Cortactin controls bone homeostasis through regulating the differentiation of osteoblasts and osteoclasts.

Cortactin, a cytoskeletal protein and substrate of src kinase, is implicated in tumor aggressiveness. However, its role in bone cell differentiation remains unknown. The current study revealed that cortactin was upregulated during osteoblast and adipocyte differentiation. Functional experiments demonstrated that cortactin promoted the differentiation of mesenchymal stem/progenitor cells into osteogenic and adipogenic lineages. Mechanistically, cortactin was able to stabilize the protein level of mechanistic target of rapamycin kinase (mTOR), leading to the activation of mTOR signaling. In-depth investigation revealed that cortactin could bind with casitas B lineage lymphoma-c (c-CBL) and counteract the function of c-CBL, a known E3 ubiquitin ligase responsible for the proteasomal degradation of mTOR. Silencing c-Cbl alleviated the impaired differentiation of osteoblasts and adipocytes caused by cortactin siRNA, while silencing mTOR mitigated the stimulation of osteoblast and adipocyte differentiation induced by cortactin overexpression. Notably, transplantation of cortactin-silenced bone marrow stromal cells (BMSCs) into the marrow of mice led to a reduction in trabecular bone mass, accompanied by a decrease in osteoblasts and an increase in osteoclasts. Furthermore, cortactin-silenced BMSCs expressed higher levels of RANKL than control BMSCs did, and promoted osteoclast differentiation when cocultured with bone marrow-derived osteoclast precursor cells. This study provides evidence that cortactin favors osteoblast differentiation by counteracting the c-CBL-induced degradation of mTOR and inhibits osteoclast differentiation by downregulating the expression of RANKL. It also suggests that maintaining an appropriate level of cortactin expression may be advantageous for maintaining bone homeostasis.

Foxk1 stimulates adipogenic differentiation via a peroxisome proliferator-activated receptor gamma 2-dependent mechanism.

Adipogenesis is a tightly regulated process, and its dysfunction has been linked to metabolic disorders such as obesity. Forkhead box k1 (Foxk1) is known to play a role in the differentiation of myogenic precursor cells and tumorigenesis of different types of cancers; however, it is not clear whether and how it influences adipocyte differentiation. Here, we found that Foxk1 was induced in mouse primary bone marrow stromal cells (BMSCs) and established mesenchymal progenitor/stromal cell lines C3H/10T1/2 and ST2 after adipogenic treatment. In addition, obese db/db mice have higher Foxk1 expression in inguinal white adipose tissue than nonobese db/m mice. Foxk1 overexpression promoted adipogenic differentiation of C3H/10T1/2, ST2 cells and BMSCs, along with the enhanced expression of CCAAT/enhancer binding protein-α, peroxisome proliferator-activated receptor γ (Pparγ), and fatty acid binding protein 4. Moreover, Foxk1 overexpression enhanced the expression levels of lipogenic factors during adipogenic differentiation in both C3H/10T1/2 cells and BMSCs. Conversely, Foxk1 silencing impaired these cells from fully differentiating. Furthermore, adipogenic stimulation induced the nuclear translocation of Foxk1, which depended on the mTOR and PI3-kinase signaling pathways. Subsequently, Foxk1 is directly bound to the Pparγ2 promoter, stimulating its transcriptional activity and promoting adipocyte differentiation. Collectively, our study provides the first evidence that Foxk1 promotes adipocyte differentiation from progenitor cells by promoting nuclear translocation and upregulating the transcriptional activity of the Pparγ2 promoter during adipogenic differentiation.

miR-196b-5p Regulates Osteoblast and Osteoclast Differentiation and Bone Homeostasis by Targeting SEMA3A.

miR-196b-5p plays a role in various malignancies. We have recently reported its function in regulating adipogenesis. However, it remains to be clarified whether and how miR-196b-5p affects bone cells and bone homeostasis. In this study, in vitro functional experiments showed an inhibitory effect of miR-196b-5p on osteoblast differentiation. Mechanistic explorations revealed that miR-196b-5p directly targeted semaphorin 3a (Sema3a) and inhibited Wnt/ß-catenin signaling. SEMA3A attenuated the impaired osteogenesis induced by miR-196b-5p. Osteoblast-specific miR-196b transgenic mice showed significant reduction of bone mass. Trabecular osteoblasts were reduced and bone formation was suppressed, whereas osteoclasts, marrow adipocytes, and serum levels of bone resorption markers were increased in the transgenic mice. The osteoblastic progenitor cells from the transgenic mice had decreased SEMA3A levels and exhibited retarded osteogenic differentiation, whereas those marrow osteoclastic progenitors exhibited enhanced osteoclastogenic differentiation. miR-196b-5p and SEMA3A oppositely regulated the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin. The calvarial osteoblastic cells expressing the transgene promoted osteoclastogenesis, whereas the osteoblasts overexpressing Sema3a inhibited it. Finally, in vivo transfection of miR-196b-5p inhibitor to the marrow reduced ovariectomy-induced bone loss in mice. Our study has identified that miR-196b-5p plays a key role in osteoblast and osteoclast differentiation and regulates bone homeostasis. Inhibition of miR-196b-5p may be beneficial for amelioration of osteoporosis. © 2023 American Society for Bone and Mineral Research (ASBMR).

Metastasis suppressor 1 interacts with protein tyrosine phosphatase receptor-δ to regulate adipogenesis.

Adipogenesis is a finely controlled process and its dysfunction may contribute to metabolic disorders such as obesity. Metastasis suppressor 1 (MTSS1) is a player in tumorigenesis and metastasis of various types of cancers. To date, it is not known whether and how MTSS1 plays a role in adipocyte differentiation. In the current study, we found that MTSS1 was upregulated during adipogenic differentiation of established mesenchymal cell lines and primary cultured bone marrow stromal cells. Gain-of-function and loss-of-function experiments uncovered that MTSS1 facilitated adipocyte differentiation from mesenchymal progenitor cells. Mechanistic explorations revealed that MTSS1 bound and interacted with FYN, a member of Src family of tyrosine kinases (SFKs), and protein tyrosine phosphatase receptor-δ (PTPRD). We demonstrated that PTPRD was capable of inducing the differentiation of adipocytes. Overexpression of PTPRD attenuated the impaired adipogenesis induced by the siRNA targeting MTSS1. Both MTSS1 and PTPRD activated SFKs by suppressing the phosphorylation of SFKs at Tyr530 and inducing the phosphorylation of FYN at Tyr419. Further investigation showed that MTSS1 and PTPRD were able to activate FYN. Collectively, our study has for the first time unraveled that MTSS1 plays a role in adipocyte differentiation in vitro through interacting with PTPRD and thereby activating SFKs such as FYN tyrosine kinase.

Tax1 binding protein 3 regulates osteogenic and adipogenic differentiation through inactivating Wnt/ß-catenin signalling.

Tax1 binding protein 3 (Tax1bp3) is a PDZ domain-containing protein that is overexpressed in cancer. Previous studies recognized Tax1bp3 as an inhibitor of ß-catenin. Till now it is not known whether Tax1bp3 regulates osteogenic and adipogenic differentiation of mesenchymal progenitor cells. In the current study, the data showed that Tax1bp3 was expressed in bone and was increased in the progenitor cells when induced toward osteoblast and adipocyte differentiation. The overexpression of Tax1bp3 in the progenitor cells inhibited osteogenic differentiation and conversely stimulated adipogenic differentiation, and the knockdown of Tax1bp3 affected the differentiation of the progenitor cells oppositely. Ex vivo experiments using the primary calvarial osteoblasts from osteoblast-specific Tax1bp3 knock-in mice also demonstrated the anti-osteogenic and pro-adipogenic function of Tax1bp3. Mechanistic investigations revealed that Tax1bp3 inhibited the activation of canonical Wnt/ß-catenin and bone morphogenetic proteins (BMPs)/Smads signalling pathways. Taken together, the current study has provided evidences demonstrating that Tax1bp3 inactivates Wnt/ß-catenin and BMPs/Smads signalling pathways and reciprocally regulates osteogenic and adipogenic differentiation from mesenchymal progenitor cells. The inactivation of Wnt/ß-catenin signalling may be involved in the reciprocal role of Tax1bp3.

Oncostatin M receptor regulates osteoblast differentiation via extracellular signal-regulated kinase/autophagy signaling.

BACKGROUND: Oncostatin M receptor (OSMR), as one of the receptors for oncostatin M (OSM), has previously been shown to mediate the stimulatory role of OSM in osteoclastogenesis and bone resorption. However, it remains to be clarified whether and how OSMR affects the differentiation of osteoblasts. METHODS: The expression level of OSMR during osteoblast and adipocyte differentiation was examined. The role of OSMR in the differentiation was investigated using in vitro gain-of-function and loss-of-function experiments. The mechanisms by which OSMR regulates bone cell differentiation were explored. Finally, in vivo function of OSMR in cell fate determination and bone homeostasis was studied after transplantation of OSMR-silenced bone marrow stromal cells (BMSCs) to the marrow of ovariectomized mice. RESULTS: OSMR was regulated during osteogenic and adipogenic differentiation of marrow stromal progenitor cells and increased in the metaphysis of ovariectomized mice. OSMR suppressed osteogenic differentiation and stimulated adipogenic differentiation of progenitor cells. Mechanistic investigations showed that OSMR inhibited extracellular signal-regulated kinase (ERK) and autophagy signaling. The downregulation of autophagy, which was mediated by ERK inhibition, suppressed osteogenic differentiation of progenitor cells. Additionally, inactivation of ERK/autophagy signaling attenuated the stimulation of osteogenic differentiation induced by Osmr siRNA. Furthermore, transplantation of BMSCs in which OSMR was silenced to the marrow of mice promoted osteoblast differentiation, attenuated fat accumulation and osteoclast differentiation, and thereby relieved the osteopenic phenotype in the ovariectomized mice. CONCLUSIONS: Our study has for the first time established the direct role of OSMR in regulating osteogenic differentiation of marrow stromal progenitor cells through ERK-mediated autophagy signaling. OSMR thus contributes to bone homeostasis through dual regulation of osteoblasts and osteoclasts. It also suggests that OSMR may be a potential target for the treatment of metabolic disorders such as osteoporosis.

Metastasis suppressor 1 controls osteoblast differentiation and bone homeostasis through regulating Src-Wnt/ß-catenin signaling.

Metastasis suppressor 1 (MTSS1) plays an inhibitory role in tumorigenesis and metastasis of a variety of cancers. To date, the function of MTSS1 in the differentiation of marrow stromal progenitor cells remains to be explored. In the current study, we investigated whether and how MTSS1 has a role in osteoblast differentiation and bone homeostasis. Our data showed that MTSS1 mRNA was upregulated during osteoblast differentiation and downregulated in the osteoblastic lineage cells of ovariectomized and aged mice. Functional studies revealed that MTSS1 promoted the osteogenic differentiation from marrow stromal progenitor cells. Mechanistic explorations uncovered that the inactivation of Src and afterward activation of canonical Wnt signaling were involved in osteoblast differentiation induced by MTSS1. The enhanced osteogenic differentiation induced by MTSS1 overexpression was attenuated when Src was simultaneously overexpressed, and conversely, the inhibition of osteogenic differentiation by MTSS1 siRNA was rescued when the Src inhibitor was supplemented to the culture. Finally, the in vivo transfection of MTSS1 siRNA to the marrow of mice significantly reduced the trabecular bone mass, along with the reduction of trabecular osteoblasts, the accumulation of marrow adipocytes, and the increase of phospho-Src-positive cells on the trabeculae. No change in the number of osteoclasts was observed. This study has unraveled that MTSS1 contributes to osteoblast differentiation and bone homeostasis through regulating Src-Wnt/ß-catenin signaling. It also suggests the potential of MTSS1 as a new target for the treatment of osteoporosis.

miR-142a-5p promoted osteoblast differentiation via targeting nuclear factor IA.

miR-142a-5p plays critical roles in multiple biological processes and diseases, such as inflammation and tumorigenesis. However, it remains to be explored if and how miR-142a-5p contributes to osteoblast differentiation. In this study, our results showed that miR-142a-5p was highly expressed in bone tissue of mice and increased during osteogenesis in preosteoblast MC3T3-E1 cells. Supplementing miR-142a-5p activity using miR-142a-5p agomir promoted osteogenic differentiation in stromal cell line ST2 and preosteoblastic line MC3T3-E1. Conversely, miR-142a-5p antagomir, an inhibitor of endogenous miR-142a-5p, could reduce osteoblast differentiation in ST2 and MC3T3-E1 cells. Nuclear factor IA (NFIA), a site-specific transcriptional factor, was demonstrated to be directly targeted by miR-142a-5p. Overexpression of NFIA inhibited miR-142a-5p-mediated osteoblast differentiation in ST2 cells. Furthermore, mechanism explorations revealed that Wnt/ß-catenin signaling transcriptionally regulated the expression of miR-142a-5p during osteogenic differentiation. ß-catenin binds to the T-cell factor/lymphoid enhancer factor binding motif within the promoter of miR-142 and positively regulates its transcriptional activity. Our findings suggested that miR-142a-5p promoted osteoblast differentiation via targeting NFIA.

A novel long noncoding RNA PGC1ß-OT1 regulates adipocyte and osteoblast differentiation through antagonizing miR-148a-3p.

Long noncoding RNAs (LncRNAs) have been implicated in the regulation of adipocyte and osteoblast differentiation. However, the functional contributions of LncRNAs to adipocyte or osteoblast differentiation remain largely unexplored. In the current study we have identified a novel LncRNA named peroxisome proliferator-activated receptor γ coactivator-1ß-OT1 (PGC1ß-OT1). The expression levels of PGC1ß-OT1 were altered during adipogenic and osteogenic differentiation from progenitor cells. 5'- and 3'-rapid amplification of cDNA ends (RACE) revealed that PGC1ß-OT1 is 1759 nt in full length. Overexpression of PGC1ß-OT1 in progenitor cells inhibited adipogenic differentiation, whereas silencing of endogenous PGC1ß-OT1 induced adipogenic differentiation. By contrast, overexpression of PGC1ß-OT1 in progenitor cells stimulated, whereas silencing of PGC1ß-OT1 inhibited osteogenic differentiation. In vivo experiment showed that silencing of endogenous PGC1ß-OT1 in marrow stimulated fat accumulation and decreased osteoblast differentiation in mice. Mechanism investigations revealed that PGC1ß-OT1 contains a functional miR-148a-3p binding site. Overexpression of the mutant PGC1ß-OT1 with mutation at the binding site failed to regulate either adipogenic or osteogenic differentiation. In vivo crosslinking combined with affinity purification studies demonstrated that PGC1ß-OT1 physically associated with miR-148a-3p through the functional miR-148a-3p binding site. Furthermore, PGC1ß-OT1 affected the expression of endogenous miR-148a-3p and its target gene lysine-specific demethylase 6b (KDM6B). Supplementation of miR-148a-3p in progenitor cells blocked the inhibitory effect of PGC1ß-OT1 on adipocyte formation. Moreover, overexpression of Kdm6b restored the osteoblast differentiation which was inhibited by silencing of endogenous PGC1ß-OT. Our studies provide evidences that the novel LncRNA PGC1ß-OT1 reciprocally regulates adipogenic and osteogenic differentiation through antagonizing miR-148a-3p and enhancing KDM6B effect.

Histone demethylase KDM7A reciprocally regulates adipogenic and osteogenic differentiation via regulation of C/EBPα and canonical Wnt signalling.

Recent emerging evidences revealed that epigenetic methylation of histone and DNA regulates the lineage commitment of mesenchymal progenitor cells. This study was undertaken to delineate the actions of histone lysine demethylase 7A (KDM7A) on osteogenic and adipogenic differentiation. Kdm7a expression was up-regulated in primary marrow stromal cells and established stromal ST2 line after adipogenic and osteogenic treatment. Silencing of endogenous Kdm7a in the cells blocked adipogenic differentiation whereas promoted osteogenic differentiation. Conversely, overexpression of wild-type Kdm7a in the progenitor cells enhanced adipogenic differentiation whereas inhibited osteogenic differentiation. However, the effect of KDM7A on cell differentiation was largely attenuated when the point mutation was made that abolishes enzymatic activity of KDM7A. Mechanism investigations revealed that silencing of Kdm7a down-regulated the expression of the CCAAT/enhancer binding protein α (C/EBPα) and secreted frizzled-related protein 1 (Sfrp1). Chromatin immunoprecipitation (ChIP) assay revealed that KDM7A directly binds to the promoters of C/EBPα and Sfrp1 and removes the histone methylation marks H3K9me2 and H3K27me2. Furthermore, silencing of Kdm7a activated canonical Wnt signalling. Thereafter, activation of canonical Wnt signalling through silencing of Sfrp1 in ST2 attenuated the stimulation of adipogenic differentiation and inhibition of osteogenic differentiation by KDM7A. Our study suggests that KDM7A balances adipogenic and osteogenic differentiation from progenitor cells through epigenetic control of C/EBPα and canonical Wnt signalling and implicates that control of KDM7A action has an epigenetic perspective of curtailing metabolic disorders like osteoporosis.

Long noncoding RNA Plnc1 controls adipocyte differentiation by regulating peroxisome proliferator-activated receptor γ.

Detailed understanding of molecular mechanisms controlling adipogenesis is of great importance to identify new targets for treating obesity. Emerging evidence suggests that long noncoding RNAs (lncRNAs) may play a pivotal role in adipogenesis. Here, we have identified a novel lncRNA, Plnc1, which is transcribed from a position ∼25,000 bp upstream of the peroxisome proliferator-activated receptor γ2 ( PPAR-γ2) gene. Plnc1 is abundantly expressed in adipose tissue, and obese mice have higher Plnc1 expression in adipose tissue than nonobese mice. Plnc1 was induced in established adipogenic lines ST2, 3T3-L1, and C3H10T1/2 as well as in bone marrow stromal cells (BMSCs) after adipogenic treatment. Plnc1 knockdown blocked differentiation of ST2 cells and BMSCs into mature adipocytes, along with the reduction of PPAR-γ, CCAAT/enhancer binding protein-α, and adipocyte protein 2. Conversely, overexpression of Plnc1 promoted ST2 cells and BMSCs to fully differentiate. Mechanism studies revealed that Plnc1 could reduce the methylation level of CpG region in the PPAR-γ2 promoter and enhance the transcriptional activity of the promoter and thereby increase PPAR-γ2 transcription. Our study suggests that Plnc1 promotes adipogenic differentiation through controlling the key adipogenic transcription factor PPAR-γ and highlights the potential of Plnc1 as a target for new therapies to control metabolic disorders like obesity.-Zhu, E., Zhang, J., Li, Y., Yuan, H., Zhou, J., Wang, B. Long noncoding RNA Plnc1 controls adipocyte differentiation by regulating peroxisome proliferator-activated receptor γ.

miR-20a-5p promotes adipogenic differentiation of murine bone marrow stromal cells via targeting Kruppel-like factor 3.

miR-20a-5p has recently been identified to induce adipogenesis of established adipogenic cell lines in our previous study. However, its role and molecular mechanisms in the regulation of adipocyte lineage commitment of bone marrow-derived stromal cells (BMSCs) still need to be explored. In this report, we demonstrated the expression of miR-20a-5p was promoted gradually during adipogenic differentiation in BMSCs. We also confirmed that miR-20a-5p has a positive function in the adipogenic differentiation of BMSCs by gain-of-function study with overexpression lentivirus or synthetic mimics of miR-20a-5p, and loss-of-function study with sponge lentivirus or synthetic inhibitor of miR-20a-5p. Dual luciferase reporter assay, GFP repression assay and Western blotting suggested Kruppel-like factor 3 (Klf3) was a direct target of miR-20a-5p. Furthermore, siRNA-mediated silencing of Klf3 recapitulated the potentiation of adipogenesis induced by miR-20a-5p overexpression, whereas enhanced expression of Klf3 attenuated the effect of miR-20a-5p. As Klf3 was reported to play an inhibitory role in adipogenesis at the initial stage of differentiation, the findings we present here indicate that miR-20a-5p promotes adipocyte differentiation from BMSCs by targeting and negatively regulating Klf3 in the early phase during the procedure of adipogenesis.

Nuclear factor I-C reciprocally regulates adipocyte and osteoblast differentiation via control of canonical Wnt signaling.

Nuclear factor I-C (NFIC) has recently been identified as an important player in osteogenesis and bone homeostasis in vivo However, the molecular mechanisms involved have yet to be defined. In the current study, Nfic expression was altered in primary marrow stromal cells and established progenitor lines after adipogenic and osteogenic treatment. Overexpression of Nfic in stromal cells ST2, mesenchymal cells C3H10T1/2, and primary marrow stromal cells inhibited adipogenic differentiation, whereas it promoted osteogenic differentiation. Conversely, silencing of endogenous Nfic in the cell lines enhanced adipogenic differentiation, whereas it blocked osteogenic differentiation. Mechanism investigations revealed that Nfic overexpression promoted nuclear translocation of ß-catenin and increased nuclear protein levels of ß-catenin and transcription factor 7-like 2 (TCF7L2). Promoter studies and the chromatin immunoprecipitation (ChIP) assay revealed that NFIC directly binds to the promoter of low-density lipoprotein receptor-related protein 5 (Lrp5) and thereafter transactivates the promoter. Finally, inactivation of canonical Wnt signaling in ST2 attenuated the inhibition of adipogenic differentiation and stimulation of osteogenic differentiation by NFIC. Our study suggests that NFIC balances adipogenic and osteogenic differentiation from progenitor cells through controlling canonical Wnt signaling and highlights the potential of NFIC as a target for new therapies to control metabolic disorders like osteoporosis and obesity.-Zhou, J., Wang, S., Qi, Q., Yang, X., Zhu, E., Yuan, H., Li, X., Liu, Y., Li, X., Wang, B. Nuclear factor I-C reciprocally regulates adipocyte and osteoblast differentiation via control of canonical Wnt signaling.

mTOR and autophagy in normal brain aging and caloric restriction ameliorating age-related cognition deficits.

Defect of autophagy is common to many neurodegenerative disorders because it serves as a major degradation pathway for the clearance of various aggregate-prone proteins. Mammalian target of rapamycin (mTOR) signaling, which is recognized as the most important negative regulator of autophagy, is also involved in neurodegenerative diseases. However, the role of mTOR and its dependent autophagy in normal brain during aging remains unknown. Furthermore, caloric restriction (CR) is frequently used as a tool to study mechanisms behind aging and age-associated diseases because CR can prevent age-related diseases and prolong lifespan in several model organisms. Inhibiting mTOR and promoting autophagy activity play roles in aging delayed by CR. However, whether CR can ameliorate age-related cognition deficits by inhibiting mTOR and activate autophagy in hippocampus needs to be further investigated. Here we showed a decline of autophagic degradation in mice hippocampus in correlation with age-dependent cognitive dysfunction, whereas the activity of mTOR and its upstream brain-derived neurotrophic factor (BDNF)/phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling was decreased with aging. In addition, facilitating the mTOR pathway successfully declines and sustains autophagic degradation with aging in hippocampus by CR treatment and is involved in CR by ameliorating age-related cognitive deficits.