Connexin 43 (Cx43) mediates osteocyte communication with other cells and with the extracellular milieu and regulates osteoblastic cell signaling and gene expression. We now report that mice lacking Cx43 in osteoblasts/osteocytes or only in osteocytes (Cx43ΔOt mice) exhibit increased osteocyte apoptosis, endocortical resorption and periosteal bone formation, resulting in higher marrow cavity and total tissue areas measured at the femoral mid-diaphysis. Blockade of resorption reversed the increased marrow cavity but not total tissue area, demonstrating that endocortical resorption and periosteal apposition are independently regulated. Anatomical mapping of apoptotic osteocytes, osteocytic protein expression, and resorption and formation, suggests that Cx43 controls osteoclast and osteoblast activity by regulating osteoprotegerin and sclerostin levels, respectively, in osteocytes located in specific areas of the cortex. Whereas empty lacunae and living osteocytes lacking osteoprotegerin were distributed throughout cortical bone in Cx43ΔOt mice, apoptotic osteocytes were preferentially located in areas containing osteoclasts, suggesting that osteoclast recruitment requires active signaling from dying osteocytes. Furthermore, Cx43 deletion in cultured osteocytic cells resulted in increased apoptosis and decreased osteoprotegerin expression. Thus, Cx43 is essential in a cell-autonomous fashion in vivo and in vitro for osteocyte survival and for controlling the expression of osteocytic genes that affect osteoclast and osteoblast function.

Sclerostin, the Wnt signaling antagonist encoded by the Sost gene, is secreted by osteocytes and inhibits bone formation by osteoblasts. Mechanical stimulation reduces sclerostin expression, suggesting that osteocytes might coordinate the osteogenic response to mechanical force by locally unleashing Wnt signaling. To investigate whether sclerostin downregulation is a pre-requisite for load-induced bone formation, we conducted experiments in transgenic mice (TG) engineered to maintain high levels of SOST expression during mechanical loading. This was accomplished by introducing a human SOST transgene driven by the 8kb fragment of the DMP1 promoter that also provided osteocyte specificity of the transgene. Right ulnae were subjected to in vivo cyclic axial loading at equivalent strains for 1 min/day at 2Hz; left ulnae served as internal controls. Endogenous murine Sost mRNA expression measured 24h after 1 loading bout was decreased by about 50% in TG and wild type (WT) littermates. In contrast, human SOST, only expressed in TG mice, remained high after loading. Mice were loaded on 3 consecutive days and bone formation was quantified 16 days after initiation of loading. Periosteal bone formation in control ulnae was similar in WT and TG mice. Loading induced the expected strain-dependent increase in bone formation in WT mice, resulting from increases in both mineralizing surface (MS/BS) and mineral apposition rate (MAR). In contrast, load-induced bone formation was reduced by 70–85% in TG mice, due to lower MS/BS and complete inhibition of MAR. Moreover, Wnt target gene expression induced by loading in WT mice was absent in TG mice. Thus, downregulation of Sost/sclerostin in osteocytes is an obligatory step in the mechanotransduction cascade that activates Wnt signaling and directs osteogenesis to where bone is structurally needed.

The periosteal and endocortical surfaces of cortical bone dictate the geometry and overall mechanical properties of bone. Yet the cellular and molecular mechanisms that regulate activity on these surfaces are far from being understood. Parathyroid hormone (PTH) has profound effects in cortical bone, stimulating periosteal expansion and at the same time accelerating intracortical bone remodeling. We report herein that transgenic mice expressing a constitutive active PTH receptor in osteocytes (DMP1-caPTHR1 mice) exhibit increased cortical bone area and an elevated rate of periosteal and endocortical bone formation. In addition, DMP1-caPTHR1 mice display a marked increase in intracortical remodeling and cortical porosity. Crossing DMP1-caPTHR1 mice with mice lacking the Wnt coreceptor, LDL-related receptor 5 (LRP5), or with mice overexpressing the Wnt antagonist Sost in osteocytes (DMP1-Sost mice) reduced or abolished, respectively, the increased cortical bone area, periosteal bone formation rate, and expression of osteoblast markers and Wnt target genes exhibited by the DMP1-caPTHR1 mice. In addition, DMP1-caPTHR1 lacking LRP5 or double transgenic DMP1-caPTHR1;DMP1-Sost mice exhibit exacerbated intracortical remodeling and increased osteoclast numbers, and markedly decreased expression of the RANK decoy receptor osteoprotegerin. Thus, whereas Sost downregulation and the consequent Wnt activation is required for the stimulatory effect of PTH receptor signaling on periosteal bone formation, the Wnt-independent increase in osteoclastogenesis induced by PTH receptor activation in osteocytes overrides the effect on Sost. These findings demonstrate that PTH receptor signaling influences cortical bone through actions on osteocytes and defines the role of Wnt signaling in PTH receptor action. © 2011 American Society for Bone and Mineral Research.

Mice with constitutive activation of parathyroid hormone (PTH) receptor signaling in osteocytes (DMP1-caPTHR1 transgenic mice) exhibit increased bone mass and remodeling, two of the recognized skeletal actions of PTH. Moreover, similar to PTH administration, DMP1-caPTHR1 mice exhibit decreased expression of the osteocyte-derived Wnt antagonist Sost/sclerostin. We now report that PTH receptor activation also regulates in vivo and in vitro the expression of fibroblast growth factor 23 (FGF23), an osteocyte product involved in inorganic phosphate (Pi) homeostasis and bone mineralization. Whole bones and osteocytes, but not osteoblasts, from DMP1-caPTHR1 mice exhibit elevated FGF23 expression, which is corrected in double transgenic mice overexpressing Sost in osteocytes. PTH, PTH related protein (PTHrP), or a cAMP stable analog, increase FGF23 transcripts in a time- and dose-dependent manner in osteocyte-containing calvarial cell cultures. Circulating FGF23 is also elevated in DMP1-caPTHR1 mice; however, plasma Pi or renal Pi reabsorption is not altered. Furthermore, the FGF23 receptor complex comprising FGFR1 and KLOTHO is expressed in osteoblastic cells; and FGFR1, GALNT3, as well as downstream targets of FGF23 signaling, are increased in osteocytes but not in osteoblasts from DMP1-caPTHR1 mice. Thus, PTH receptor signaling has the potential to modulate the endocrine and auto/paracrine functions of osteocytes by regulating FGF23 through cAMP- and Wnt-dependent mechanisms.

Background Physicians treating COVID-19 patients increasingly believe that the hyperinflammatory acute stage of COVID-19 results in a cytokine storm. The circulating biomarkers seen across the spectrum of COVID-19 have not been characterized compared to healthy controls, but such analyses are likely to yield insights into the pursuit of interventions that adequately reduce the burden of these cytokine storms. Objective To identify and characterize the host inflammatory response to SARS-CoV-2 infection, we assessed levels of proteins related to immune responses and cardiovascular disease, in patients stratified as mild, moderate, and severe, versus matched healthy controls. Methods Blood samples from adult patients hospitalized with COVID-19 were analyzed using high-throughput and ultrasensitive proteomic platforms and compared with age- and sex-matched healthy controls to provide insights into differential regulation of 185 markers. Results Results indicate a dominant hyperinflammatory milieu in the circulation and vascular endothelial damage markers within COVID-19 patients, and strong biomarker association with patient response as measured by Ordinal scale. As patients progress, we observe statistically significant dysregulation of IFNγ, IL-1RA, IL-6, IL-10, IL-19, MCP-1, -2, -3, CXCL9, CXCL10, CXCL5, ENRAGE and PARP-1. Furthermore, in a limited series of patients who were sampled frequently confirming reliability and reproducibility of our assays, we demonstrate that intervention with baricitinib attenuates these circulating biomarkers associated with the cytokine storm. Conclusion These wide-ranging circulating biomarkers show an association with increased disease severity and may help stratify patients and selection of therapeutic options. They also provide insights into mechanisms of SARS-CoV-2 pathogenesis and the host response.