Intervertebral disc-intrinsic Hedgehog signaling maintains disc cell phenotypes and prevents disc degeneration through both cell autonomous and non-autonomous mechanisms.

Intervertebral disc degeneration is closely related to abnormal phenotypic changes in disc cells. However, the mechanism by which disc cell phenotypes are maintained remains poorly understood. Here, Hedgehog-responsive cells were found to be specifically localized in the inner annulus fibrosus and cartilaginous endplate of postnatal discs, likely activated by Indian Hedgehog. Global inhibition of Hedgehog signaling using a pharmacological inhibitor or Agc1-CreERT2-mediated deletion of Smo in disc cells of juvenile mice led to spontaneous degenerative changes in annulus fibrosus and cartilaginous endplate accompanied by aberrant disc cell differentiation in adult mice. In contrast, Krt19-CreER-mediated deletion of Smo specifically in nucleus pulposus cells led to healthy discs and normal disc cell phenotypes. Similarly, age-related degeneration of nucleus pulposus was accelerated by genetic inactivation of Hedgehog signaling in all disc cells, but not in nucleus pulposus cells. Furthermore, inactivation of Gli2 in disc cells resulted in partial loss of the vertebral growth plate but otherwise healthy discs, whereas deletion of Gli3 in disc cells largely corrected disc defects caused by Smo ablation in mice. Taken together, our findings not only revealed for the first time a direct role of Hedgehog-Gli3 signaling in maintaining homeostasis and cell phenotypes of annuls fibrosus and cartilaginous endplate, but also identified disc-intrinsic Hedgehog signaling as a novel non-cell-autonomous mechanism to regulate nucleus pulposus cell phenotype and protect mice from age-dependent nucleus pulposus degeneration. Thus, targeting Hedgehog signaling may represent a potential therapeutic strategy for the prevention and treatment of intervertebral disc degeneration.

Piperlongumine, a Piper longum-derived amide alkaloid, protects mice from ovariectomy-induced osteoporosis by inhibiting osteoclastogenesis via suppression of p38 and JNK signaling.

Postmenopausal osteoporosis (PMOP) is a metabolic bone disease that results from overproduction and hyperactivation of osteoclasts caused by insufficient estrogen in women after menopause. Current therapeutic strategies are mainly focused on treating PMOP patients who have already developed severe bone loss or even osteoporotic fractures. Obviously, a better strategy is to prevent PMOP from occurring in the first place. However, such reagents are largely lacking. Piperlongumine (PLM), an amide alkaloid extracted from long pepper Piper longum, exhibits the anti-osteoclastogenic effect in normal bone marrow macrophages (BMMs) and the protective effect against osteolysis induced by titanium particles in mice. This study examined the preventive effect of PLM on PMOP and explored the potential mechanism of this effect using both ovariectomized mice and their primary cells. The result showed that PLM (5 and 10 mg kg-1) administered daily for 6 weeks ameliorated ovariectomy-induced bone loss and osteoclast formation in mice. Further cell experiments showed that PLM directly suppressed osteoclast formation, F-actin ring formation, and osteoclastic resorption pit formation in BMMs derived from osteoporotic mice, but did not obviously affect osteogenic differentiation of bone marrow stromal cells (BMSCs) from these mice. Western blot analysis revealed that PLM attenuated maximal activation of p38 and JNK pathways by RANKL stimulation without affecting acute activation of NF-κB, AKT, and ERK signaling. Furthermore, PLM inhibited expression of key osteoclastogenic transcription factors NFATc1/c-Fos and their target genes (Dcstamp, Atp6v0d2, Acp5, and Oscar). Taken together, our findings suggest that PLM inhibits osteoclast formation and function by suppressing RANKL-induced activation of the p38/JNK-cFos/NFATc1 signaling cascade, thereby preventing ovariectomy-induced osteoporosis in mice. Thus, PLM can potentially be used as an anti-resorption drug or dietary supplement for the prevention of PMOP.

Pharmacological inhibition of protein S-palmitoylation suppresses osteoclastogenesis and ameliorates ovariectomy-induced bone loss.

Background: Excessive osteoclast formation disrupts bone homeostasis, thereby significantly contributing to pathological bone loss associated with a variety of diseases. Protein S-palmitoylation is a reversible post-translational lipid modification catalyzed by ZDHHC family of palmitoyl acyltransferases, which plays an important role in various physiological and pathological processes. However, the role of palmitoylation in osteoclastogenesis has never been explored. Consequently, it is unclear whether this process can be targeted to treat osteolytic bone diseases that are mainly caused by excessive osteoclast formation. Materials and methods: In this study, we employed acyl-biotin exchange (ABE) assay to reveal protein S-palmitoylation in differentiating osteoclasts (OCs). We utilized 2-bromopalmitic acid (2-BP), a pharmacological inhibitor of protein S-palmitoylation, to inhibit protein palmitoylation in mouse bone marrow-derived macrophages (BMMs), and tested its effect on receptor activator of nuclear factor κß ligand (RANKL)-induced osteoclast differentiation and activity by TRAP staining, phalloidin staining, qPCR analyses, and pit formation assays. We also evaluated the protective effect of 2-BP against estrogen deficiency-induced bone loss and bone resorption in ovariectomized (OVX) mice using µCT, H&E staining, TRAP staining, and ELISA assay. Furthermore, we performed western blot analyses to explore the molecular mechanism underlying the inhibitory effect of 2-BP on osteoclastogenesis. Results: We found that many proteins were palmitoylated in differentiating OCs and that pharmacological inhibition of palmitoylation impeded RANKL-induced osteoclastogenesis, osteoclast-specific gene expression, F-actin ring formation and osteoclastic bone resorption in vitro, and to a lesser extent, osteoblast formation from MC3T3-E1 cells. Furthermore, we demonstrated that administration of 2-BP protected mice from ovariectomy-induced osteoporosis and bone resorption in vivo. Mechanistically, we showed that 2-BP treatment inhibited osteoclastogenesis partly by downregulating the expression of c-Fos and NFATc1 without overtly affecting RANKL-induced activation of osteoclastogenic AKT, MAPK, and NF-κB pathways. Conclusion: Pharmacological inhibition of palmitoylation potently suppresses RANKL-mediated osteoclast differentiation in vitro and protects mice against OVX-induced osteoporosis in vivo. Mechanistically, palmitoylation regulates osteoclast differentiation partly by promoting the expression of c-Fos and NFATc1. Thus, palmitoylation plays a key role in promoting osteoclast differentiation and activity, and could serve as a potential therapeutic target for the treatment of osteoporosis and other osteoclast-related diseases. The translational potential of this article: The translation potential of this article is that we first revealed palmitoylation as a key mechanism regulating osteoclast differentiation, and therefore provided a potential therapeutic target for treating osteolytic bone diseases.

Suppressor of fused-restrained Hedgehog signaling in chondrocytes is critical for epiphyseal growth plate maintenance and limb elongation in juvenile mice.

Hedgehog (Hh) signaling plays multiple critical roles in regulating chondrocyte proliferation and differentiation during epiphyseal cartilage development. However, it is still unclear whether Hh signaling in chondrocytes is required for growth plate maintenance during juvenile growth, and whether sustained activation of Hh signaling in chondrocytes promotes limb elongation. In this study, we first utilized Hh reporter mice to reveal that Hh signaling was activated in resting and columnar chondrocytes in growth plates of juvenile and adult mice. Next, we genetically modulated Hh signaling by conditionally deleting Smo or Sufu in all or a subpopulation of growth plate chondrocytes, and found that ablation of either Smo or Sufu in chondrocytes of juvenile mice caused premature closure of growth plates and shorter limbs, whereas Osx-Cre-mediated deletion of either of these two genes in prehypertrophic chondrocytes did not lead to obvious growth plate defects, indicating that Hh signaling mainly functions in resting and/or columnar chondrocytes to maintain growth plates at the juvenile stage. At the cellular level, we found that chondrocyte-specific ablation of Smo or Sufu accelerated or suppressed chondrocyte hypertrophy, respectively, whereas both decreased chondrocyte proliferation and survival. Thus, our study provided the first genetic evidence to establish the essential cell-autonomous roles for tightly-regulated Hh signaling in epiphyseal growth plate maintenance and limb elongation during juvenile growth.

HDAC inhibitor quisinostat prevents estrogen deficiency-induced bone loss by suppressing bone resorption and promoting bone formation in mice.

Postmenopausal osteoporosis (PMOP) is a metabolic skeletal disorder characterized by reduced bone mass and impaired bone microarchitecture resulting in increased bone fragility and fracture risk. PMOP is primarily caused by excessive osteoclastogenesis induced by estrogen deficiency. Quisinostat (Qst) is a potent hydroxamate-based second-generation inhibitor of histone deacetylases (HDACs) that can inhibit osteoclast differentiation in vitro, and protect mice from titanium particle-induced osteolysis in vivo. However, whether Qst has therapeutic potential against PMOP remains unclear. In the present study, we evaluated the therapeutic efficacy of Qst on PMOP, using a murine model of ovariectomy (OVX)-induced osteoporosis. We examined the body weight, femur length, and histology of major organs, and showed that Qst did not cause obvious toxicity in mice. Micro-computed tomography and histological analyses revealed that Qst treatment prevented OVX-induced trabecular bone loss both in femurs and vertebrae. Moreover, ELISA showed that Qst decreased the serum levels of the osteoclastic bone resorption marker CTX-1, whereas increased the levels of the osteoblastic bone formation marker Osteocalcin in OVX mice. Consistent with the CTX-1 results, TRAP staining showed that Qst suppressed OVX-induced osteoclastogenesis. Mechanistically, we showed that Qst suppressed RANKL-induced osteoclast differentiation in part by inhibiting p65 nuclear translocation. Collectively, our results demonstrated that Qst can ameliorate estrogen deficiency-induced osteoporosis by inhibiting bone resorption and promoting bone formation in vivo. In summary, our study provided the first preclinical evidence to support Qst as a potential therapeutic agent for PMOP prevention and treatment.

Hedgehog Signaling Controls Bone Homeostasis by Regulating Osteogenic/Adipogenic Fate of Skeletal Stem/Progenitor Cells in Mice.

Skeletal stem/progenitor cells (SSPCs) can differentiate into osteogenic or adipogenic lineage. The mechanism governing lineage allocation of SSPCs is still not completely understood. Hedgehog (Hh) signaling plays an essential role in specifying osteogenic fate of mesenchymal progenitors during embryogenesis. However, it is still unclear whether Hh signaling is required for lineage allocation of SSPCs in postnatal skeleton, and whether its dysregulation is related to age-related osteoporosis. Here, we demonstrated that Hh signaling was activated in metaphyseal SSPCs during osteogenic differentiation in the adult skeleton, and its activity decreased with aging. Inactivation of Hh signaling by genetic ablation of Smo, a key molecule in Hh signaling, in Osx-Cre-targeted SSPCs and hypertrophic chondrocytes led to decreased bone formation and increased bone marrow adiposity, two key pathological features of age-related osteoporosis. Moreover, we found that the bone-fat imbalance phenotype caused by Smo deletion mainly resulted from aberrant allocation of SSPCs toward adipogenic lineage at the expense of osteogenic differentiation, but not due to accelerated transdifferentiation of chondrocytes into adipocytes. Mechanistically, we found that Hh signaling regulated osteoblast versus adipocyte fate of SSPCs partly through upregulating Wnt signaling. Thus, our results indicate that Hh signaling regulates bone homeostasis and age-related osteoporosis by acting as a critical switch of cell fate decisions of Osx-Cre-targeted SSPCs in mice and suggest that Hh signaling may serve as a potential therapeutic target for the treatment of osteoporosis and other metabolic bone diseases. © 2021 American Society for Bone and Mineral Research (ASBMR).

Characterization of Cre recombinase mouse lines enabling cell type-specific targeting of postnatal intervertebral discs.

Cre/loxP technology is an important tool for studying cell type-specific gene functions. Cre recombinase mouse lines, including Agc1-CreERT2 , Col2a1-Cre; Col2a1-CreERT2 , Shh-Cre, Shh-CreERT2 , and Osx-Cre, have been proven to be valuable tools to elucidate the biology of long bones, yet the information for their activity in postnatal intervertebral disc (IVD) tissues was very limited. In this study, we used R26-mTmG fluorescent reporter to systematically analyze cell specificity and targeting efficiency of these six mouse lines in IVD tissues at postnatal growing and adult stages. We found that Agc1-CreERT2 is effective to direct recombination in all components of IVDs, including annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplate (CEP), upon tamoxifen induction at either 2 weeks or 2 months of ages. Moreover, Col2a1-Cre targets most of the cells in IVDs, except for some cells in the outer AF (OAF) and NP. In contrast, the activity of Col2a1-CreERT2 is mainly limited to the IAF of IVD tissues at either stage of tamoxifen injection. Similarly, Shh-Cre directs recombination specifically in all NP cells, whereas Shh-CreERT2 is active only in a few NP cells when tamoxifen is administered at either stage. Finally, Osx-Cre targets cells in the CEP, but not in the NP or AF of IVDs tissues at these two stages. Thus, our data demonstrated that all these Cre lines can direct recombination in IVD tissues at postnatal stages with different cell type specificity and/or targeting efficiency, and can, therefore, serve as valuable tools to dissect cell type-specific gene functions in IVD development and homeostasis.

A dual role of cholesterol in osteogenic differentiation of bone marrow stromal cells.

Osteoblasts, the chief bone-forming cells, are differentiated from mesenchymal stromal/stem cells. Disruption of this differentiation process can cause osteoporosis, a bone disease characterized by low bone mass and deteriorated bone structure. Cholesterol has been implicated in pathogenesis of osteoporosis, and was recently identified as an endogenous activator of Hedgehog (Hh) signaling. However, its pathological and physiological roles in osteoblast differentiation are still poorly understood. Moreover, it is unclear whether these potential roles played by cholesterol are related to its capability to modulate Hh pathway. In this study, we investigated the role of exogenous versus endogenous cholesterol in osteogenesis and Hh pathway activation using ST2 cells, a bone marrow stromal cell line. We found that exogenous cholesterol significantly inhibited alkaline phosphatase (ALP) activity and messenger RNA expression of osteoblast markers genes (Alpl, Sp7, and Ibsp) while modestly activating expression of Gli1 (a readout of Hh signaling) under both basal osteogenic culture condition and Wnt3a treatment. Similarly, exogenous cholesterol suppressed osteogenic response of ST2 cells to sonic Hh (Shh) or purmorphamine (Purmo) treatment, which, however, was accompanied by diminished induction of Gli1, indicating the involvement of a Hh-dependent mechanism. Interestingly, depletion of endogenous cholesterol also reduced Shh-induced ALP activity and Gli1 expression. Likewise, cholesterol depletion inhibited osteogenic response to Purmo, although it did not affect Gli1 induction. Taken together, our findings have demonstrated that cholesterol plays a dual role in osteoblast differentiation likely through both Hh-dependent and -independent mechanisms.

Proinflammatory macrophages promote degenerative phenotypes in rat nucleus pulpous cells partly through ERK and JNK signaling.

Intervertebral disc (IVD) degeneration is the major contributor to low back pain, a highly prevalent musculoskeletal problem that represents the leading cause of disability. Proinflammatory M1 macrophages were identified in degenerated IVDs. However, their role in the pathogenesis of IVD degeneration and the underlying mechanism was largely unknown. In this study, we explored the combined effects of molecules secreted by M1 macrophages on nucleus pulposus cells, by treating rat nucleus pulposus cells (rNP) with the conditioned medium collected from M1-polarized RAW264.7 cells (MФCM). We found that MФCM caused molecular changes associated with IVD degeneration, including increased expression of key matrix catabolic genes (Adamts4, Adamts5, Mmp3, and Mmp13), reduced the expression of major matrix-associated anabolic genes ( Sox9, Acan, and Col2a1), and upregulated transcription of inflammation-related genes ( IL-1b, IL-6, Ccl2, and Ccl3), in rNP cells. Moreover, we found that MФCM activated both ERK and JNK pathways in these cells, and that inhibition of JNK pathway attenuated MФCM-induced expression of both catabolic and inflammatory genes, whereas ERK inhibition only suppressed induction of catabolic, but not inflammatory genes. Together, our data demonstrated that proinflammatory macrophages promoted the degenerative phenotypes in rNP cells in part through ERK and JNK signaling, and suggested that inhibition of these pathways may serve as a potential therapeutic approach for the treatment of IVD degeneration.