Calmodulin-dependent kinase 1β is expressed in the epiphyseal growth plate and regulates proliferation of mouse calvarial osteoblasts in vitro

Abstract: The Ca2+/Calmodulin-dependent protein kinase (CaMK) family is activated in response to elevation of intracellular Ca2+, and includes CaMK1 (as well as CaMK2 and CaMK4), which exists as different isoforms (α, β, γ and δ). CaMK1 is present in several cell types and may be involved in various cellular processes, but its role in bone is unknown. In situ hybridization was used to determine the spatial and temporal expression of CaMK1β during endochondral bone development in mouse embryos and newborn pups. The cellular and subcellular distribution of CaMK1 was assessed by quantitative immunogold electron microscopy (EM). The role of CaMK1β in mouse calvarial osteoblasts was investigated by using small interfering RNA (siRNA) to silence its expression, while in parallel monitoring cell proliferation and levels of skeletogenic transcripts. cRNA in situ hybridization and EM studies show that CaMK1β is mainly located in developing long bones and vertebrae (from ED14.5 until day 10 after birth), with highest expression in epiphyseal growth plate hypertrophic chondrocytes. By RT-PCR, we show that CaMK1β2 (but not β1) is expressed in mouse hind limbs (in vivo) and mouse calvarial osteoblasts (in vitro), and also in primary human articular chondrocyte cultures. Silencing of CaMK1β in mouse calvarial osteoblasts by siRNA significantly decreases osteoblast proliferation and c-Fos gene expression (approx. 50%), without affecting skeletogenic markers for more differentiated osteoblasts (i.e. Cbfa1/Runx2, Osterix (Osx), Osteocalcin (Oc), Alkaline phosphatase (Alp) and Osteopontin (Opn)). These results identify CaMK1β as a novel regulator of osteoblast proliferation, via mechanisms that may at least in part involve c-Fos, thus implicating CaMK1β in the regulation of bone and cartilage development. [Copyright &y& Elsevier]