Abstract
Connexin (Cx) proteins are essential for cell differentiation, function, and survival in all tissues with Cx43 being the most studied in bone. We now report that Cx37, another member of the connexin family of proteins, is expressed in osteoclasts, osteoblasts, and osteocytes. Mice with global deletion of Cx37 (Cx37(-/-)) exhibit higher bone mineral density, cancellous bone volume, and mechanical strength compared with wild type littermates. Osteoclast number and surface are significantly lower in bone of Cx37(-/-) mice. In contrast, osteoblast number and surface and bone formation rate in bones from Cx37(-/-) mice are unchanged. Moreover, markers of osteoblast activity ex vivo and in vivo are similar to those of Cx37(+/+) littermates. sRANKL/M-CSF treatment of nonadherent Cx37(-/-) bone marrow cells rendered a 5-fold lower level of osteoclast differentiation compared with Cx37(+/+) cell cultures. Further, Cx37(-/-) osteoclasts are smaller and have fewer nuclei per cell. Expression of RANK, TRAP, cathepsin K, calcitonin receptor, matrix metalloproteinase 9, NFATc1, DC-STAMP, ATP6v0d1, and CD44, markers of osteoclast number, fusion, or activity, is lower in Cx37(-/-) osteoclasts compared with controls. In addition, nonadherent bone marrow cells from Cx37(-/-) mice exhibit higher levels of markers for osteoclast precursors, suggesting altered osteoclast differentiation. The reduction of osteoclast differentiation is associated with activation of Notch signaling. We conclude that Cx37 is required for osteoclast differentiation and fusion, and its absence leads to arrested osteoclast maturation and high bone mass in mice. These findings demonstrate a previously unrecognized role of Cx37 in bone homeostasis that is not compensated for by Cx43 in vivo.
Highlights
Connexin proteins are essential for cell differentiation, function, and survival
Cx37 and Cx43 mRNA expression measured by Quantitative RT-PCR (qPCR) and corrected by GAPDH in murine lumbar vertebra, OB-6, and immortalized calvaria osteoblastic cells and MLO-Y4 osteocytic cells (a) and in osteoblasts and osteocytes isolated from murine calvaria and osteoclasts obtained in vitro by treatment of bone marrow nonadherent cells with M-CSF and sRANKL (b)
Cx37 Is Expressed in Osteoblasts, Osteocytes, and Osteoclasts— Gene expression analysis shows that Cx37 is expressed in whole bone at approximately four times lower levels than Cx43 (Fig. 1a)
Summary
Connexin proteins are essential for cell differentiation, function, and survival. Results: Global deletion of Cx37 results in increased bone mass caused by reduced osteoclast maturation. Nonadherent bone marrow cells from Cx37؊/؊ mice exhibit higher levels of markers for osteoclast precursors, suggesting altered osteoclast differentiation. We conclude that Cx37 is required for osteoclast differentiation and fusion, and its absence leads to arrested osteoclast maturation and high bone mass in mice. These findings demonstrate a previously unrecognized role of Cx37 in bone homeostasis that is not compensated for by Cx43 in vivo. Cx43 is the main connexin expressed in osteoblasts, osteocytes, and osteoclasts, and it has been the focus of research on the role of this family of proteins in the skeleton [1].
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