were analyzed using one-way ANOVA (GraphPad Prism6). A = <0.05 (from day 0), and B = <0.05 (from day 1).</p

The expression level of “classic” Cldns during ascorbic acid induced osteoblast differentiation.

<p>The expression level of “classic” Cldns during primary osteoblast differentiation was measured by real time RT-PCR, at different time points (0, 4, 6, 8, 13, 19, 24 days). A) The expression level of cluster A of the “classic” Cldns, which includes Cldn-3, -4, -5, -6, -8, -9, and -17. B) The expression level of cluster B of the “classic” Cldns, which includes Cldn-1, -2, -7, -14, and -19. C) The expression level of cluster C of the “classic” Cldns, which includes Cldn-10 and -15. Values (means ± SEM; n = 6) are presented as fold change from day 0 (untreated cells), data were analyzed using one-way ANOVA (GraphPad Prism6). A = <0.05 (from day 0), B = <0.05 (from day 4), C = <0.05 (from day 6), D = <0.05 (from day 8), E = <0.05 (from day 13), and F = <0.05 (from day 19).</p

Cldn-1 protein levels during early stages of ascorbic acid induced osteoblast differentiation.

<p>Primary osteoblasts isolated from calvarias were treated with βGP ± AA for 6 days, before Cldn-1 expression was evaluated by western blotting. Values (means ± SEM; n = 4). A = <0.05 vs. untreated cells (βGP alone).</p

The expression level of “classic” Cldns during RANKL induced osteoclast differentiation.

<p>The expression level of “classic” Cldns (Cldn 1-8, -14, -15, and -17) during primary osteoclast differentiation was evaluated by real time RT-PCR, at different time points (0, 1, and 6 days). Values (means ± SEM; n = 4) are presented as fold change from day 0 (untreated cells)), data were analyzed using one-way ANOVA (GraphPad Prism6). A = <0.05 (from day 0), and B = <0.05 (from day 1).</p

The regulation of Cldn-1 expression.

<p>MC3T3-E1 cells were treated with vehicle, BMP-7, IGF-1, vitamin-D3 (Vit-D3), and Wnt3a for 72 hrs, before the expression level of Cldn-1 was examined by real time RT-PCR. Values (means ± SEM; n = 4) are represented as % vehicle-treated control. A = <0.05 vs. vehicle control.</p

The expression level of osteoblastogenic marker genes during ascorbic acid induced osteoblast differentiation.

<p>A) The expression of alkaline phosphatase (ALP), an early stage maker gene of osteoblast differentiation, as determined by real time RT-PCR. B) The expression of osteoclacin, a late stage maker gene of osteoblast differentiation, as determined by real time RT-PCR. Values (means ± SEM; n = 6) are presented as fold change from day 0 (untreated cells), data were analyzed using one-way ANOVA (GraphPad Prism6). A = <0.05 (from day 0), B = <0.05 (from day 4), C = <0.05 (from day 6), D = <0.05 (from day 8), E = <0.05 (from day 13), and F = <0.05 (from day 19).</p

The effect of Cldn-1 knockdown on osteoblast differentiation in MC3T3-E1 cells.

<p>A) ALP activity was determined in MC3T3-E1 cells transduced with control or Cldn-1 shRNA and treated with βGP ± AA for 72 hrs. Values (means ± SEM; n = 8). A = <0.05 vs. βGP treated, and B = <0.05 vs. control shRNA at the corresponding treatment. B) ALP staining was determined on MC3T3-E1 cells transduced with control or Cldn-1 shRNA and treated with βGP ± AA for 6 days, followed by ALP activity staining. Values (means ± SEM; n = 5) are represented as % ALP stained area. A = <0.05 vs. βGP treated, and B = <0.05 vs. control shRNA at the corresponding treatment. C) The expression of osteogenic master transcription factor genes (osterix and Runx-2) and osteogenic marker genes (ALP, bone sialoprotein (BSP), and osteocalcin) was evaluated in control and Cldn-1 shRNA MC3T3-E1 treated β-glycerophosphate (βGP) with or without ascorbic acid (AA) for 24 hrs and 6 days using real time RT-PCR. Values (means ± SEM; n = 4) are presented as % of control shRNA. A = <0.05 vs. control shRNA cells.</p

Primer sequences used in real-time RT-PCR.

<p>Primer sequences used in real-time RT-PCR.</p

The expression level of tartrate resistant acid phosphatase (TRAP), an osteoclastogenic marker gene, during RANKL induced osteoclast differentiation.

<p>The expression level of TRAP during primary osteoclast differentiation was evaluated by real time RT-PCR, at different time points (0, 1, and 6 days). Values are (means ± SEM; n = 4) presented as fold change from day 0 (untreated cells); data were analyzed using one-way ANOVA (GraphPad Prism6). A = <0.05 (from day 0), and B = <0.05 (from day 1).</p