Osteoblast-lineage calcium/calmodulin-dependent kinase 2 delta and gamma regulates bone mass and quality.

Bone regulates its mass and quality in response to diverse mechanical, hormonal, and local signals. The bone anabolic or catabolic responses to these signals are often received by osteocytes, which then coordinate the activity of osteoblasts and osteoclasts on bone surfaces. We previously established that calcium/calmodulin-dependent kinase 2 (CaMKII) is required for osteocytes to respond to some bone anabolic cues in vitro. However, a role for CaMKII in bone physiology in vivo is largely undescribed. Here, we show that conditional codeletion of the most abundant isoforms of CaMKII (delta and gamma) in mature osteoblasts and osteocytes [Ocn-cre:Camk2d/Camk2g double-knockout (dCKO)] caused severe osteopenia in both cortical and trabecular compartments by 8 wk of age. In addition to having less bone mass, dCKO bones are of worse quality, with significant deficits in mechanical properties, and a propensity to fracture. This striking skeletal phenotype is multifactorial, including diminished osteoblast activity, increased osteoclast activity, and altered phosphate homeostasis both systemically and locally. These dCKO mice exhibited decreased circulating phosphate (hypophosphatemia) and increased expression of the phosphate-regulating hormone fibroblast growth factor 23. Additionally, dCKO mice expressed less bone-derived tissue nonspecific alkaline phosphatase protein than control mice. Consistent with altered phosphate homeostasis, we observed that dCKO bones were hypo-mineralized with prominent osteoid seams, analogous to the phenotypes of mice with hypophosphatemia. Altogether, these data reveal a fundamental role for osteocyte CaMKIIδ and CaMKIIγ in the maintenance of bone mass and bone quality and link osteoblast/osteocyte CaMKII to phosphate homeostasis.

Nitric oxide contributes to rapid sclerostin protein loss following mechanical load.

In response to mechanical loading of bone, osteocytes produce nitric oxide (NO•) and decrease sclerostin protein expression, leading to an increase in bone mass. However, it is unclear whether NO• production and sclerostin protein loss are mechanistically linked, and, if so, the nature of their hierarchical relationship within an established mechano-transduction pathway. Prior work showed that following fluid-shear stress (FSS), osteocytes produce NOX2-derived reactive oxygen species, inducing calcium (Ca2+) influx. Increased intracellular Ca2+ results in calcium-calmodulin dependent protein kinase II (CaMKII) activation, which regulates the lysosomal degradation of sclerostin protein. Here, we extend our discoveries, identifying NO• as a regulator of sclerostin degradation downstream of mechano-activated CaMKII. Pharmacological inhibition of nitric oxide synthase (NOS) activity in Ocy454 osteocyte-like cells prevented FSS-induced sclerostin protein loss. Conversely, short-term treatment with a NO• donor in Ocy454 cells or isolated murine long bones was sufficient to induce the rapid decrease in sclerostin protein abundance, independent of changes in Sost gene expression. Ocy454 cells express all three NOS genes, and transfection with siRNAs targeting eNOS/Nos3 was sufficient to prevent FSS-induced loss of sclerostin protein, while siRNAs targeting iNOS/Nos2 mildly blunted the loss of sclerostin but did not reach statistical significance. Similarly, siRNAs targeting both eNOS/Nos3 and iNOS/Nos2 prevented FSS-induced NO• production. Together, these data show iNOS/Nos2 and eNOS/Nos3 are the primary producers of FSS-dependent NO•, and that NO• is necessary and sufficient for sclerostin protein control. Further, selective inhibition of elements within this sclerostin-controlling mechano-transduction pathway indicated that NO• production occurs downstream of CaMKII activation. Targeting Camk2d and Camk2g with siRNA in Ocy454 cells prevented NO• production following FSS, indicating that CaMKII is needed for NO• production. However, NO• donation (1min) resulted in a significant increase in CaMKII activation, suggesting that NO• may have the ability to tune CaMKII response. Together, these data support that CaMKII is necessary for, and may be modulated by NO•, and that the interaction of these two signals is involved in the control of sclerostin protein abundance, consistent with a role in bone anabolic responses.

Connexin43 regulates osteoprotegerin expression via ERK1/2 -dependent recruitment of Sp1.

In bone, connexin43 expression in cells of the osteoblast lineage plays an important role in restraining osteoclastogenesis and bone resorption. While there is a consensus around the notion that the anti-osteoclastogenic factor, osteoprotegerin, is a driver of this effect, how connexin43 regulates osteoprotegerin gene expression is unclear. Here, we show that loss of connexin43 decreased osteoprotegerin gene expression and reduced ERK1/2 activation. Conversely, overexpression of connexin43 increased osteoprotegerin expression and enhanced ERK1/2 activation. This increase in phospho-ERK1/2 is required for connexin43 to induce transcription from the osteoprotegerin proximal promoter. Connexin43 increased promoter activity via a specific 200 base pair region of the osteoprotegerin promoter located at -1486 to -1286 with respect to the transcriptional start site, a region which includes four Sp1 binding elements. Further, activation of this promoter region required an intact functional connexin43, as hypomorphic or dominant negative connexin43 mutant constructs, including one with increased hemichannel activity, were unable to stimulate osteoprotegerin expression as strongly as wild type connexin43. Using chromatin immunoprecipitations, we show that connexin43 expression enhanced the recruitment of Sp1, but not Runx2, to the osteoprotegerin proximal promoter. In total, these data show that connexin43-dependent gap junctional communication among osteoblast cells permits efficient ERK1/2 activation. ERK1/2 signaling promotes the recruitment of the potent transcriptional activator, Sp1, to the osteoprotegerin proximal promoter, resulting in robust transcription of anti-osteoclastogenic factor, osteoprotegerin.

Age-associated changes in lineage composition of the enteric nervous system regulate gut health and disease.

The enteric nervous system (ENS), a collection of neural cells contained in the wall of the gut, is of fundamental importance to gastrointestinal and systemic health. According to the prevailing paradigm, the ENS arises from progenitor cells migrating from the neural crest and remains largely unchanged thereafter. Here, we show that the lineage composition of maturing ENS changes with time, with a decline in the canonical lineage of neural-crest derived neurons and their replacement by a newly identified lineage of mesoderm-derived neurons. Single cell transcriptomics and immunochemical approaches establish a distinct expression profile of mesoderm-derived neurons. The dynamic balance between the proportions of neurons from these two different lineages in the post-natal gut is dependent on the availability of their respective trophic signals, GDNF-RET and HGF-MET. With increasing age, the mesoderm-derived neurons become the dominant form of neurons in the ENS, a change associated with significant functional effects on intestinal motility which can be reversed by GDNF supplementation. Transcriptomic analyses of human gut tissues show reduced GDNF-RET signaling in patients with intestinal dysmotility which is associated with reduction in neural crest-derived neuronal markers and concomitant increase in transcriptional patterns specific to mesoderm-derived neurons. Normal intestinal function in the adult gastrointestinal tract therefore appears to require an optimal balance between these two distinct lineages within the ENS.

The cytoskeleton and connected elements in bone cell mechano-transduction.

Bone is a mechano-responsive tissue that adapts to changes in its mechanical environment. Increases in strain lead to increased bone mass acquisition, whereas decreases in strain lead to a loss of bone mass. Given that mechanical stress is a regulator of bone mass and quality, it is important to understand how bone cells sense and transduce these mechanical cues into biological changes to identify druggable targets that can be exploited to restore bone cell mechano-sensitivity or to mimic mechanical load. Many studies have identified individual cytoskeletal components - microtubules, actin, and intermediate filaments - as mechano-sensors in bone. However, given the high interconnectedness and interaction between individual cytoskeletal components, and that they can assemble into multiple discreet cellular structures, it is likely that the cytoskeleton as a whole, rather than one specific component, is necessary for proper bone cell mechano-transduction. This review will examine the role of each cytoskeletal element in bone cell mechano-transduction and will present a unified view of how these elements interact and work together to create a mechano-sensor that is necessary to control bone formation following mechanical stress.

Aging, Osteo-Sarcopenia, and Musculoskeletal Mechano-Transduction.

The decline in the mass and function of bone and muscle is an inevitable consequence of healthy aging with early onset and accelerated decline in those with chronic disease. Termed osteo-sarcopenia, this condition predisposes the decreased activity, falls, low-energy fractures, and increased risk of co-morbid disease that leads to musculoskeletal frailty. The biology of osteo-sarcopenia is most understood in the context of systemic neuro-endocrine and immune/inflammatory alterations that drive inflammation, oxidative stress, reduced autophagy, and cellular senescence in the bone and muscle. Here we integrate these concepts to our growing understanding of how bone and muscle senses, responds and adapts to mechanical load. We propose that age-related alterations in cytoskeletal mechanics alter load-sensing and mechano-transduction in bone osteocytes and muscle fibers which underscores osteo-sarcopenia. Lastly, we examine the evidence for exercise as an effective countermeasure to osteo-sarcopenia.

In vitro Fluid Shear Stress Induced Sclerostin Degradation and CaMKII Activation in Osteocytes.

Bone is a dynamic tissue that adapts to changes in its mechanical environment. Mechanical stimuli pressurize interstitial fluid in the lacunar-canalicular system within the bone matrix, causing fluid shear stress (FSS) across bone embedded, mechano-sensitive osteocytes. Therefore, modeling this mechanical stimulus in vitro is vital for identifying mechano-transduction cascades that contribute to the regulation of mechano-responsive proteins, such as the Wnt/ß-catenin antagonist, sclerostin, which is reduced in response to FSS. Recently, we reported the rapid post-translational degradation of sclerostin protein in bone cells following FSS. Given the fundamental nature of sclerostin to bone physiology and the nuances of studying its rapid post-translational control, here, we detail our FSS protocol, and adaptations that can be made, to stimulate Ocy454 osteocyte-like cells to study sclerostin protein in vitro. While this protocol is optimized for detecting sclerostin degradation by western blot, this protocol can be adapted to examine transcriptional changes with RT-qPCR, cellular dynamics with live cell imaging, or secreted factors in the FSS buffer. This protocol utilizes 3D-printed FSS tips that are compatible with commercially available 96-well plates, allowing for high experimental accessibility, versatility, and throughput. However, this protocol can be adapted for any FSS chamber. It can also be combined with pharmacological inhibitors or genetic manipulations to interrogate the role of specific cellular components. In all, this experimental set-up and protocol is highly adaptable to allow for many experimental outcomes to examine many aspects of cell mechano-transduction.

Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein.

The downregulation of sclerostin in osteocytes mediates bone formation in response to mechanical cues and parathyroid hormone (PTH). To date, the regulation of sclerostin has been attributed exclusively to the transcriptional downregulation of the Sost gene hours after stimulation. Using mouse models and rodent cell lines, we describe the rapid, minute-scale post-translational degradation of sclerostin protein by the lysosome following mechanical load and PTH. We present a model, integrating both new and established mechanically and hormonally activated effectors into the regulated degradation of sclerostin by lysosomes. Using a mouse forelimb mechanical loading model, we find transient inhibition of lysosomal degradation or the upstream mechano-signaling pathway controlling sclerostin abundance impairs subsequent load-induced bone formation by preventing sclerostin degradation. We also link dysfunctional lysosomes to aberrant sclerostin regulation using human Gaucher disease iPSCs. These results reveal how bone anabolic cues post-translationally regulate sclerostin abundance in osteocytes to regulate bone formation.

TRPV4 calcium influx controls sclerostin protein loss independent of purinergic calcium oscillations.

Skeletal remodeling is driven in part by the osteocyte's ability to respond to its mechanical environment by regulating the abundance of sclerostin, a negative regulator of bone mass. We have recently shown that the osteocyte responds to fluid shear stress via the microtubule network-dependent activation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species and subsequent opening of TRPV4 cation channels, leading to calcium influx, activation of CaMKII, and rapid sclerostin protein downregulation. In addition to the initial calcium influx, purinergic receptor signaling and calcium oscillations occur in response to mechanical load and prior to rapid sclerostin protein loss. However, the independent contributions of TRPV4-mediated calcium influx and purinergic calcium oscillations to the rapid sclerostin protein downregulation remain unclear. Here, we showed that NOX2 and TRPV4-dependent calcium influx is required for calcium oscillations, and that TRPV4 activation is both necessary and sufficient for sclerostin degradation. In contrast, calcium oscillations are neither necessary nor sufficient to acutely decrease sclerostin protein abundance. However, blocking oscillations with apyrase prevented fluid shear stress induced changes in osterix (Sp7), osteoprotegerin (Tnfrsf11b), and sclerostin (Sost) gene expression. In total, these data provide key mechanistic insights into the way bone cells translate mechanical cues to target a key effector of bone formation, sclerostin.