Bone mass peaks in early adulthood before declining with age and is influenced by many factors, such as activity, reproduction, nutrition, and disease. In the post-developmental skeleton, changes in bone mass are due to the activity of osteoclasts, which secrete proteases and hydrogen protons to demineralize and remove existing bone, and osteoblasts, which deposit organic matrix and facilitate its mineralization in order to produce new bone. In the healthy adult, these two processes, resorption and deposition, continually occur to maintain the skeleton and adapt it to changing needs. The cycle of removal and replacement of bone by osteoclasts and osteoblasts is coordinated by osteocytes, which form a tightly-interconnected system of local and systemic signaling. Osteocytes act to receive, incorporate, and transmit anabolic and catabolic signals down to bone-resident cells and their progenitors to tune the balance of bone removal and replacement.

When osteocytes are mechanically stimulated, as by fluid shear stress, several anabolic pathways are activated. We have recently defined one such pathway in which mechanical strain deforms the microtubule network, leading to the production of reactive oxygen species (ROS) by NOX2. ROS induces TRPV4 calcium channels to open, leading to calcium influx and an elevation in intercellular calcium. This causes activation of calcium/calmodulin-dependent protein kinase II (CaMKII), which leads to lysosomal degradation of sclerostin protein, a powerful catabolic signal. As a consequence of this mechanically-sensitive pathway activation, reduced sclerostin abundance unleashes osteoblast differentiation and new bone deposition.

Osteocytes have long been known to produce nitric oxide during mechanical stimulation, however the biological effect of this elevation has been uncertain. Our recent work demonstrated that exogenous nitric oxide is sufficient to suppress sclerostin abundance. Here, we demonstrate that nitric oxide production participates directly in the above-mentioned pathway to suppress sclerostin expression and that nitric oxide production is necessary for the loss of sclerostin abundance. We will also show that the endothelial (eNOS, NOS1) and inducible (iNOS, NOS2) isoforms of nitric oxide synthase are most relevant to this process.

We will also discuss CaMKII, another osteocytic mediator of anabolic signaling. We have previously demonstrated that two isoforms of CaMKII, CaMKIIδ and CaMKIIγ, are expressed in primary osteocytes. We conditionally deleted both isoforms from osteoblast-lineage cells using the Osteocalcin Cre driver. Camk2delta/gamma double cKO mice express a profound reduction in bone mass and quality, which appears to be mediated through FGF23 overexpression and hypophosphatemia. Here, we will characterize independent models of CaMKIIδ or CaMKIIγ conditional deletion to determine whether these isoforms may fulfil redundant or separate roles and to better understand CaMK2’s function in post-developmental bone maintenance.