Estrogen Differentially Regulates Protein Abundance in Exosomes Released From Immortalized Kisspeptin Neurons in Vitro

Estrogen (E2) is essential for multiple physiological effects in females, ensuring maximum reproductive fitness and maintaining skeletal homeostasis. E2 has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), an effect widely accepted to be mediated directly at bone. A recent landmark study (Herber et al., Nat Commun 2019) demonstrated bone density increases in female mice harboring ERα-deletions specifically in arcuate Kiss-1 neurons. In this study, bone from transgenic females showed higher osteoblast functioning and increases in the expression of sp7 and runx2, positing a direct neural-bone regulatory axis altered by circulating E2 acting in brain. Our laboratory has used two immortalized Kisspeptin (Kiss1)-expressing and -secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) as models to explore the role of Kiss-1 in multiple physiological regulatory contexts. We recently determined that factors in the media of female ARC-derived KTaR-1 cells can affect parameters of osteoblast function in vitro, including increases in sp7 and runx2 expression, and formation of bone matrix (evaluated by Alizarin Red assay). Exposure of canine osteosarcoma cells to conditioned media from KTaR-1 cells led to increases in sp7 expression in an E2-dependent manner, and 24h E2-deprivation of these neurons stimulated secretion of osteogenic factors. In this current study, we have used LCMS-MS proteomic analysis to determine the contents of exosomes isolated from Kisspeptin neurons under varying E2 exposure conditions in vitro. Preliminary results reveal ~150-170 proteins up-regulated by E2 exposure and ~200-220 proteins downregulated by E2 exposure in exosomes of both KTaR-1 and KTaV-3 Kisspeptin neurons. Estrogen-regulated Kiss-1 exosomal proteins include several candidates involved in bone remodeling (pentraxin, osteonectin, osteoclast-stimulating factor-1) and neuronal synaptic plasticity and signaling (annexins, semaphorins, connexins). Current work is exploring the effects of exposure of purified exosomes on morphology and gene expression in immortalized GnRH neurons and osteoblasts. While further study is required, initial results suggest that exosomes may represent additional cellular communication pathways utilized by Kisspeptin neurons to elicit changes in brain and bone.

PTH Protects Osteocytes From Oxidative Stress and Cellular Senescence

Age-induced osteoporosis is characterized by a progressive decline in bone formation and increase in bone resorption with uncoupled activities of osteoblasts and osteoclasts. Parathyroid hormone (PTH) is used in the clinic to treat osteoporosis due to its anabolic actions on bone via binding to the PTH receptor (PPR). The receptor is highly expressed in cells of the osteoblastic lineage, including osteocytes. Osteocytes are the most abundant cells in bone and serve as a key regulator of bone remodeling. Despite the significant role of PPR signaling in skeletal homeostasis, its function in osteocytes during aging remains unclear. We have gathered preliminary data demonstrating that mice lacking PPR predominantly in osteocytes (Dmp1-PPRKO) have marked age-induced bone loss due to increased bone resorption and suppressed bone formation. These mice, with aging, develop characteristics of skeletal senescence: a decrease in osteoprogenitors and an increase in bone marrow adiposity and p16Ink4a/Cdkn2a expression in bone. Since senescence of cells in the bone microenvironment has been reported as a cause of age-induced bone loss, we hypothesized that PPR signaling protects osteocytes from senescence. To test this hypothesis, we generated osteocytes (Ocy454-12H), in which the PPR expression was ablated using CRISPR/Cas9 technique. Ocy454-12H-PPRKO and Ocy454-12H-PPRCtrl cells were treated with PTH followed by an exposure to hydrogen peroxide (H2O2). High levels of intracellular reactive oxygen species (ROS), including H2O2, promote protein and DNA oxidation, resulting in cell death and senescence. PTH treatment significantly suppressed the increase in H2O2-induced cell death, measured by resazurin-based assays, in PPRCtrl but not in PPRKO cells. We analyzed intracellular ROS levels using a fluorescent probe and found that PTH treatment significantly suppressed the increase in ROS upon H2O2 exposure, suggesting an antioxidant function of PTH in osteocytes. To further investigate if PTH prevents osteocytes from oxidative stress-induced senescence, we examined senescence-associated β-galactosidase (SA β-gal) activity in cells that were treated with PTH followed by an exposure to low doses of H2O2. Compared to untreated and PPRKO groups, treatment with PTH significantly decreased the number of SA β-gal positive cells, demonstrating that PPR signaling protects osteocytes, and possibly other osteoblastic cells, from H2O2-induced cellular senescence. PTH treatment reduced mRNA expression of p21/Cdkn1a. Taken together these results demonstrate that PPR signaling is important to protect osteocytes from cellular senescence.

Improved Bone Quality and Bone Healing of Dystrophic Mice by Parabiosis

Duchenne muscular dystrophy (DMD) is a degenerative muscle disorder characterized by a lack of dystrophin expression in the sarcolemma of muscle fibers. DMD patients acquire bone abnormalities including osteopenia, fragility fractures, and scoliosis indicating a deficiency in skeletal homeostasis. The dKO (dystrophin/Utrophin double knockout) is a more severe mouse model of DMD than the mdx mouse (dystrophin deficient), and display numerous clinically-relevant manifestations, including a spectrum of degenerative changes outside skeletal muscle including bone, articular cartilage, and intervertebral discs. To examine the influence of systemic factors on the bone abnormalities and healing in DMD, parabiotic pairing between dKO mice and mdx mice was established. Notably, heterochronic parabiosis with young mdx mice significantly increased bone mass and improved bone micro-structure in old dKO-hetero mice, which showed progressive bone deterioration. Furthermore, heterochronic parabiosis with WT C56/10J mice significantly improved tibia bone defect healing in dKO-homo mice. These results suggest that systemic blood-borne factor(s) and/or progenitors from WT and young mdx mice can influence the bone deficiencies in dKO mice. Understanding these circulating factors or progenitor cells that are responsible to alleviate the bone abnormalities in dKO mice after heterochronic parabiosis might be useful for the management of poor bone health in DMD.

Osteoblast-Specific Wnt Secretion is Required for Skeletal Homeostasis and Loading-Induced Bone Formation in Adult Mice

Wnt signaling is critical to many aspects of skeletal regulation, but the importance of Wnt ligands in adult bone homeostasis and the anabolic response to mechanical loading is not well documented. We inhibited Wnt ligand secretion in adult (5-mo) mice using a systemic (drug) and a bone-targeted (genetic) approach, and subjected them to axial tibial loading to induce lamellar bone formation. Mice treated with the porcupine inhibitor WNT974 exhibited a decrease in bone formation in non-loaded limbs as well as a 54% decline in the periosteal bone formation response to tibial loading. Similarly, within 1-2 weeks of Wls deletion in osteoblasts (Osx-CreERT2;WlsF/F mice), skeletal homeostasis was altered with decreased bone formation and increased resorption, and the anabolic response to loading was reduced 65% compared to control (WlsF/F). These findings establish a requirement for Wnt ligand secretion by osteoblasts for adult bone homeostasis and the anabolic response to mechanical loading.

Cytokine “fine tuning” of enthesis tissue homeostasis as a pointer to spondyloarthritis pathogenesis with a focus on relevant TNF and IL-17 targeted therapies

A curious feature of axial disease in ankylosing spondylitis (AS) and related non-radiographic axial spondyloarthropathy (nrAxSpA) is that spinal inflammation may ultimately be associated with excessive entheseal tissue repair with new bone formation. Other SpA associated target tissues including the gut and the skin have well established paradigms on how local tissue immune responses and proven disease relevant cytokines including TNF and the IL-23/17 axis contribute to tissue repair. Normal skeletal homeostasis including the highly mechanically stressed entheseal sites is subject to tissue microdamage, micro-inflammation and ultimately repair. Like the skin and gut, healthy enthesis has resident immune cells including ILCs, γδ T cells, conventional CD4+ and CD8+ T cells and myeloid lineage cells capable of cytokine induction involving prostaglandins, growth factors and cytokines including TNF and IL-17 that regulate these responses. We discuss how human genetic studies, animal models and translational human immunology around TNF and IL-17 suggest a largely redundant role for these pathways in physiological tissue repair and homeostasis. However, disease associated immune system overactivity of these cytokines with loss of tissue repair “fine tuning” is eventually associated with exuberant tissue repair responses in AS. Conversely, excessive biomechanical stress at spinal enthesis or peripheral enthesis with mechanically related or degenerative conditions is associated with a normal immune system attempts at cytokine fine tuning, but in this setting, it is commensurate to sustained abnormal biomechanical stressing. Unlike SpA, where restoration of aberrant and excessive cytokine “fine tuning” is efficacious, antagonism of these pathways in biomechanically related disease may be of limited or even no value.

Interleukin-17A Interweaves the Skeletal and Immune Systems

The complex crosstalk between the immune and the skeletal systems plays an indispensable role in the maintenance of skeletal homeostasis. Various cytokines are involved, including interleukin (IL)-17A. A variety of immune and inflammatory cells produces IL-17A, especially Th17 cells, a subtype of CD4+ T cells. IL-17A orchestrates diverse inflammatory and immune processes. IL-17A induces direct and indirect effects on osteoclasts. The dual role of IL-17A on osteoclasts partly depends on its concentrations and interactions with other factors. Interestingly, IL-17A exerts a dual role in osteoblasts in vitro. IL-17A is a bone-destroying cytokine in numerous immune-mediated bone diseases including postmenopausal osteoporosis (PMOP), rheumatoid arthritis (RA), psoriatic arthritis (PsA) and axial spondylarthritis (axSpA). This review will summarize and discuss the pathophysiological roles of IL-17A on the skeletal system and its potential strategies for application in immune-mediated bone diseases.

Targeted Ptpn11 deletion in mice reveals the essential role of SHP2 in osteoblast differentiation and skeletal homeostasis

The maturation and function of osteoblasts (OBs) rely heavily on the reversible phosphorylation of signaling proteins. To date, most of the work in OBs has focused on phosphorylation by tyrosyl kinases, but little has been revealed about dephosphorylation by protein tyrosine phosphatases (PTPases). SHP2 (encoded by PTPN11) is a ubiquitously expressed PTPase. PTPN11 mutations are associated with both bone and cartilage manifestations in patients with Noonan syndrome (NS) and metachondromatosis (MC), although the underlying mechanisms remain elusive. Here, we report that SHP2 deletion in bone gamma-carboxyglutamate protein-expressing (Bglap+) bone cells leads to massive osteopenia in both trabecular and cortical bones due to the failure of bone cell maturation and enhanced osteoclast activity, and its deletion in Bglap+ chondrocytes results in the onset of enchondroma and osteochondroma in aged mice with increased tubular bone length. Mechanistically, SHP2 was found to be required for osteoblastic differentiation by promoting RUNX2/OSTERIX signaling and for the suppression of osteoclastogenesis by inhibiting STAT3-mediated RANKL production by osteoblasts and osteocytes. These findings are likely to explain the compromised skeletal system in NS and MC patients and to inform the development of novel therapeutics to combat skeletal disorders.