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

Alshbool FZ, Mohan S - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Musculoskeletal Disease Center, Jerry L Pettis VA Med Ctr, Loma Linda, CA 92357, United States of America; Department of Pharmacology, Loma Linda University, Loma Linda, CA 92354, United States of America.

ABSTRACT
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.

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The expression level of “non-classic” Cldns during ascorbic acid induced osteoblast differentiation.The expression level of “non-classic” Cldns during primary osteoblast differentiation was evaluated by real time RT-PCR at different time points (0, 4, 6, 8, 13, 19, 24 days). 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).
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pone-0114357-g006: The expression level of “non-classic” Cldns during ascorbic acid induced osteoblast differentiation.The expression level of “non-classic” Cldns during primary osteoblast differentiation was evaluated by real time RT-PCR at different time points (0, 4, 6, 8, 13, 19, 24 days). 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).

Mentions: Regarding the “non-classic Cldns”, our results indicate that they exhibit a diverse expression profile. While the expression levels of Cldn-11, -13, -22, and -23 were increased significantly during osteoblast differentiation and peaked during either early or late stages (Fig. 6), those of Cldn-12 decreased significantly (Fig. 6). Moreover, no change in the expression level of Cldn-20 was observed during osteoblast differentiation (Fig. 6). Of note, the mRNA of Cldn-16 was found to be undetectable by real-time RT-PCR, at least under our experimental conditions. Collectively, these data suggest that the regulation of expression of Cldns during osteoblast differentiation is complex and differentiation stage dependent.


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

Alshbool FZ, Mohan S - PLoS ONE (2014)

The expression level of “non-classic” Cldns during ascorbic acid induced osteoblast differentiation.The expression level of “non-classic” Cldns during primary osteoblast differentiation was evaluated by real time RT-PCR at different time points (0, 4, 6, 8, 13, 19, 24 days). 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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4257558&req=5

pone-0114357-g006: The expression level of “non-classic” Cldns during ascorbic acid induced osteoblast differentiation.The expression level of “non-classic” Cldns during primary osteoblast differentiation was evaluated by real time RT-PCR at different time points (0, 4, 6, 8, 13, 19, 24 days). 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).
Mentions: Regarding the “non-classic Cldns”, our results indicate that they exhibit a diverse expression profile. While the expression levels of Cldn-11, -13, -22, and -23 were increased significantly during osteoblast differentiation and peaked during either early or late stages (Fig. 6), those of Cldn-12 decreased significantly (Fig. 6). Moreover, no change in the expression level of Cldn-20 was observed during osteoblast differentiation (Fig. 6). Of note, the mRNA of Cldn-16 was found to be undetectable by real-time RT-PCR, at least under our experimental conditions. Collectively, these data suggest that the regulation of expression of Cldns during osteoblast differentiation is complex and differentiation stage dependent.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Musculoskeletal Disease Center, Jerry L Pettis VA Med Ctr, Loma Linda, CA 92357, United States of America; Department of Pharmacology, Loma Linda University, Loma Linda, CA 92354, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus