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.

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.