Splicing factor YBX1 regulates bone marrow stromal cell fate during aging.

Senescence and altered differentiation potential of bone marrow stromal cells (BMSCs) lead to age-related bone loss. As an important posttranscriptional regulatory pathway, alternative splicing (AS) regulates the diversity of gene expression and has been linked to induction of cellular senescence. However, the role of splicing factors in BMSCs during aging remains poorly defined. Herein, we found that the expression of the splicing factor Y-box binding protein 1 (YBX1) in BMSCs decreased with aging in mice and humans. YBX1 deficiency resulted in mis-splicing in genes linked to BMSC osteogenic differentiation and senescence, such as Fn1, Nrp2, Sirt2, Sp7, and Spp1, thus contributing to BMSC senescence and differentiation shift during aging. Deletion of Ybx1 in BMSCs accelerated bone loss in mice, while its overexpression stimulated bone formation. Finally, we identified a small compound, sciadopitysin, which attenuated the degradation of YBX1 and bone loss in old mice. Our study demonstrated that YBX1 governs cell fate of BMSCs via fine control of RNA splicing and provides a potential therapeutic target for age-related osteoporosis.

Protective effects of mitochondria-targeted antioxidants and statins on cholesterol-induced osteoarthritis.

The contribution of metabolic factors on the severity of osteoarthritis (OA) is not fully appreciated. This study aimed to define the effects of hypercholesterolemia on the progression of OA. Apolipoprotein E-deficient (ApoE-/-) mice and rats with diet-induced hypercholesterolemia (DIHC) rats were used to explore the effects of hypercholesterolemia on the progression of OA. Both models exhibited OA-like changes, characterized primarily by a loss of proteoglycans, collagen and aggrecan degradation, osteophyte formation, changes to subchondral bone architecture, and cartilage degradation. Surgical destabilization of the knees resulted in a dramatic increase of degradative OA symptoms in animals fed a high-cholesterol diet compared with controls. Clinically relevant doses of free cholesterol resulted in mitochondrial dysfunction, overproduction of reactive oxygen species (ROS), and increased expression of degenerative and hypertrophic markers in chondrocytes and breakdown of the cartilage matrix. We showed that the severity of diet-induced OA changes could be attenuated by treatment with both atorvastatin and a mitochondrial targeting antioxidant. The protective effects of the mitochondrial targeting antioxidant were associated with suppression of oxidative damage to chondrocytes and restoration of extracellular matrix homeostasis of the articular chondrocytes. In summary, our data show that hypercholesterolemia precipitates OA progression by mitochondrial dysfunction in chondrocytes, in part by increasing ROS production and apoptosis. By addressing the mitochondrial dysfunction using antioxidants, we were able attenuate the OA progression in our animal models. This approach may form the basis for novel treatment options for this OA risk group in humans.-Farnaghi, S., Prasadam, I., Cai, G., Friis, T., Du, Z., Crawford, R., Mao, X., Xiao, Y. Protective effects of mitochondria-targeted antioxidants and statins on cholesterol-induced osteoarthritis.

YBX1 promotes type H vessel-dependent bone formation in an m5C-dependent manner.

RNA-binding proteins (RBPs) interact with RNA and ubiquitously regulate RNA transcripts during their life cycle, playing a fundamental role in the progression of angiogenesis-related diseases. In the skeletal system, endothelium-dependent angiogenesis is indispensable for bone formation. However, the role of RBPs in endothelium-dependent bone formation is unclear. Here, we show that RBP-Y-box-binding protein 1 (YBX1) was strongly reduced in the bone vasculature of ovariectomy (OVX) mice. Endothelial cell-specific deletion of Ybx1 impaired CD31-high, endomucin-high (CD31hiEMCNhi) endothelium morphology and resulted in low bone mass whereas Ybx1 overexpression promoted angiogenesis-dependent osteogenesis and ameliorated bone loss. Mechanistically, YBX1 deletion disrupted CD31, EMCN, and bone morphogenetic protein 4 (BMP4) stability in an m5C-dependent manner and blocked endothelium-derived BMP4 release, thereby inhibiting osteogenic differentiation of bone mesenchymal stromal cells. Administration of recombinant BMP4 protein restored impaired bone formation in Ybx1 deletion mice. Tail vein injection of CD31-modified polyethylene glycol-poly (lactic-co-glycolic acid) carrying sciadopitysin, a natural YBX1 agonist, pharmacologically partially reversed CD31hiEMCNhi vessels' decline and improved bone mass in both OVX and aging animals. These findings demonstrated the role of RBP-YBX1 in angiogenesis-dependent bone formation and provided a therapeutic approach for ameliorating osteoporosis.

Excessive mechanical loading promotes osteoarthritis development by upregulating Rcn2.

Exposure of articular cartilage to excessive mechanical loading is closely related to the pathogenesis of osteoarthritis (OA). However, the exact molecular mechanism by which excessive mechanical loading drives OA remains unclear. In vitro, primary chondrocytes were exposed to cyclic tensile strain at 0.5 Hz and 10 % elongation for 30 min to simulate excessive mechanical loading in OA. In vivo experiments involved mice undergoing anterior cruciate ligament transection (ACLT) to model OA, followed by interventions on Rcn2 expression through adeno-associated virus (AAV) injection and tamoxifen-induced gene deletion. 10 µL AAV2/5 containing AAV-Rcn2 or AAV-shRcn2 was administered to the mice by articular injection at 1 week post ACLT surgery, and Col2a1-creERT: Rcn2flox/flox mice were injected with tamoxifen intraperitoneally to obtain Rcn2-conditional knockout mice. Finally, we explored the mechanism of Rcn2 affecting OA. Here, we identified reticulocalbin-2 (Rcn2) as a mechanosensitive factor in chondrocytes, which was significantly elevated in chondrocytes under mechanical overloading. PIEZO type mechanosensitive ion channel component 1 (Piezo1) is a critical mechanosensitive ion channel, which mediates the effect of mechanical loading on chondrocytes, and we found that increased Rcn2 could be suppressed through knocking down Piezo1 under excessive mechanical loading. Furthermore, chondrocyte-specific deletion of Rcn2 in adult mice alleviated OA progression in the mice receiving the surgery of ACLT. On the contrary, articular injection of Rcn2-expressing adeno-associated virus (AAV) accelerated the progression of ACLT-induced OA in mice. Mechanistically, Rcn2 accelerated the progression of OA through promoting the phosphorylation and nuclear translocation of signal transducer and activator of transcription 3 (Stat3).

Myeloid-derived grancalcin instigates obesity-induced insulin resistance and metabolic inflammation in male mice.

The crosstalk between the bone and adipose tissue is known to orchestrate metabolic homeostasis, but the underlying mechanisms are largely unknown. Herein, we find that GCA + (grancalcin) immune cells accumulate in the bone marrow and release a considerable amount of GCA into circulation during obesity. Genetic deletion of Gca in myeloid cells attenuates metabolic dysfunction in obese male mice, whereas injection of recombinant GCA into male mice causes adipose tissue inflammation and insulin resistance. Mechanistically, we found that GCA binds to the Prohibitin-2 (PHB2) receptor on adipocytes and activates the innate and adaptive immune response of adipocytes via the PAK1-NF-κB signaling pathway, thus provoking the infiltration of inflammatory immune cells. Moreover, we show that GCA-neutralizing antibodies improve adipose tissue inflammation and insulin sensitivity in obese male mice. Together, these observations define a mechanism whereby bone marrow factor GCA initiates adipose tissue inflammation and insulin resistance, showing that GCA could be a potential target to treat metainflammation.

Long noncoding RNA Gm31629 promotes bone regeneration by maintaining bone marrow mesenchymal stem cells activity.

Background: Long noncoding RNA Gm31629 can regulate hypothalamic neural stem cells (htNSCs) senescence and the aging process. However, the effect of Gm31629 on the senescence of bone marrow mesenchymal stem cells (BMSCs) and bone regeneration is unclear. In the present study, we investigated the effects of Gm31629 on the senescence of BMSCs and bone regeneration. Methods: Gm31629 knockout (Gm31629-KO) and wild-type (WT) mice were used to establish a bone regeneration model. The Brdu labelling, CCK8 assay, wound healing assay, ß-gal staining and osteogenic differentiation assay were used to assess the effects of Gm31629 on the functions of BMSCs. Micro-computed tomography (CT), histochemical and immunohistochemical staining were used to evaluate the ability of bone regeneration. The mimic of Gm31629, theaflavin 3-gallate, was used to investigate its role on the senescence of BMSCs and bone regeneration. Results: The expression of Gm31629 reduced in BMSCs of middle-aged mice was compared with that of young mice. The deletion of Gm31629 was sufficient to drive the senescence of BMSCs, resulting in impaired bone regeneration in mice. Mechanistically, Gm31629 could interact with Y-box protein 1(YB-1) and delay its degradation, decreasing the transcription of p16INK4A of BMSCs. We also found that theaflavin 3-gallate could alleviate the senescence of BMSCs and promote bone regeneration in middle-aged mice. Conclusion: These results indicated that Gm31629 played an important role on BMSCs senescence and bone regeneration and provided a therapeutic target to promote bone regeneration.

Alkbh1-mediated DNA N6-methyladenine modification regulates bone marrow mesenchymal stem cell fate during skeletal aging.

OBJECTIVES: DNA N6-methyladenine (N6-mA) demethylase Alkbh1 participates in regulating osteogenic differentiation of mesenchymal stem cell (MSCs) and vascular calcification. However, the role of Alkbh1 in bone metabolism remains unclear. MATERIALS AND METHODS: Bone marrow mesenchymal stem cells (BMSCs)-specific Alkbh1 knockout mice were used to investigate the role of Alkbh1 in bone metabolism. Western blot, qRT-PCR, and immunofluorescent staining were used to evaluate the expression of Alkbh1 or optineurin (optn). Micro-CT, histomorphometric analysis, and calcein double-labeling assay were used to evaluate bone phenotypes. Cell staining and qRT-PCR were used to evaluate the osteogenic or adipogenic differentiation of BMSCs. Dot blotting was used to detect the level of N6-mA in genomic DNA. Chromatin immunoprecipitation (Chip) assays were used to identify critical targets of Alkbh1. Alkbh1 adeno-associated virus was used to overexpress Alkbh1 in aged mice. RESULTS: Alkbh1 expression in BMSCs declined during aging. Knockout of Alkbh1 promoted adipogenic differentiation of BMSCs while inhibited osteogenic differentiation. BMSC-specific Alkbh1 knockout mice exhibited reduced bone mass and increased marrow adiposity. Mechanistically, we identified optn as the downstream target through which Alkbh1-mediated DNA m6A modification regulated BMSCs fate. Overexpression of Alkbh1 attenuated bone loss and marrow fat accumulation in aged mice. CONCLUSIONS: Our findings demonstrated that Alkbh1 regulated BMSCs fate and bone-fat balance during skeletal aging and provided a potential target for the treatment of osteoporosis.

Impaction Bone Grafting with Low Dose Irradiated Freeze-Dried Allograft Bone for Acetabular Reconstruction.

OBJECTIVE: Reconstruction of acetabular defects has been extremely challenging in both primary and revision total hip arthroplasty (THA). Impaction bone grafting (IBG) can restore the acetabulum bone mass and anatomically reconstruct the acetabulum. Our study aimed to report the short and medium-term clinical and radiographic outcomes of IBG for acetabular reconstruction in the cemented THA in the Chinese population. METHODS: This was a single-center retrospective review enrolling 57 patients between May 2013 and July 2019. The patients with acetabular defects were treated with IBG, using low dose irradiated freeze-dried allograft bone with or without autograft bone, in the cemented THA performed by one senior surgeon. Harris hip score (HHS), standard pelvis anterior-posterior radiograph and lateral hip radiograph were obtained before operation and at 1 week, 3 months, 12 months, and yearly. Graft osteointegration was evaluated by Oswestry's criteria, and complication was documented at the last follow-up. Independent sample ANOVA test and Pearson chi-square tests are used for statistical analysis. RESULTS: There were 61 hips in 57 patients. The average follow-up time was 35.59 months (5-77 months). According to AAOS classification, a total of 18 hips were identified as segmental bone deficiency (type I), with 21 and 22 hips for cavitary bone deficiency (type II) and the combined bone deficiency (type III), respectively. The average HHS was improved from 44.49 (range: 32-58) preoperatively to 86.98 (range: 78-93) postoperatively. Graft osteointegration was satisfactory (Oswestry score ≥2) in all patients. No dislocation occurred in the 57 patients (61 hips) during follow-up. Although one cup migrated, no revision, re-revision, radiographic loosening, graft bone lysis, or postoperative complications were detected at the final follow-up. CONCLUSIONS: IBG with low-dose irradiated freeze-dried allograft bone in acetabular bone defect reconstruction is a reliable technique for restoring acetabular bone defects in THA.

UBE2E3 regulates cellular senescence and osteogenic differentiation of BMSCs during aging.

BACKGROUND: Osteoporosis has gradually become a public health problem in the world. However, the exact molecular mechanism of osteoporosis still remains unclear. Senescence and osteogenic differentiation inhibition of bone marrow mesenchymal stem cells (BMSCs ) are supposed to play an important part in osteoporosis. METHODS: We used two gene expression profiles (GSE35956 and GSE35958) associated with osteoporosis and selected the promising gene Ubiquitin-conjugating enzyme E2 E3 (UBE2E3). We then verified its function and mechanism by in vitro experiments. RESULTS: UBE2E3 was highly expressed in the bone marrow and positively associated with osteogenesis related genes. Besides, UBE2E3 expression reduced in old BMSCs compared with that in young BMSCs. In in vitro experiments, knockdown of UBE2E3 accelerated cellular senescence and inhibited osteogenic differentiation of young BMSCs. On the other hand, overexpression of UBE2E3 attenuated cellular senescence as well as enhanced osteogenic differentiation of old BMSCs. Mechanistically, UBE2E3 might regulate the nuclear factor erythroid 2-related factor (Nrf2) and control its function, thus affecting the senescence and osteogenic differentiation of BMSCs. CONCLUSION: UBE2E3 may be potentially involved in the pathogenesis of osteoporosis by regulating cellular senescence and osteogenic differentiation of BMSCs.