scholarly journals RAB37 Promotes Adipogenic Differentiation of hADSCs via the TIMP1/CD63/Integrin Signaling Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Haili Huang ◽  
Anran Li ◽  
Jin Li ◽  
Dan Sun ◽  
Ling Liang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Early Stage ◽  
Adipogenic Differentiation ◽  
Critical Role ◽  
Proximity Ligation Assay ◽  
Integrin Β1 ◽  
Protein Levels ◽  
Proximity Ligation ◽  
Matrix Metalloproteinase 1

The adipogenic differentiation ability of human adipose-derived mesenchymal stem cells (hADSCs) is critical for the construction of tissue engineering adipose, which shows promising applications in plastic surgery and regenerative medicine. RAB37 is a member of the small RabGTPase family and plays a critical role in vesicle trafficking. However, the role of RAB37 in adipogenic differentiation of hADSCs remains unclear. Here, we report that both the mRNA and protein levels of RAB37 fluctuated during adipogenic differentiation. Upregulation of RAB37 was observed at the early stage of adipogenic differentiation, which was accompanied by increased expression of transcription factors PPARγ2 and C/EBPα, and lipoprotein lipase (LPL). Overexpression of RAB37 promoted adipogenesis of hADSCs, as revealed by Oil Red O staining and increased expression of PPARγ2, C/EBPα, and LPL. Several upregulated cytokines related to RAB37-mediated adipogenic differentiation were identified using a cytokine array, including tissue inhibitor of matrix metalloproteinase 1 (TIMP1). ELISA confirmed that upregulation of RAB37 increased the secretion of TIMP1 by hADSCs. Proximity ligation assay showed that RAB37 interacts with TIMP1 directly. Knockdown of TIMP1 compromised RAB37-mediated adipogenic differentiation. In addition, TIMP1 binds membrane receptor CD63 and integrin β1. RAB37 promotes Tyr397 phosphorylation of FAK, an important protein kinase of the integrin β1 signaling. Moreover, both knockdown of CD63 and inhibitor of FAK impeded RAB37-mediated adipogenic differentiation. In conclusion, RAB37 positively regulates adipogenic differentiation of hADSCs via the TIMP1/CD63/integrin β1 signaling pathway.

scholarly journals Role of Cathepsin S in Periodontal Inflammation and Infection

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
S. Memmert ◽  
A. Damanaki ◽  
A. V. B. Nogueira ◽  
S. Eick ◽  
M. Nokhbehsaim ◽  
...  
Keyword(s):  
Periodontal Diseases ◽  
Interleukin 1 ◽  
Critical Role ◽  
Gingival Tissue ◽  
Cathepsin S ◽  
Protein Levels ◽  
Periodontal Inflammation

Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1β and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1β and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.

scholarly journals Lipotoxicity-Induced mtDNA Release Promotes Diabetic Cardiomyopathy by Activating the cGAS-STING Pathway in Obesity Related Diabetes

2021 ◽  
Author(s):  
Xiu Mei Ma ◽  
Kang Geng ◽  
Betty Yuen-Kwan Law ◽  
Peng Wang ◽  
Yue Li Pu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Signaling Pathway ◽  
Diabetic Cardiomyopathy ◽  
Cell Model ◽  
Critical Role ◽  
Kidney Tissue ◽  
H9c2 Cells ◽  
Downstream Targets ◽  
Sting Pathway

Abstract Diabetic cardiomyopathy (DCM) is characterized by lipid accumulation, mitochondrial dysfunction, and aseptic inflammatory activation. Mitochondria-derived cytosolic DNA has been reported to induce inflammation by activating cyclic GMP-AMP synthase (cGAS)/the stimulator of interferon genes (STING) pathway in the adipose, liver, and kidney tissue. However, the role of cytosolic mtDNA in the progression of DCM is unclear. In this study, with an obesity-related DCM mouse model established by feeding db/db mice with a high-fat diet (HFD), we observed increased mtDNA in the cytosol and activated cGAS-STING signaling pathway during DCM, as well as the downstream targets, IRF3, NF-κB, IL-18, and IL-1β. In further study with a palmitic acid (PA)-induced lipotoxic cell model established in H9C2 cells, we revealed that the cytosolic mtDNA was resulted from PA-induced overproduction of mitochondrial ROS, which also led to the activation of the cGAS/STING system and its downstream targets. Notably, treatment of extracted mtDNA alone was sufficient to activate the cGAS-STING signaling pathway in cultured H9C2 cells. Besides, both knockdown of STING in PA-induced H9C2 cells and inhibition of STING by C-176 injection in the DCM mouse model could remarkably block the inflammation and apoptosis of cardiomyocytes. In conclusion, our study elucidated the critical role of cytosolic mtDNA-induced cGAS-STING activation in the pathogenesis of obesity-related DCM and provided preclinical validation for using a STING inhibitor as a new potential therapeutic strategy for the treatment of DCM.

scholarly journals Nox4 Mediates RANKL-induced ER-Phagy and Osteoclastogenesis via Activating ROS/PERK/eIF-2α/ATF4 Pathway

2020 ◽  
Author(s):  
Jing Sun ◽  
wugui chen ◽  
Songtao Li ◽  
Sizhen Yang ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Bone Resorption ◽  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Nuclear Factor Κb ◽  
Protein Levels ◽  
Unfolded Protein ◽  
Regulatory Effects ◽  
Protein Response

Abstract Background: Receptor activator of nuclear factor-κB ligand (RANKL) has been found to induce osteoclastogenesis and bone resorption. However, the underlying molecular mechanisms remain unclear. Methods: Osteoclastogenesis was evaluated by number of TRAP-positive multinuclear (≥3) osteoclasts, bone resorption pits and expression levels of related genes. Autophagy activity were evaluated by LC3-II/LC3-I ratio, number of autophagic vacuoles and adenovirus-mRFP-GFP-tagged LC3 reporting system; Inhibitor chloroquine (CQ) was used to verified the role of autophagy in RANKL-induced osteoclastogenesis; Via downregulating Nox4 with inhibitor (DPI) and retrovirus-conveyed shRNA, we further explored the importance of Nox4 in RANKL-induced autophagy and osteoclastogenesis, as well as the regulatory effects of Nox4 on nonmitochondrial reactive oxygen species (ROS) and PERK/eIF-2α/ATF4 pathway. Intracellular ROS scavenger (NAC), mitochondrial-targeted antioxidant (MitoTEMPO) and inhibitor of PERK (GSK2606414) were also employed to investigate the role of ROS and PERK/eIF-2α/ATF4 pathway in RANKL-induced autophagy and osteoclastogenesis. Results: RANKL markedly increased autophagy, while CQ treatment caused reduction of RANKL-induced autophagy and osteoclastogenesis. Consistent with the increased autophagy, the protein levels of Nox4 were significantly increased, and Nox4 was selectively localized within the endoplasmic reticulum (ER) after RANKL stimulation. DPI and shRNA efficiently decreased the protein level and (or) activity of Nox4 in the ER and inhibited RANKL-induced autophagy and osteoclastogenesis. Mechanistically, we found that Nox4 regulates RANKL-induced autophagy activation and osteoclastogenesis by stimulating the production of nonmitochondrial ROS. Additionally, Nox4-derived nonmitochondrial ROS dramatically activate PERK/eIF-2α/ATF4, which is a critical unfolded protein response (UPR)-related signaling pathway during ER stress. Blocking the activation of the PERK/eIF-2α/ATF4 signaling pathway either by Nox4 shRNA, ROS antioxidant or PERK inhibitor (GSK2606414) treatment significantly inhibited endoplasmic reticulum autophagy (ER-phagy) during RANKL-induced osteoclastogenesis. Conclusions: Our findings provide new insights into the processes of RANKL-induced osteoclastogenesis and will help the development of new therapeutic strategies for osteoclastogenesis-related diseases.

scholarly journals Metformin Reverses the Enhanced Myocardial SR/ER–Mitochondria Interaction and Impaired Complex I-Driven Respiration in Dystrophin-Deficient Mice

2021 ◽  
Vol 8 ◽  
Author(s):  
Claire Angebault ◽  
Mathieu Panel ◽  
Mathilde Lacôte ◽  
Jennifer Rieusset ◽  
Alain Lacampagne ◽  
...  
Keyword(s):  
Complex I ◽  
Early Stage ◽  
Intrinsic Activity ◽  
Proximity Ligation Assay ◽  
Proximity Ligation ◽  
Skeletal Muscle Dysfunction ◽  
Species Production ◽  
Therapeutic Perspective ◽  
Deficient Mice

Besides skeletal muscle dysfunction, Duchenne muscular dystrophy (DMD) exhibits a progressive cardiomyopathy characterized by an impaired calcium (Ca2+) homeostasis and a mitochondrial dysfunction. Here we aimed to determine whether sarco-endoplasmic reticulum (SR/ER)–mitochondria interactions and mitochondrial function were impaired in dystrophic heart at the early stage of the pathology. For this purpose, ventricular cardiomyocytes and mitochondria were isolated from 3-month-old dystrophin-deficient mice (mdx mice). The number of contacts points between the SR/ER Ca2+ release channels (IP3R1) and the porine of the outer membrane of the mitochondria, VDAC1, measured using in situ proximity ligation assay, was greater in mdx cardiomyocytes. Expression levels of IP3R1 as well as the mitochondrial Ca2+ uniporter (MCU) and its regulated subunit, MICU1, were also increased in mdx heart. MICU2 expression was however unchanged. Furthermore, the mitochondrial Ca2+ uptake kinetics and the mitochondrial Ca2+ content were significantly increased. Meanwhile, the Ca2+-dependent pyruvate dehydrogenase phosphorylation was reduced, and its activity significantly increased. In Ca2+-free conditions, pyruvate-driven complex I respiration was decreased whereas in the presence of Ca2+, complex I-mediated respiration was boosted. Further, impaired complex I-mediated respiration was independent of its intrinsic activity or expression, which remains unchanged but is accompanied by an increase in mitochondrial reactive oxygen species production. Finally, mdx mice were treated with the complex I modulator metformin for 1 month. Metformin normalized the SR/ER-mitochondria interaction, decreased MICU1 expression and mitochondrial Ca2+ content, and enhanced complex I-driven respiration. In summary, before any sign of dilated cardiomyopathy, the DMD heart displays an aberrant SR/ER-mitochondria coupling with an increase mitochondrial Ca2+ homeostasis and a complex I dysfunction. Such remodeling could be reversed by metformin providing a novel therapeutic perspective in DMD.

FC 006PP2A PHOSPHATASE INHIBITION IS ANTI-FIBROTIC THROUGH SER77 PHOSPHORYLATION-MEDIATED ARNT/ARNT HOMODIMER FORMATION

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Gunsmaa Nyamsuren ◽  
Gregor Christof Rapp ◽  
Björn Tampe ◽  
Michael Zeisberg
Keyword(s):  
Molecular Mechanisms ◽  
Therapeutic Potential ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Receptor Expression ◽  
Proximity Ligation Assay ◽  
Proximity Ligation ◽  
Aryl Hydrocarbon ◽  
Ureter Obstruction

Abstract Background and Aims Aryl hydrocarbon receptor nuclear translocator (ARNT) mediates anti-fibrotic activity in kidney and liver through induction of ALK3-receptor expression and subsequently increased Smad1/5/8 signaling. While expression of ARNT can be pharmacologically induced by sub-immunosuppressive doses of FK506 or by GPI1046, its anti-fibrotic activity is only realized when ARNT-ARNT homodimers form, as opposed to formation of ARNT-AHR or ARNT-HIF1α heterodimers. Mechanisms underlying ARNTs dimerization decision to specifically form ARNT-ARNT homodimers and possible cues to specifically induce ARNT homodimerization have been previously unknown. We here aimed to elucidate the molecular mechanisms underlying control of ARNT dimerization decision and to explore its therapeutic potential. Method We analyzed dimerization of recombinant and native ARNT by immunoprecipitation, MALDI-TOF mass spectrometry, and LS-MS/MS analysis and proximity ligation assay. Phosphorylation sites were mapped through generation of phosphorylation site mutants and through pharmacological inhibition. For in vivo analysis we challenged mice with model of unilateral ureter obstruction and carbon tetrachloride to induce fibrosis in kidney and liver. Results Here we report that inhibition of PP2A phosphatase activity increases intracellular accumulation of ARNT-ARNT homodimers. This effect is dependent on enhanced ARNT-ARNT homodimerization and decreased ARNT proteolytic degradation, but independent of ARNT transcription (which remains unchanged upon PP2A inhibition). We further identify that Ser77 phosphorylation plays a critical role in ARNT homodimerization, as ARNT-ARNT homodimers do not form with Ser77/Asp-mutant ARNT proteins. In light of previous studies which identified anti-fibrotic activity upon increased ARNT expression, we further demonstrate attenuated fibrosis upon monotherapy with the PP2A inhibitor LB100, and additive anti-fibrotic activities upon combination with pharmacological inducers of ARNT expression FK506 or GPI1046 in murine models of kidney and liver fibrosis. Conclusion Our study provides additional evidence for the anti-fibrotic activity of ARNT and reveals Ser77 phosphorylation as a novel pharmacological target to realize the therapeutic potential of increased ARNT transactivation activity.

Abstract P207: Role of (Pro)Renin Receptor in the Pathogenesis of Colon Cancer

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Akira Nishiyama ◽  
Juan Wang ◽  
Shinichi Yachida ◽  
Genevieve Nguyen ◽  
Takuo Hirose ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Signaling Pathway ◽  
Western Blotting ◽  
Cell Number ◽  
Ductal Adenocarcinoma ◽  
Mrna Levels ◽  
Protein Levels ◽  
Colon Cancer Cell Lines ◽  
Cancer Tissues

(Pro)renin receptor ((P)RR) is a component of the Wnt receptor complex (Science, 2010). We have recently demonstrated that (P)RR plays an important role in the tumorigenesis of pancreatic ductal adenocarcinoma via the activation of Wnt/β-catenin signaling pathway (Shibayama et al. Sci Rep. 2015). Since the patients with colon cancer often show aberrantly activated Wnt/β-catenin-dependent signaling pathway by the mutations of its components, we investigated the possible role of (P)RR and Wnt/β-catenin signaling pathway in carcinogenesis of colon cancer. Real-time PCR was used for measuring mRNA levels of (P)RR. Protein levels of (P)RR was determined by Western blotting and immunohistochemistry. Activated β-catenin levels were determined by Western blotting. Cell proliferative ability was evaluated by counting the cell number in cultured colon cancer cell lines, HCT116 and DLD-1 cells. As compared to normal colon tissues (n=6), mRNA and protein levels of (P)RR were increased by 2.6- and 2.2-fold, respectively, in colon cancer tissues (n=9), which were associated with increased activated β-catenin levels (by 2.8-fold, P<0.05). However, plasma soluble (P)RR levels were not changed in patients with colon cancer (n=9). (P)RR and activated β-catenin levels were also increased in HCT116 (by 2.2- and 2.7-fold, n=5, respectively) and DLD-1 cells (by 1.9- and 2.8-fold, n=5, respectively). In these cells, inhibiting (P)RR with an siRNA attenuated the activity of β-catenin and reduced the proliferative abilities (n=5, P<0.05, respectively). These data suggest that (P)RR contributes to the tumorigenesis of colon cancer through the activation of Wnt/β-catenin signaling pathway.

scholarly journals Syk and Hrs Regulate TLR3-Mediated Antiviral Response in Murine Astrocytes

2019 ◽  
Vol 2019 ◽  
pp. 1-21 ◽  
Author(s):  
Matylda B. Mielcarska ◽  
Magdalena Bossowska-Nowicka ◽  
Karolina P. Gregorczyk-Zboroch ◽  
Zbigniew Wyżewski ◽  
Lidia Szulc-Dąbrowska ◽  
...  
Keyword(s):  
Immune Responses ◽  
Signaling Pathway ◽  
Critical Role ◽  
Antiviral Response ◽  
Proteolytic Processing ◽  
Viral Dsrna ◽  
Kinase Substrate ◽  
Tlr3 Signaling ◽  
The Brain

Toll-like receptors (TLRs) sense the presence of pathogen-associated molecular patterns. Nevertheless, the mechanisms modulating TLR-triggered innate immune responses are not yet fully understood. Complex regulatory systems exist to appropriately direct immune responses against foreign or self-nucleic acids, and a critical role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), endosomal sorting complex required for transportation-0 (ESCRT-0) subunit, has recently been implicated in the endolysosomal transportation of TLR7 and TLR9. We investigated the involvement of Syk, Hrs, and STAM in the regulation of the TLR3 signaling pathway in a murine astrocyte cell line C8-D1A following cell stimulation with a viral dsRNA mimetic. Our data uncover a relationship between TLR3 and ESCRT-0, point out Syk as dsRNA-activated kinase, and suggest the role for Syk in mediating TLR3 signaling in murine astrocytes. We show molecular events that occur shortly after dsRNA stimulation of astrocytes and result in Syk Tyr-342 phosphorylation. Further, TLR3 undergoes proteolytic processing; the resulting TLR3 N-terminal form interacts with Hrs. The knockdown of Syk and Hrs enhances TLR3-mediated antiviral response in the form of IFN-β, IL-6, and CXCL8 secretion. Understanding the role of Syk and Hrs in TLR3 immune responses is of high importance since activation and precise execution of the TLR3 signaling pathway in the brain seem to be particularly significant in mounting an effective antiviral defense. Infection of the brain with herpes simplex type 1 virus may increase the secretion of amyloid-β by neurons and astrocytes and be a causal factor in degenerative diseases such as Alzheimer’s disease. Errors in TLR3 signaling, especially related to the precise regulation of the receptor transportation and degradation, need careful observation as they may disclose foundations to identify novel or sustain known therapeutic targets.

scholarly journals Reduced Expression of Glutathione S-Transferase α4 Promotes Vascular Neointimal Hyperplasia in CKD

2017 ◽  
Vol 29 (2) ◽  
pp. 505-517 ◽  
Author(s):  
Jinlong Luo ◽  
Guang Chen ◽  
Ming Liang ◽  
Aini Xie ◽  
Qingtian Li ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Neointimal Hyperplasia ◽  
Critical Role ◽  
Mapk Signaling ◽  
Neointima Formation ◽  
Glutathione S Transferase ◽  
Protein Levels ◽  
Underlying Mechanisms ◽  
Arteries And Veins

Neointima formation is the leading cause of arteriovenous fistula (AVF) failure. We have shown that CKD accelerates this process by transforming the vascular smooth muscle cells (SMCs) lining the AVF from a contractile to the synthetic phenotype. However, the underlying mechanisms affecting this transformation are not clear. Previous studies have shown that the α-class glutathione transferase isozymes have an important role in regulating 4-hydroxynonenal (4-HNE)–mediated proliferative signaling of cells. Here, using both the loss- and gain-of-function approaches, we investigated the role of glutathione S-transferase α4 (GSTA4) in modulating cellular 4-HNE levels for the transformation and proliferation of SMCs. Compared with non-CKD controls, mice with CKD had downregulated expression of GSTA4 at the mRNA and protein levels, with concomitant increase in 4-HNE in arteries and veins. This effect was associated with upregulated phosphorylation of MAPK signaling pathway proteins in proliferating SMCs. Overexpressing GSTA4 blocked 4-HNE–induced SMC proliferation. Additionally, inhibitors of MAPK signaling inhibited the 4-HNE–induced responses. Compared with wild-type mice, mice lacking GSTA4 exhibited increased CKD-induced neointima formation in AVF. Transient expression of an activated form of GSTA4, achieved using a combined Tet-On/Cre induction system in mice, lowered levels of 4-HNE and reduced the proliferation of SMCs. Together, these results demonstrate the critical role of GSTA4 in blocking CKD-induced neointima formation and AVF failure.

scholarly journals VBP1 modulates Wnt/β-catenin signaling by mediating the stability of the transcription factors TCF/LEFs

2020 ◽  
Vol 295 (49) ◽  
pp. 16826-16839
Author(s):  
Haifeng Zhang ◽  
Xiaozhi Rong ◽  
Caixia Wang ◽  
Yunzhang Liu ◽  
Ling Lu ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Protein Stability ◽  
Signaling Pathway ◽  
Cultured Cells ◽  
Critical Role ◽  
Family Members ◽  
Von Hippel Lindau ◽  
T Cell Factor ◽  
Protein Levels ◽  
The Stability

The Wnt/β-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the β-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/β-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/β-catenin signaling pathway activity.

scholarly journals Hedgehog Inhibitors Suppress Osteoclastogenesis in In Vitro Cultures, and Deletion of Smo in Macrophage/Osteoclast Lineage Prevents Age-Related Bone Loss

2020 ◽  
Vol 21 (8) ◽  
pp. 2745
Author(s):  
Yukihiro Kohara ◽  
Ryuma Haraguchi ◽  
Riko Kitazawa ◽  
Yuuki Imai ◽  
Sohei Kitazawa
Keyword(s):  
Bone Resorption ◽  
Bone Loss ◽  
Signaling Pathway ◽  
Early Stage ◽  
Osteoclast Differentiation ◽  
Osteoclast Formation ◽  
Age Related ◽  
Hh Signaling

The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on Hh functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0–48 h (early stage of osteoclast differentiation) or 48–96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.

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