Identification of Postn+ periosteal progenitor cells with bone regenerative potential.

Bone contains multiple pools of skeletal stem/progenitor cells (SSPCs), and SSPCs in periosteal compartments are known to exhibit higher regenerative potential than those in BM and endosteal compartments. However, the in vivo identity and hierarchical relationships of periosteal SSPCs (P-SSPCs) remain unclear due to a lack of reliable markers to distinguish BM SSPCs and P-SSPCs. Here, we found that periosteal mesenchymal progenitor cells (P-MPs) in periosteum can be identified based on Postn-CreERT2 expression. Postn-expressing periosteal subpopulation produces osteolineage descendants that fuel bones to maintain homeostasis and support regeneration. Notably, Postn+ P-MPs are likely derived from Gli1+ skeletal stem cells (SSCs). Ablation of Postn+ cells results in impairments in homeostatic cortical bone architecture and defects in fracture repair. Genetic deletion of Igf1r in Postn+ cells dampens bone fracture healing. In summary, our study provides a mechanistic understanding of bone regeneration through the regulation of region-specific Postn+ P-MPs.

Pharmacologic or genetic interference with atrogene signaling protects from glucocorticoid-induced musculoskeletal and cardiac disease.

Despite their beneficial actions as immunosuppressants, glucocorticoids (GC) have devastating effects on the musculoskeletal and cardiac systems, as long-term treated patients exhibit high incidence of falls, bone fractures, and cardiovascular events. Herein, we show that GC upregulate simultaneously in bone, skeletal muscle, and the heart, the expression of E3 ubiquitin ligases (atrogenes), known to stimulate the proteasomal degradation of proteins. Activation of Vitamin D receptor (VDR) signaling with the VDR ligands 1,25D3 (calcitriol, 1,25-dihydroxyvitamin D3) or ED (eldecalcitol, 2ß-(3-hydroxypropyloxy)-1,25-dihydroxyvitamin D3) prevented GC-induced atrogene upregulation in vivo and ex vivo in bone/muscle organ cultures and preserved tissue structure/mass and function of three tissues in vivo. Direct pharmacologic inhibition of the proteasome with carfilzomib also conferred musculoskeletal protection. Genetic loss of the atrogene MuRF1-mediated protein ubiquitination in ∆RING mice afforded temporary or sustained protection from GC excess in bone, or skeletal and heart muscle, respectively. We conclude that the atrogene pathway downstream of MuRF1 underlies GC action in bone, muscle, and the heart, and it can be pharmacologically or genetically targeted to confer protection against the damaging actions of GC simultaneously in the three tissues.

Identification of LRP1+CD13+ human periosteal stem cells that require LRP1 for bone repair.

Human periosteal skeletal stem cells (P-SSCs) are critical for cortical bone maintenance and repair. However, their in vivo identity, molecular characteristics, and specific markers remain unknown. Here, single-cell sequencing revealed human periosteum contains SSC clusters expressing known SSC markers, PDPN and PDGFRA. Notably, human P-SSCs, but not bone marrow SSCs (BM-SSCs), selectively expressed newly identified markers, LRP1 and CD13. These LRP1+CD13+ human P-SSCs were perivascular cells with high osteochondrogenic but minimal adipogenic potential. Upon transplantation into bone injuries in mice, they preserved self-renewal capability in vivo. Single-cell analysis of mouse periosteum further supported the preferential expression of LRP1 and CD13 in Prx1+ P-SSCs. When Lrp1 was conditionally deleted in Prx1-lineage cells, it led to severe bone deformity, short statue, and periosteal defects. By contrast, local treatment with a LRP1 agonist at the injury sites induced early P-SSC proliferation and bone healing. Thus, human and mouse periosteum contains unique osteochondrogenic stem cell subsets, and these P-SSCs express specific markers, LRP1 and CD13, with regulatory mechanism through LRP1 that enhances P-SSC function and bone repair.

The Role of Bone Sialoprotein in Bone Healing.

Bone sialoprotein (BSP) is a multi-functional extracellular matrix (ECM) protein associated with mineralized tissues, particularly bone and cementum. The amino acid sequence of BSP includes three evolutionarily conserved functional domains which contribute to functions of the protein: an N-terminal collagen-binding domain, polyglutamic acid (polyE) sequences involved in hydroxyapatite nucleation and crystal growth, and a C-terminal arginine-glycine-aspartic acid (RGD) integrin-binding domain. BSP promotes attachment and differentiation of osteogenic and osteoclastic cells. Genetic ablation of BSP in mice results in skeletal and dental developmental defects and impaired bone healing in both appendicular bone and alveolar bone of the jaw. Several studies demonstrated positive effects of BSP on bone healing in rodent models, though other experiments show negligible results. Native (harvested from rat bones) BSP cross-linked to collagen induced slight improvements in calvarial bone healing in rats. Recombinant BSP and collagen delivered in a polylactide (PLA) cylinder improved bone defect healing in rat femurs. Both native and recombinant BSP delivered in a collagen gel improved alveolar bone healing in wild-type and BSP-deficient mice. These advances suggest BSP is a new player in bone healing that has potential to be an alternative or complimentary to other bioactive factors. Future studies are necessary to understand mechanisms of how BSP influences bone healing and optimize delivery and dose in different types of bone defects and injuries.

Metabolic rewiring during bone development underlies tRNA m7G-associated primordial dwarfism.

Translation of mRNA to protein is tightly regulated by transfer RNAs (tRNAs), which are subject to various chemical modifications that maintain structure, stability, and function. Deficiency of tRNA N7-methylguanosine (m7G) modification in patients causes a type of primordial dwarfism, but the underlying mechanism remains unknown. Here we report that the loss of m7G rewires cellular metabolism, leading to the pathogenesis of primordial dwarfism. Conditional deletion of the catalytic enzyme Mettl1 or missense mutation of the scaffold protein Wdr4 severely impaired endochondral bone formation and bone mass accrual. Mechanistically, Mettl1 knockout decreased abundance of m7G-modified tRNAs and inhibited translation of mRNAs relating to cytoskeleton and Rho GTPase signaling. Meanwhile, Mettl1 knockout enhanced cellular energy metabolism despite incompetent proliferation and osteogenic commitment. Further exploration revealed that impairment of Rho GTPase signaling upregulated the level of branched-chain amino acid transaminase 1 (BCAT1) that rewired cell metabolism and restricted intracellular α-ketoglutarate (αKG). Supplementation of αKG ameliorated the skeletal defect of Mettl1-deficient mice. In addition to the selective translation of metabolism-related mRNAs, we further revealed that Mettl1 knockout globally regulated translation via integrated stress response (ISR) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Restoring translation by targeting either ISR or mTORC1 aggravated bone defects of Mettl1-deficient mice. Overall, our study unveils a critical role of m7G tRNA modification in bone development by regulation of cellular metabolism and indicates suspension of translation initiation as a quality control mechanism in response to tRNA dysregulation.

S1P regulates intervertebral disc aging by mediating endoplasmic reticulum - mitochondrial calcium ion homeostasis.

Mechanistically, S1P deficiency impeded COP II-mediated transport vesicles formation, which leads to proteins retention in the endoplasmic reticulum (ER) and subsequently ER distension. ER distension increased the contact between the ER and mitochondria, disrupting ER-to-mitochondria calcium flow, resulting in mitochondrial dysfunction and energy metabolism disturbance. Finally, using 2-APB to inhibit calcium ion channels and the senolytic drug dasatinib and quercetin (D + Q) partially rescued the aging and degenerative phenotypes caused by S1P deficiency. In conclusion, our findings suggest that S1P is a critical factor in causing IVDD in the process of aging and highlight the potential of targeting S1P as a therapeutic approach for age-related IVDD.

Itm2a expression marks periosteal skeletal stem cells that contribute to bone fracture healing.

The periosteum contains skeletal stem/progenitor cells that contribute to bone fracture healing. However, the in vivo identity of periosteal skeletal stem cells (P-SSCs) remains unclear, and membrane protein markers of P-SSCs that facilitate tissue engineering are needed. Here, we identified integral membrane protein 2A (Itm2a) enriched in SSCs using single-cell transcriptomics. Itm2a+ P-SSCs displayed clonal multipotency and self-renewal and sat at the apex of their differentiation hierarchy. Lineage-tracing experiments showed that Itm2a selectively labeled the periosteum and that Itm2a+ cells were preferentially located in the outer fibrous layer of the periosteum. The Itm2a+ cells rarely expressed CD34 or Osx, but expressed periosteal markers such as Ctsk, CD51, PDGFRA, Sca1, and Gli1. Itm2a+ P-SSCs contributed to osteoblasts, chondrocytes, and marrow stromal cells upon injury. Genetic lineage tracing using dual recombinases showed that Itm2a and Prrx1 lineage cells generated spatially separated subsets of chondrocytes and osteoblasts during fracture healing. Bone morphogenetic protein 2 (Bmp2) deficiency or ablation of Itm2a+ P-SSCs resulted in defects in fracture healing. ITM2A+ P-SSCs were also present in the human periosteum. Thus, our study identified a membrane protein marker that labels P-SSCs, providing an attractive target for drug and cellular therapy for skeletal disorders.

Deciphering the spatial distribution of Gli1-lineage cells in dental, oral, and craniofacial regions.

The craniofacial bone, crucial for protecting brain tissue and supporting facial structure, undergoes continuous remodeling through mesenchymal (MSCs) or skeletal stem cells (SSCs) in their niches. Gli1 is an ideal marker for labeling MSCs and osteoprogenitors in this region, and Gli1-lineage cells are identified as pivotal for bone growth, development, repair, and regeneration. Despite its significance, the distribution of Gli1-lineage cells across the dental, oral, and craniofacial (DOC) regions remains to be systematically explored. Utilizing tissue-clearing and light sheet fluorescence microscopy (LSFM) with a Gli1CreER; tdTomatoAi14 mouse model, we mapped the spatial distribution of Gli1-lineage cells throughout the skull, focusing on calvarial bones, sutures, bone marrow, teeth, periodontium, jaw bones, and the temporomandibular joint (TMJ). We found Gli1-lineage cells widespread in these areas, underscoring their significance in DOC regions. Additionally, we observed their role in repairing calvarial bone defects, providing novel insights into craniofacial biology and stem cell niches and enhancing our understanding of stem cells and their progeny's behavior in vivo.

This study investigates the presence and role of a specific stem cell population, known as Gli1-lineage cells, in various parts of the skull and facial bones. Using advanced imaging techniques, we found that these cells are widely distributed across the dental, oral, and craniofacial regions, especially in the cranial sutures, teeth, and jaw. Notably, Gli1-lineage cells migrate to the injury site, which is essential in bone repair and regeneration. These findings enhance our understanding of how stem cells contribute to healing and development in the craniofacial region.

m6Am Methyltransferase PCIF1 Regulates Periodontal Inflammation.

N6,2'-O-dimethyladenosine (m6Am), a common mRNA modification in eukaryotic capped mRNAs, plays a pivotal role in cellular functions and disease progression. However, its involvement in host inflammation remains elusive. Here, we demonstrate that loss of m6Am methyltransferase phosphorylated CTD interacting factor 1 (PCIF1) attenuates periodontal inflammation in whole-body and myeloid lineage-specific knockout mouse models. Pcif1 deletion inhibits macrophage phagocytosis and migration through m6Am-Csf1r signaling. In addition, colony-stimulating factor-1 receptor (CSF1R) is identified as a potential target for the treatment of periodontitis. We thus reveal a previously unrecognized role for PCIF1-mediated m6Am modification in governing macrophage responses and periodontal inflammation.

Macrophage Polarization during MRONJ Development in Mice.

Macrophages are important regulators of bone remodeling, and M1 polarization is observed in the setting of medication-related osteonecrosis of the jaws (MRONJ). Here, we characterize the phenotype of macrophages during early stages of MRONJ development in zoledronate (ZA)-treated mice with periodontal disease and explore the role of rosiglitazone, a drug that has been reported to lower the M1/M2 macrophage ratio, in MRONJ burden. Mice received ZA, and experimental periodontal disease (EPD) was induced around their second left maxillary molar. The mice were euthanized 1, 2, or 4 wk later. Micro-computed tomography and histologic and immunohistochemical analyses were carried out. In a separate experiment, mice were treated with ZA in the absence or presence of rosiglitazone, EPD was induced for 5 wk, and the MRONJ burden was assessed. An M1 predilection was noted in ZA versus vehicle (Veh) mice at 1, 2, or 4 wk after ligature placement. M1 cells were found to be positive for MMP-13, and their presence coincided with disruption of the surrounding collagen network in ZA mice. Rosiglitazone caused a reversal in the M1/M2 polarization in Veh and ZA mice. Rosiglitazone did not cause significant radiographic changes 5 wk after EPD in Veh or ZA animals. Importantly, percentage osteonecrosis and bone exposure were decreased in the rosiglitazone-treated versus nontreated ZA sites 5 wk after EPD. Our data point to an important role of M1 macrophage polarization with an overexpression of MMP-13 in the early phases of MRONJ development and provide insight into the use of interventional approaches promoting an M2 phenotype as a preventative means to alleviate MRONJ burden.

Proton-activated chloride channel increases endplate porosity and pain in a mouse spine degeneration model.

Chronic low back pain (LBP) can severely affect daily physical activity. Aberrant osteoclast-mediated resorption leads to porous endplates, which allow the sensory innervation that causes LBP. Here, we report that expression of the proton-activated chloride (PAC) channel was induced during osteoclast differentiation in the porous endplates via a RANKL/NFATc1 signaling pathway. Extracellular acidosis evoked robust PAC currents in osteoclasts. An acidic environment of porous endplates and elevated PAC activation-enhanced osteoclast fusion provoked LBP. Furthermore, we found that genetic knockout of the PAC gene Pacc1 significantly reduced endplate porosity and spinal pain in a mouse LBP model, but it did not affect bone development or homeostasis of bone mass in adult mice. Moreover, both the osteoclast bone-resorptive compartment environment and PAC traffic from the plasma membrane to endosomes to form an intracellular organelle Cl channel had a low pH of approximately 5.0. The low pH environment activated the PAC channel to increase sialyltransferase St3gal1 expression and sialylation of TLR2 in the initiation of osteoclast fusion. Aberrant osteoclast-mediated resorption is also found in most skeletal disorders, including osteoarthritis, ankylosing spondylitis, rheumatoid arthritis, heterotopic ossification, and enthesopathy. Thus, elevated Pacc1 expression and PAC activity could be a potential therapeutic target for the treatment of LBP and osteoclast-associated pain.

HIF1 activation safeguards cortical bone formation against impaired oxidative phosphorylation.

Energy metabolism, through pathways such as oxidative phosphorylation (OxPhos) and glycolysis, plays a pivotal role in cellular differentiation and function. Our study investigates the impact of OxPhos disruption in cortical bone development by deleting mitochondrial transcription factor A (TFAM). TFAM controls OxPhos by regulating the transcription of mitochondrial genes. The cortical bone, constituting the long bones' rigid shell, is sheathed by the periosteum, a connective tissue layer populated with skeletal progenitors that spawn osteoblasts, the bone-forming cells. TFAM-deficient mice presented with thinner cortical bone, spontaneous midshaft fractures, and compromised periosteal cell bioenergetics, characterized by reduced ATP levels. Additionally, they exhibited an enlarged periosteal progenitor cell pool with impaired osteoblast differentiation. Increasing hypoxia-inducible factor 1a (HIF1) activity within periosteal cells substantially mitigated the detrimental effects induced by TFAM deletion. HIF1 is known to promote glycolysis in all cell types. Our findings underscore the indispensability of OxPhos for the proper accrual of cortical bone mass and indicate a compensatory mechanism between OxPhos and glycolysis in periosteal cells. The study opens new avenues for understanding the relationship between energy metabolism and skeletal health and suggests that modulating bioenergetic pathways may provide a therapeutic avenue for conditions characterized by bone fragility.

Exploring mechanobiology network of bone and dental tissue based on Natural Language Processing.

Bone and cartilage tissues are physiologically dynamic organs that are systematically regulated by mechanical inputs. At cellular level, mechanical stimulation engages an intricate network where mechano-sensors and transmitters cooperate to manipulate downstream signaling. Despite accumulating evidence, there is a notable underutilization of available information, due to limited integration and analysis. In this context, we conceived an interactive web tool named MechanoBone to introduce a new avenue of literature-based discovery. Initially, we compiled a literature database by sourcing content from Pubmed and processing it through the Natural Language Toolkit project, Pubtator, and a custom library. We identified direct co-occurrence among entities based on existing evidence, archiving in a relational database via SQLite. Latent connections were then quantified by leveraging the Link Prediction algorithm. Secondly, mechanobiological pathway maps were generated, and an entity-pathway correlation scoring system was established through weighted algorithm based on our database, String, and KEGG, predicting potential functions of specific entities. Additionally, we established a mechanical circumstance-based exploration to sort genes by their relevance based on big data, revealing the potential mechanically sensitive factors in bone research and future clinical applications. In conclusion, MechanoBone enables: 1) interpreting mechanobiological processes; 2) identifying correlations and crosstalk among molecules and pathways under specific mechanical conditions; 3) connecting clinical applications with mechanobiological processes in bone research. It offers a literature mining tool with visualization and interactivity, facilitating targeted molecule navigation and prediction within the mechanobiological framework of bone-related cells, thereby enhancing knowledge sharing and big data analysis in the biomedical realm.

High-fat and high-carbohydrate diets increase bone fragility through TGF-ß-dependent control of osteocyte function.

Obesity can increase the risk of bone fragility, even when bone mass is intact. This fragility stems from poor bone quality, potentially caused by deficiencies in bone matrix material properties. However, cellular and molecular mechanisms leading to obesity-related bone fragility are not fully understood. Using male mouse models of obesity, we discovered TGF-ß signaling plays a critical role in mediating the effects of obesity on bone. High-carbohydrate and high-fat diets increase TGF-ß signaling in osteocytes, which impairs their mitochondrial function, increases cellular senescence, and compromises perilacunar/canalicular remodeling and bone quality. By specifically inhibiting TGF-ß signaling in mouse osteocytes, some of the negative effects of high-fat and high-carbohydrate diets on bones, including the lacunocanalicular network, perilacunar/canalicular remodeling, senescence, and mechanical properties such as yield stress, were mitigated. DMP1-Cre-mediated deletion of TGF-ß receptor II also blunted adverse effects of high-fat and high-carbohydrate diets on energy balance and metabolism. These findings suggest osteocytes are key in controlling bone quality in response to high-fat and high-carbohydrate diets. Calibrating osteocyte function could mitigate bone fragility associated with metabolic diseases while reestablishing energy balance.

Sclerostin antibody corrects periodontal disease in type 2 diabetic mice.

Type 2 diabetes (T2D) is on the rise worldwide and is associated with various complications in the oral cavity. Using an adult-onset diabetes preclinical model, we demonstrated profound periodontal alterations in T2D mice, including inflamed gingiva, disintegrated periodontal ligaments (PDLs), marked alveolar bone loss, and unbalanced bone remodeling due to decreased formation and increased resorption. Notably, we observed elevated levels of the Wnt signaling inhibitor sclerostin in the alveolar bone of T2D mice. Motivated by these findings, we investigated whether a sclerostin-neutralizing antibody (Scl-Ab) could rescue the compromised periodontium in T2D mice. Administering Scl-Ab subcutaneously once a week for 4 weeks, starting 4 weeks after T2D induction, led to substantial increases in bone mass. This effect was attributed to the inhibition of osteoclasts and promotion of osteoblasts in both control and T2D mice, effectively reversing the bone loss caused by T2D. Furthermore, Scl-Ab stimulated PDL cell proliferation, partially restored the PDL fibers, and mitigated inflammation in the periodontium. Our study thus established a T2D-induced periodontitis mouse model characterized by inflammation and tissue degeneration. Scl-Ab emerged as a promising intervention to counteract the detrimental effects of T2D on the periodontium, exhibiting limited side effects on other craniofacial hard tissues.

FoxO1-modulated macrophage polarization regulates osteogenesis via PPAR-γ signaling.

Periodontitis, a common chronic inflammatory disease, epitomizes a significant impairment in the host immune system and an imbalance of bone metabolism. Macrophage polarization, a dynamic process dictated by the microenvironment, intricately contributes to the interplay between the immune system and bone remodeling, namely the osteoimmune system. Forkhead box protein O1 (FoxO1) has been shown to play a dramatic role in mediating oxidative stress, bone mass, as well as cellular metabolism. Nevertheless, the function and underlying mechanisms of FoxO1 in regulating macrophage polarization-mediated osteogenesis in periodontitis remain to be further elucidated. Here, we found that FoxO1 expression was closely linked to periodontitis, accompanied by aggravated inflammation. Notably, FoxO1 knockdown skewed macrophage polarization from M1 to the antiinflammatory M2 phenotype under inflammatory conditions, which rescued the impaired osteogenic potential. Mechanistically, we revealed that the enhancement of the transcription of peroxisome proliferator-activated receptor (PPAR) signaling in FoxO1-knockdown macrophages. In agreement with this contention, GW9662, a specific inhibitor of PPAR-γ signaling, greatly aggravated macrophage polarization from M2 to the M1 phenotype and attenuated osteogenic potential under inflammatory conditions. Additionally, PPAR-γ signaling agonist rosiglitazone (RSG) was applied to address ligature-induced periodontitis with attenuated inflammation. Our data lend conceptual credence to the function of FoxO1 in mediating macrophage polarization-regulated osteogenesis which serves as a novel therapeutic target for periodontitis.

Spatial transcriptomics implicates impaired BMP signaling in NF1 fracture pseudarthrosis in murine and patient tissues.

The neurofibromatosis type 1 (NF1) RASopathy is associated with persistent fibrotic nonunions (pseudarthrosis) in human and mouse skeletal tissue. Here, we performed spatial transcriptomics to define the molecular signatures occurring during normal endochondral healing following fracture in mice. Within the control fracture callus, we observed spatially restricted activation of morphogenetic pathways, such as TGF-ß, WNT, and BMP. To investigate the molecular mechanisms contributing to Nf1-deficient delayed fracture healing, we performed spatial transcriptomic analysis on a Postn-cre;Nf1fl/- (Nf1Postn) fracture callus. Transcriptional analyses, subsequently confirmed through phospho-SMAD1/5/8 immunohistochemistry, demonstrated a lack of BMP pathway induction in Nf1Postn mice. To gain further insight into the human condition, we performed spatial transcriptomic analysis of fracture pseudarthrosis tissue from a patient with NF1. Analyses detected increased MAPK signaling at the fibrocartilaginous-osseus junction. Similar to that in the Nf1Postn fracture, BMP pathway activation was absent within the pseudarthrosis tissue. Our results demonstrate the feasibility of delineating the molecular and tissue-specific heterogeneity inherent in complex regenerative processes, such as fracture healing, and reconstructing phase transitions representing endochondral bone formation in vivo. Furthermore, our results provide in situ molecular evidence of impaired BMP signaling underlying NF1 pseudarthrosis, potentially informing the clinical relevance of off-label BMP2 as a therapeutic intervention.

Explainable deep learning and biomechanical modeling for TMJ disorder morphological risk factors.

Clarifying multifactorial musculoskeletal disorder etiologies supports risk analysis, development of targeted prevention, and treatment modalities. Deep learning enables comprehensive risk factor identification through systematic analyses of disease data sets but does not provide sufficient context for mechanistic understanding, limiting clinical applicability for etiological investigations. Conversely, multiscale biomechanical modeling can evaluate mechanistic etiology within the relevant biomechanical and physiological context. We propose a hybrid approach combining 3D explainable deep learning and multiscale biomechanical modeling; we applied this approach to investigate temporomandibular joint (TMJ) disorder etiology by systematically identifying risk factors and elucidating mechanistic relationships between risk factors and TMJ biomechanics and mechanobiology. Our 3D convolutional neural network recognized TMJ disorder patients through participant-specific morphological features in condylar, ramus, and chin. Driven by deep learning model outputs, biomechanical modeling revealed that small mandibular size and flat condylar shape were associated with increased TMJ disorder risk through increased joint force, decreased tissue nutrient availability and cell ATP production, and increased TMJ disc strain energy density. Combining explainable deep learning and multiscale biomechanical modeling addresses the "mechanism unknown" limitation undermining translational confidence in clinical applications of deep learning and increases methodological accessibility for smaller clinical data sets by providing the crucial biomechanical context.

Protein kinase G2 activation restores Wnt signaling and bone mass in glucocorticoid-induced osteoporosis in mice.

Osteoporotic fractures are a major complication of long-term glucocorticoid therapy. Glucocorticoids transiently increase bone resorption, but they predominantly inhibit bone formation and induce osteocyte apoptosis, leading to bone loss. Current treatments of glucocorticoid-induced osteoporosis aim mainly at reducing bone resorption and are, therefore, inadequate. We previously showed that signaling via the NO/cGMP/protein kinase G pathway plays a key role in skeletal homeostasis. Here, we show that pharmacological PKG activation with the guanylyl cyclase-1 activator cinaciguat or expression of a constitutively active, mutant PKG2R242Q restored proliferation, differentiation, and survival of primary mouse osteoblasts exposed to dexamethasone. Cinaciguat treatment of WT mice or osteoblast-specific expression of PKG2R242Q in transgenic mice prevented dexamethasone-induced loss of cortical bone mass and strength. These effects of cinaciguat and PKG2R242Q expression were due to preserved bone formation parameters and osteocyte survival. The basis for PKG2's effects appeared to be through recovery of Wnt/ß-catenin signaling, which was suppressed by glucocorticoids but critical for proliferation, differentiation, and survival of osteoblast-lineage cells. Cinaciguat reduced dexamethasone activation of osteoclasts, but this did not occur in the PKG2R242Q transgenic mice, suggesting a minor role in osteoprotection. We propose that existing PKG-targeting drugs could represent a novel therapeutic approach to prevent glucocorticoid-induced osteoporosis.

Chromatin profiling identifies chondrocyte-specific Sox9 enhancers important for skeletal development.

The transcription factor SRY-related HMG box 9 (Sox9) is essential for chondrogenesis. Mutations in and around SOX9 cause campomelic dysplasia (CD) characterized by skeletal malformations. Although the function of Sox9 in this context is well studied, the mechanisms that regulate Sox9 expression in chondrocytes remain to be elucidated. Here, we have used genome-wide profiling to identify 2 Sox9 enhancers located in a proximal breakpoint cluster responsible for CD. Enhancer activity of E308 (located 308 kb 5' upstream) and E160 (located 160 kb 5' upstream) correlated with Sox9 expression levels, and both enhancers showed a synergistic effect in vitro. While single deletions in mice had no apparent effect, simultaneous deletion of both E308 and E160 caused a dwarf phenotype, concomitant with a reduction of Sox9 expression in chondrocytes. Moreover, bone morphogenetic protein 2-dependent chondrocyte differentiation of limb bud mesenchymal cells was severely attenuated in E308/E160 deletion mice. Finally, we found that an open chromatin region upstream of the Sox9 gene was reorganized in the E308/E160 deletion mice to partially compensate for the loss of E308 and E160. In conclusion, our findings reveal a mechanism of Sox9 gene regulation in chondrocytes that might aid in our understanding of the pathophysiology of skeletal disorders.