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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 κβ 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.

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.

Chlamydiae are very important pathogens which could cause several types of diseases in human, but little is known about its pathogenic mechanism. In order to elucidate host inflammatory response and the signal pathway induced by Chlamydial lipoproteins, the predicted lipoproteins of Chlamydia trachomatis were tested for their ability to induce the release of proinflammatory cytokines by mouse macrophages or human TLR (Toll-Like Receptor) expressing cell lines. The results showed that recombinant proteins of C. trachomatis D381, D541, D067, and D775 displayed a strong ability to induce the release of IL-8 in TLR expressing cell line. The signal pathways involved TLR1/2 and TLR2/CD14 but not TLR4. Moreover, except D067, the proinflammatory cytokine induction by D381, D541, and D775 required the thioacylation site (cysteine) for lipid modification and the induction was through MyD88-mediated pathway. Our data supported that lipoproteins played a vital role in pathogenesis of C. trachomatis-induced inflammatory responses via TLR pathway. It was the first study to characterize other chlamydial lipoproteins after identifying the role of MIP (D541) on pathogenesis of Chlamydial diseases. [ABSTRACT FROM AUTHOR]