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Bone mass declines with age and its maintenance is tightly linked to osteoblasts (crucial bone-building cells). Although disruption of the peripheral circadian clock is involved in various pathologies including aging-related diseases, evidence regarding how the peripheral clock regulates bone mass remains elusive. In the present study, we aimed to elucidate the effects of Bmal1 (the key activator of the peripheral circadian clock system) knockdown by lentivirus-mediated shRNA on osteoblast differentiation and its related mechanisms. We found that the expression of osteogenic markers, alkaline phosphatase activity, and mineralization were decreased, whereas apoptosis and inflammatory response were increased in Bmal1 knockdown osteoblasts. In addition, Bmal1 knockdown promoted ERK and JNK phosphorylation, as well as mTOR activity, whereas mTOR inhibition by rapamycin abrogated Bmal1 knockdown-mediated effects on osteoblast differentiation and mineralization capacity. Remarkably, Bmal1 knockdown in osteoblasts inhibited GSK3β/β-catenin signaling with decreased β-catenin expression and GSK-3β phosphorylation at serine 9, while GSK3β inhibition with TDZD-8, but not WNT3a or SKL2001, rescued Bmal1 knockdown-induced defects in osteoblast differentiation. Moreover, rapamycin partly nullified the suppression of Bmal1 knockdown on β-catenin expression and GSK-3β phosphorylation. Collectively, overall data indicated that circadian gene Bmal1 regulated osteoblast differentiation and inflammatory response in an mTOR/GSK3β/β-catenin-dependent manner, and thereby may contribute to the mineralization process and bone modeling/remodeling.

Taking advantage of the remarkable processivity and membrane penetrability, the gold nanoparticle (AuNP)-based three-dimensional (3D) DNA walking nanomachine has induced tremendous promise in molecular diagnostics and cancer therapy, whereas the executive ability of this nanomachine was eventually limited because of the disordered assembly between the walker and the track. Therefore, we developed a well-directed 3D DNA walking nanomachine by employing a DNA dendrimer as the track for intracellular imaging with high directionality and controllability. The nanomachine was constructed on a DNA dendrimer decorated with a substrate strand serving as the DNA track and a DNAzyme restrained by a locking strand as the walker. In this system, the distribution of the substrate strand and DNAzyme on the DNA dendrimer could be precisely regulated to achieve expected goals because of the specificity and predictability of the Watson-Crick base pairing, paving an explicit route for each walker to move along the track. Moreover, such a DNA dendrimer-based nanomachine owned prominent stability and anti-interference ability. By choosing microRNA-21 as a model analyte, the nanomachine was applied for the imaging of microRNA-21 in different cell lines and the monitoring of the dynamic microRNA-21 expression level in cancer cells. Therefore, we believe that this directed DNA walking nanomachine will have a variety of applications in molecular diagnostics and biological function modulation.

The etiology of lumbocrural pain is closely related to intervertebral disc degeneration (IDD). Long noncoding RNAs (lncRNAs) serve crucial roles in IDD progression. This study investigated the effe...

Cervical cancer is a common gynecologic cancer and a frequent cause of death. In this study, we investigated the role of MELK (maternal embryonic leucine zipper kinase) in cervical cancer. We found that HPV 18 E6/E7 promoted MELK expression by activating E2F1. MELK knockdown blocked cancer cells growth. Furthermore, we used MELK-8A to inhibit the kinase activity of MELK and caused the G2/M phase arrest of cancer cells. Under the treatment of inhibitors, Hela cells formed multipolar spindles and eventually underwent apoptosis. We also found that MELK is involved in protein translation and folding during cell division through the MELK interactome and the temporal proteomic analysis under inhibition with MELK-8A. Altogether, these results suggest that MELK may play a vital role in cancer cell proliferation and indicate a potential therapeutic target for cervical cancer.

OBJECTIVE: To assess impact of personal protective equipment (PPE) on healthcare providers (HCPs) in caring for COVID-19 patients. METHODS: A cross-sectional survey was conducted over 50 hospitals in China. Descriptive analyses and Chi-square tests were performed on the collected data. RESULTS: All 104 frontline HCPs report negative impacts of PPE on their clinical performance, 97% of them experienced discomfort and injuries caused by wearing PPE for long hours. Frontline HCPs provided suggestions to alleviate the negative impacts and to enhance communication between healthcare staff and patients. Two hundred eighty two non-frontline HCPs also revealed similar problems; however, we recorded a few discrepancies between answers given by frontline and non-frontline HCPs. CONCLUSIONS: Wearing PPE for long hours degrades health performance. Measures were suggested to improve the design of PPE for protecting HCPs and enhancing their services to COVID patients.

Background The initiation and progression of colorectal cancer (CRC) are a multistep complex process regulated by multiple factors. Previous evidence indicated that microRNA-802 (miR-802) participated in tumorigenesis of numerous solid cancers; however, the potential roles and underlying mechanisms of miR‑802 in CRC still need further exploration. Methods Quantitative real-time PCR (qRT-PCR) was employed to evaluate miR-802 levels in human CRC tissues and cell lines. In vitro proliferation, apoptosis, migration and invasion assays, and in vivo subcutaneous mouse xenograft model were utilized to examine the effects of miR-802 on the malignant behaviors of CRC cells. Then, bioinformatics prediction, dual-luciferase reporter, qRT-PCR, and Western blot was conducted to confirm the down-stream target of miR-802. Results MiR-802 was frequently down-regulated in CRC tissues and cells. Further analyses showed that the low expression of miR-802 in CRC tissues was significantly correlated with tumor progression and poor patients' prognosis. Overexpression of miR-802 profoundly inhibited proliferation, migration and invasion but promoted apoptosis of CRC cells, by contrast, miR-802 silencing exhibited opposite effects in vitro. Further animal experiment demonstrated that miR-802 could suppress tumor growth via inhibiting the proliferation and promoting the apoptosis of CRC cells in vivo. Mechanistically, miR-802 functioned as a tumor suppressor through inhibiting the expression of Ubinuclein-2 (UBN2) on post-transcriptional level. Moreover, upregulation of UBN2 expression could reverse the biological effects of CRC cells induced by miR-802 overexpression. Conclusion Our study demonstrates that miR-802 inhibits the proliferation, migration and invasion while promotes the apoptosis of CRC cells via directly suppressing UBN2 expression. These findings provide a promising biomarker and potential treatment target for CRC.

A large number of studies in recent years indicated the involvement of peripheral circadian clock in varied pathologies. However, evidence regarding how peripheral clocks regulate bone metabolism is still very limited. The present study aimed to investigate the direct role of Bmal1 (the key activator of peripheral circadian clock system) in vivo during bone developmental and remodeling stages using inducible osteoblast-specific Bmal1 knockout mice. Unexpectedly, the removal of Bmal1 in osteoblasts caused multiple abnormalities of bone metabolism, including a progressive increase in trabecular bone mass in as early as 8 weeks, manifested by an 82.3% increase in bone mineral density and 2.8-fold increase in bone volume per tissue volume. As mice age, an increase in trabecular bone mass persists while cortical bone mass decreases by about 33.7%, concomitant with kyphoscoliosis and malformed intervertebral disk. The increased trabecular bone mass is attributed to increased osteoblast number and osteoblast activity coupled with decreased osteoclastogenesis. Remarkably, the ablation of Bmal1 in osteoblasts promoted the expression level of Bmp2 and phosphorylation of SMAD1, whereas the attenuation of BMP2/SMAD1 signaling partially alleviated the effects of Bmal1 deficiency on osteoblast differentiation and activity. The results revealed that Bmal1 was a transcriptional silencer of Bmp2 by targeting the Bmp2 promoter. The peripheral clock gene Bmal1 in osteoblasts was crucial to coordinate differential effects on trabecular and cortical bones through regulating BMP2/SMAD1 during bone development, thus providing novel insights into a key role of osteoblast Bmal1 in homeostasis and integrity of adult bones. © 2020 American Society for Bone and Mineral Research.

We aimed to analyze the computed tomography (CT) imaging signs of bowel wall ischemia in patients with acute intestinal obstruction and construct an imaging prediction model to guide clinical treatment. The CT imaging signs of patients with acute intestinal obstruction diagnosed in our center in recent 6 years were collected for retrospective analysis. The etiology of intestinal obstruction and incidence rate of bowel wall ischemia were recorded, and the specific CT findings of bowel wall ischemia, including mesenteric edema, bowel wall thickening, and fish tooth sign, were analyzed. Among the 302 patients selected, 130 surgically treated patients were eligible for analysis. Bowel wall ischemia in acute intestinal obstruction showed an incidence rate of 14.90%, and the incidence rates of bowel wall ischemia in intra-abdominal hernia, intussusception, incarcerated external abdominal hernia, and volvulus were about 92.30%, 50%, 35.71%, 33.33%, and 12.59%, respectively. The incidence rate of bowel wall ischemia in simple adhesive intestinal obstruction was about 12.59%, and that in malignancy-induced intestinal obstruction was about 6.56%. Univariate analysis revealed 5 factors with statistical significance, including bowel wall thickening, mesenteric edema, bowel wall pneumatosis, ascites, and fish tooth sign. Multivariate logistic regression analysis indicated that fish tooth sign, bowel wall thickening, and mesenteric edema were able to predict bowel wall ischemia, and the corresponding partial regression coefficients were 2.164, 1.129, and 1.173, odds ratios (ORs) were 8.707, 3.093, and 3.232, sensitivity was 0.356, 0.400, and 0.844, and specificity was 0.859, 0.835, and 0.529, respectively. Imaging signs of bowel wall thickening, mesenteric edema, and fish tooth sign are valuable in predicting bowel wall ischemia, among which bowel wall thickening and mesenteric edema have relatively high specificity and fish tooth sign has a relatively high sensitivity. Furthermore, a fish tooth sign has the most favorable predictive value for bowel wall ischemia in acute intestinal obstruction, followed by bowel wall thickening and mesenteric edema.

A growing body of evidence implies a pivotal role of sirtuin-1 (Sirt1) in chondrocyte function and homeostasis; however, its underlying mechanisms mediating chondrogenesis, which is an essential process for physiological skeletal growth, are still poorly understood. In the current study, we generated TamCartSirt1−/− [Sirt1 conditional knockout (cKO)] mice to explore the role of Sirt1 during postnatal endochondral ossification. Compared with control mice, cKO mice exhibited growth retardation associated with inhibited chondrocyte proliferation and hypertrophy, as well as activated apoptosis. These effects were regulated by hyperactivation of mammalian target of rapamycin complex 1 (mTORC1) signaling, and thereby inhibition of autophagy and induction of endoplasmic reticulum stress in growth plate chondrocytes. IP injection of the mTORC1 inhibitor rapamycin to mice with Sirt1 deletion partially neutralized such inhibitory effects of Sirt1 ablation on longitudinal bone growth, indicating the causative link between SIRT1 and mTORC1 signaling in the growth plate. Mechanistically, SIRT1 interacted with tuberous sclerosis complex 2 (TSC2), a key upstream negative regulator of mTORC1 signaling, and loss of Sirt1 inhibited TSC2 expression, resulting in hyperactivated mTORC1 signaling in chondrocytes. In conclusion, our findings suggest that loss of Sirt1 may trigger mTORC1 signaling in growth plate chondrocytes and contributes to growth retardation, thus indicating that SIRT1 is an important regulator during chondrogenesis and providing new insights into the clinical potential of SIRT1 in bone development.

Non-alcoholic fatty liver disease (NAFLD) is always characterized by hepatic steatosis and insulin resistance. Dysregulated long noncoding RNAs regulate pathogenesis of NAFLD. However, the role of Mirt2 (long noncoding RNA myocardial infraction associated transcript 2) in NAFLD remains unclear. This original study aims to investigate the role of Mirt2 in hepatic steatosis and insulin resistance. Mirt2 was decreased in the livers of high-fat diet (HFD) mice, Ob/Ob, Db/Db, and fasting mice. Hepatic Mirt2 restoration attenuated hyperglycemia, insulin resistance and steatosis in the livers of obese mice, and Mirt2 inhibition promoted fasting hyperglycemia and lipid droplets accumulation in normal mouse livers. Furthermore, overexpression of Mirt2 resulted in suppression of miR-34a-5p, whereas knockdown of Mirt2 exerted opposite effects in the livers. Then, miR-34a-5p was a positive regulator of NAFLD by targeting USP10, which serves as a negative regulator of NAFLD. Overexpression of Mirt2 made miR-34a-5p mimic fail to reduce luciferase activity of USP10 3'-UTR, and this regulation was also demonstrated by Western blot. Similarly, overexpression of miR-34a-5p significantly let Mirt2 lost the ability to elevate USP10 protein level. Thus, Mirt2 can function as the sponge of miR-34a-5p. Moreover, Mirt2-mediated upregulation of USP10 protein expression can be reversed by silencing USP10. USP10 inhibition could abolish Mirt2 overexpression-induced suppression of glucose production and lipogenesis in hepatocytes. In conclusion, the decrease of Mirt2 expression contributed to hepatic insulin resistance and steatosis in obese mice, and Mirt2/miR-34a-5p/USP10 was involved in NAFLD development. Overexpression of Mirt2 might be a promising strategy for treatment of NAFLD.

How to restore seismic observation signals as soon as possible when there is a failure in a seismic station's instrument or transmission channel has become a challenge that needs to be solved urgently in our seismic observation work. By taking enhanced single-chip microcomputer (SCM) as the core of hardware, and the third/fourth-generation (3G /4G) of industrial-level mobile communication modules as a communication bridge for information exchange, this study proposed an instrument that allows us to carry out real-time monitoring for a seismic station's equipment running status via its network interface and serial interface, thus realizing a remote control over the powering-up and restart of the equipment, and switching between the main equipment and standby equipment, meanwhile, when there is a failure in the transmission channel, the 3G/4G wireless data transmission function can be invoked so that seismic observation data can be transmitted timely to the seismic station network center, thus an uninterrupted transmission of seismic observation signals is ensured.

The etiology of lumbocrural pain is closely related to intervertebral disc degeneration (IDD). Long noncoding RNAs (lncRNAs) serve crucial roles in IDD progression. This study investigated the effect of lncRNA

The dysfunction of chondrocytes is thought to play a role in the initiation and progression of osteoarthritis (OA). Brucine possesses wide pharmacological activities. But the protective mechanism of the brucine on chondrocytes remains unclear. This study is aimed to determine the therapeutic effects of brucine on the mouse chondrocyte OA model by sodium nitroprusside (SNP). The primary chondrocytes were obtained from the knee articular cartilage of a healthy suckling mouse donor. The cultured chondrocytes were divided into the control group, SNP group, brucine group, brucine-SNP group, brucine-SNP-GSK-3β antagonist group (brucine-SNP

IntroductionmicroRNAs (miRs) have been reported to contribute to sepsis-associated acute kidney injury (AKI). Herein, the roles of miR-361-3p and the underlying mechanism were investigated in lipopolysaccharide (LPS)-induced podocyte injury.Material and methodsAfter podocyte exposure to LPS (1 mg/ml), proinflammatory cytokines were measured using enzyme-linked immunosorbent assay (ELISA); cell apoptosis was evaluated by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining. In vivo septic mice, miR-361-3p agonist, agomir-miR-361-3p (20 nM/0.1 ml), was used to treat LPS-induced AKI.ResultsOur findings reveal a significant elevation of proinflammatory cytokine production, in addition to the development of podocyte apoptosis after LPS stimulation. However, knockdown of myeloid differentiation primary response protein 88 (MyD88) significantly prevents podocyte death and suppresses the inflammatory response in LPS-stimulated podocytes. More importantly, MyD88 is a direct target of miR-361-3p that can counteract LPS-induced inflammation and podocyte dysfunction via post-transcriptional repression of MyD88. The rescue experiments provide a confrontational phenomenon between miR-361-3p and MyD88 in LPS-stimulated podocytes, reflecting that overexpression of MyD88 neutralizes the protective effect of miR-361-3p on LPS-stimulated podocyte inflammation and apoptosis. Our results also demonstrate that miR-361-3p agonist alleviates LPS-induced renal injury and activation of the toll-like receptor 4 (TLR4)/MyD88/nuclear factor-kappa B (NF-κB) axis.ConclusionsmiR-361-3p suppresses LPS-induced inflammation, podocyte apoptosis and AKI by targeting the MyD88/NF-κB pathway. This may provide a novel strategy for the treatment of sepsis-associated AKI.

Pulmonary vascular endothelial cell (PVEC) injury following acute lung injury or acute respiratory distress syndrome seriously affects disease development. Recently, accumulating evidence has suggested that long noncoding RNA (lncRNA) exerts significant effects in vascular endothelial cell injury. However, PRNCR1, a novel lncRNA, remains scarcely understood in terms of its functions in PVEC injury. Both in vivo and in vitro models of PVEC injury were constructed by lipopolysaccharide (LPS) administration. The relative expressions of PRNCR1, miR-330-5p, and TLR4 were detected by quantitative reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry. Besides, gain and loss assays of PRNCR1/miR-330-5p were conducted to verify their effects on LPS-induced PVEC injury. Cell Counting Kit-8 assay used to measure cell viability and flow cytometry was used to detect apoptosis. Besides, the protein levels of caspase 3, nuclear factor-κB (NF-κB), and inflammatory cytokines (including tumor necrosis factor-α, interleukin-1β [IL-1β], and IL-6) were evaluated via Western blot and enzyme-linked immunosorbent assay. Moreover, a dual-luciferase activity experiment and RNA immunoprecipitation were applied to confirm the targeting relationship between PRNCR1 and miR-330-5p, miR-330-5p, and TLR4. PRNCR1 and TLR4 levels were significantly upregulated in LPS-treated PVEC, both in vivo and in vitro, while miR-330-5p were downregulated. Inhibiting PRNCR1 or overexpressing miR-330-5p markedly attenuated LPS-induced PVEC injury, expressions of TLR4, NF-κB, and inflammatory cytokines. Mechanistically, PRNCR1 functioned as a competitive endogenous RNA by sponging miR-330-5p and then promoting TLR4 expression. PRNCR1 was upregulated in LPS-induced PVEC and aggravated its injury via modulating the miR-330-5p/TLR4 axis.

Proepithelin, a previously unrecognized growth factor in cartilage, has recently emerged as an important regulator for cartilage formation and function. In the present study, we provide several lines of evidences in proepithelin-mediated induction of cell proliferation, differentiation, and apoptosis in the metatarsal growth plate. Proepithelin-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by pyrrolidine dithiocarbamate, a known NF-κB inhibitor. In rat growth plate chondrocytes, proepithelin induced NF-κB-p65 nuclear translocation, and nuclear NF-κB-p65 initiated its target gene cyclin D1 to regulate chondrocyte functions. The inhibition of NF-κB-p65 expression and activity (by p65 short interfering RNA (siRNA) and pyrrolidine dithiocarbamate, respectively) in chondrocytes reversed the proepithelin-mediated induction of cell proliferation and differentiation and the proepithelin-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of proepithelin on NF-κB activation. Finally, using siRNA and antisense strategies, we demonstrated that endogenously produced proepithelin by chondrocytes is important for chondrocyte growth in serum-deprived conditions. These results support the hypothesis that the induction of NF-κB activity of in growth plate chondrocytes is critical in proepithelin-mediated growth plate chondrogenesis and longitudinal bone growth.

Background The initiation and progression of colorectal cancer (CRC) are a multistep complex process regulated by multiple factors. Previous evidence indicated that microRNA-802 (miR-802) participated in tumorigenesis of numerous solid cancers; however, the potential roles and underlying mechanisms of miR‑802 in CRC still need further exploration. Methods Quantitative real-time PCR (qRT-PCR) was employed to evaluate miR-802 levels in human CRC tissues and cell lines. In vitro proliferation, apoptosis, migration and invasion assays, and in vivo subcutaneous mouse xenograft model were utilized to examine the effects of miR-802 on the malignant behaviors of CRC cells. Then, bioinformatics prediction, dual-luciferase reporter, qRT-PCR, and Western blot was conducted to confirm the down-stream target of miR-802. Results MiR-802 was frequently down-regulated in CRC tissues and cells. Further analyses showed that the low expression of miR-802 in CRC tissues was significantly correlated with tumor progression and poor patients’ prognosis. Overexpression of miR-802 profoundly inhibited proliferation, migration and invasion but promoted apoptosis of CRC cells, by contrast, miR-802 silencing exhibited opposite effects in vitro. Further animal experiment demonstrated that miR-802 could suppress tumor growth via inhibiting the proliferation and promoting the apoptosis of CRC cells in vivo. Mechanistically, miR-802 functioned as a tumor suppressor through inhibiting the expression of Ubinuclein-2 (UBN2) on post-transcriptional level. Moreover, upregulation of UBN2 expression could reverse the biological effects of CRC cells induced by miR-802 overexpression. Conclusion Our study demonstrates that miR-802 inhibits the proliferation, migration and invasion while promotes the apoptosis of CRC cells via directly suppressing UBN2 expression. These findings provide a promising biomarker and potential treatment target for CRC.

Glioma is one of the most common brain tumors, suggesting the importance of investigating the molecular mechanism of gliomas. We studied the roles of Ribonucleotide Reductase Regulatory Subunit M2 (RRM2) in glioma. Expressions of RRM2 are higher in glioma tissues evidenced by TCGA data, western blot and immunohistochemistry. RRM2 is negatively correlated with glioma patient's survival. RNA-seq showed that genes involved in apoptosis, proliferation, cell adhesion and negative regulation of signaling were up-regulated upon RNAi-mediated knock-down of RRM2. Cell phenotypes specific for stably knocking down RRM2 were determined using stable transfection in vitro. In an in vivo model, knock-down of RRM2 inhibited tumor growth and caused suppression of AKT and ERK1/2 signalings. Interfering RRM2 also down-regulated the expression of cyclin A, cyclin B1, cyclin D1, Vimentin, and N-cadherin, and elevated E-cadherin expression. Moreover, overexpression of RRM2 failed to increase the expression of cyclin B1, cyclin D1, and N-cadherin when phosphorylation of AKT and ERK1/2 was suppressed by LY294002 or PD98059. These findings indicated that RRM2 is a positive regulator of glioma progression which contributes to the migration and proliferation of glioma cells through ERK1/2 and AKT signalings and might be a novel prognostic indicator for glioma patients.

Aquaporin-5 (AQP5), a water channel protein, has been reported to possess oncogenic potential in multiple types of malignancies, including colorectal cancer (CRC). However, its effect on the chemosensitivity of CRC cells remains elusive. Hence, this study investigated the effect of AQP5 silencing in CRC cells on 5-fluorouracil (5-FU) sensitivity and attempted to elucidate the underlying mechanisms. A short hairpin RNA construct targeting AQP5 was transfected into HCT116 or HT29 cells to generate stable AQP5-silenced cell lines. The effects of AQP5 knockdown on cell viability, apoptosis, tumor growth, and 5-FU chemoresistance were evaluated. Relative protein levels of Wnt–β-catenin pathway effectors were also measured. The results showed that silencing of AQP5 increased the chemosensitivity of CRC cells to 5-FU, facilitated 5-FU-mediated apoptosis, suppressed tumor growth, and reduced 5-FU chemoresistance in vivo. Furthermore, the effect of AQP5 on 5-FU chemosensitivity was mediated by the Wnt–β-catenin pathway. Silencing of AQP5 inhibited Wnt–β-catenin signaling, whereas overexpression of the degradation-resistant mutant of β-catenin (S33Y) reversed apoptosis induced by AQP5 silencing. Taken together, these results suggest that AQP5 silencing enhances the sensitivity of CRC cells to 5-FU, and the underlying mechanism is related to inhibition of the Wnt–β-catenin pathway. AQP5 could be a useful therapeutic target for CRC treatment.

Macroautophagy/autophagy is a highly regulated process involved in the turnover of cytosolic components, however its pivotal role in maintenance of bone homeostasis remains elusive. In the present study, we investigated the direct role of ATG7 (autophagy related 7) during developmental and remodeling stages in vivo using osteoblast-specific Atg7 conditional knockout (cKO) mice. Atg7 cKO mice exhibited a reduced bone mass at both developmental and adult age. The trabecular bone volume of Atg7 cKO mice was significantly lower than that of controls at 5 months of age. This phenotype was attributed to decreased osteoblast formation and matrix mineralization, accompanied with an increased osteoclast number and the extent of the bone surface covered by osteoclasts as well as an elevated secretion of TNFSF11/RANKL (tumor necrosis factor [ligand] superfamily, member 11), and a decrease in TNFRSF11B/OPG (tumor necrosis factor receptor superfamily, member 11b [osteoprotegerin]). Remarkably, Atg7 deficiency in osteoblasts triggered endoplasmic reticulum (ER) stress, whereas attenuation of ER stress by administration of phenylbutyric acid in vivo abrogated Atg7 ablation-mediated effects on osteoblast differentiation, mineralization capacity and bone formation. Consistently, Atg7 deficiency impeded osteoblast mineralization and promoted apoptosis partially in DDIT3/CHOP (DNA-damage-inducible transcript 3)- and MAPK8/JNK1 (mitogen-activated protein kinase 8)-SMAD1/5/8-dependent manner in vitro, while reconstitution of Atg7 could improve ER stress and restore skeletal balance. In conclusion, our findings provide direct evidences that autophagy plays crucial roles in regulation of bone homeostasis and suggest an innovative therapeutic strategy against skeletal diseases.

The NAD(+)-dependent deacetylase sirtuin-1 (SIRT1) has emerged as an important regulator of chondrogenesis and cartilage homeostasis, processes that are important for physiological skeletal growth and that are dysregulated in osteoarthritis. However, the functional role and underlying mechanism by which SIRT1 regulates chondrogenesis remain unclear. Using cultured rat metatarsal bones and chondrocytes isolated from rat metatarsal rudiments, here we studied the effects of the SIRT1 inhibitor EX527 or of SIRT1 siRNA on chondrocyte proliferation, hypertrophy, and apoptosis. We show that EX527 or SIRT1 siRNA inhibits chondrocyte proliferation and hypertrophy and induces apoptosis. We also observed that SIRT1 inhibition mainly induces the PERK–eIF-2α–CHOP axis of the endoplasmic reticulum (ER) stress response in growth-plate chondrocytes. Of note, EX527- or SIRT1 siRNA–mediated inhibition of metatarsal growth and growth-plate chondrogenesis were partly neutralized by phenylbutyric acid, a chemical chaperone that attenuates ER stress. Moreover, EX527-mediated impairment of chondrocyte function (i.e. of chondrocyte proliferation, hypertrophy, and apoptosis) was partly reversed in CHOP(−/−) cells. We also present evidence that SIRT1 physically interacts with and deacetylates PERK. Collectively, our findings indicate that SIRT1 deacetylates PERK and attenuates the PERK–eIF-2α–CHOP axis of the unfolded protein response pathway and thereby promotes growth-plate chondrogenesis and longitudinal bone growth.