Xin-Feng Zheng

Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER–ERK–mTOR pathway

Estradiol could protect osteoblast against apoptosis, and apoptosis and autophagy were extensively and intimately connected. The aim of the present study was to test the hypothesis that autophagy was present in osteoblasts under serum deprivation and estrogen protected against osteoblast apoptosis via promotion of autophagy. MC3T3-E1 osteoblastic cells were cultured in a serum-free and phenol red-free minimal essential medium (α-MEM). Ultrastructural analysis, lysosomal activity assessment and monodansycadaverine (MDC) staining were employed to determine the presence of autophagy, and real time PCR was used to evaluate the expression of autophagic markers. Meanwhile, the osteoblasts were transferred in a serum-free and phenol red-free α-MEM containing either vehicle or estradiol. Apoptosis and autophagy was assessed by using the techniques of real-time PCR, Western blot, immunofluorescence assay, and flow cytometry. The possible pathway through which estrogen promoted autophagy in the serum-deprived osteoblasts was also investigated. Real-time PCR demonstrated the expression of LC3, beclin1 and ULK1 genes in osteoblasts under serum deprivation, and immunofluorescence assay verified high expression of proteins of these three autophagic bio-markers. Lysosomes and autolysosomes accumulated in the cytoplasm of osteoblasts were also detected under transmission electron microscopy, MDC staining and lysosomal activity assessment. Meanwhile, estradiol significantly decreased the expression of proteins of the bio-markers of apoptosis, and at the same time increased the expression of proteins of the bio-markers of autophagy in the serum-deprived osteoblasts. Furthermore, the estradiol-promoted autophagy in serum-deprived osteoblasts could be blocked by estrogen receptor (ER) antagonist (ICI 182780), and estradiol failed to rescue the cells pretreated with an inhibitor of vacuolar ATPase (bafilomycin A) from apoptosis. Serum deprivation resulted in apoptosis through activation of Caspase-3 and induced autophagy through inhibition of phospho-mammalian target of rapamycin (p-mTOR). Both 3-methyladenine (3MA) and U0126 led to increase of apoptosis in osteoblasts with serum deprivation. Estradiol failed to over-ride the inhibitory effect of 3MA on phosphorylation of AKT but directly led to dephosphorylation of mTOR and upregulation of LC3 protein expression. However, the estradiol-enhanced LC3 protein expression was significantly suppressed by U0126 through inhibition of phosphorylation of extracellular signal-regulated kinase (ERK). Estradiol rescued osteoblast apoptosis via promotion of autophagy through the ER–ERK–mTOR pathway.

Increased activity of osteocyte autophagy in ovariectomized rats and its correlation with oxidative stress status and bone loss.

OBJECTIVES The objectives of the present study were to investigate ovariectomy on autophagy level in the bone and to examine whether autophagy level is associated with bone loss and oxidative stress status. METHODS 36 female Sprague-Dawley rats were randomly divided into sham-operated (Sham), and ovariectomized (OVX) rats treated either with vehicle or 17-β-estradiol. At the end of the six-week treatment, bone mineral density (BMD) and bone micro-architecture in proximal tibias were assessed by micro-CT. Serum 17β-estradiol (E2) level were measured. Total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, catalase (CAT) activity in proximal tibia was also determined. The osteocyte autophagy in proximal tibias was detected respectively by Transmission Electron Microscopy (TEM), immunofluorescent histochemistry (IH), realtime-PCR and Western blot. In addition, the spearman correlation between bone mass, oxidative stress status, serum E2 and autophagy were analyzed. RESULTS Ovariectomy increased Atg5, LC3, and Beclin1 mRNA and proteins expressions while decreased p62 expression. Ovariectomy also declined the activities of T-AOC, CAT, and SOD. Treatment with E2 prevented the reduction in bone mass as well as restored the autophagy level. Furthermore, LC3-II expression was inversely correlated with T-AOC, CAT, and SOD activities. A significant inverse correlation between LC3-II expression and BV/TV, Tb.N, BMD in proximal tibias was found. CONCLUSIONS Ovariectomy induced oxidative stress, autophagy and bone loss. Autophagy of osteocyte was inversely correlated with oxidative stress status and bone loss.

Increasing expression of substance P and calcitonin gene-related peptide in synovial tissue and fluid contribute to the progress of arthritis in developmental dysplasia of the hip

IntroductionDevelopmental dysplasia of the hip (DDH) is a common musculoskeletal disorder that has pain and loss of joint function as major pathological features. In the present study, we explored the mechanisms of possible involvement and regulation of substance P (SP) and calcitonin gene-related peptide (CGRP) in the pathological and inflammatory processes of arthritis in DDH.MethodsBlood, synovial tissue and fluid samples were collected from patients diagnosed with different severities of DDH and from patients with femoral neck fracture. Levels of SP, CGRP and inflammatory cytokines in synovium and synovial fluid (SF) in the different groups were evaluated by immunohistochemistry, real-time PCR and enzyme-linked immunosorbent assay (ELISA). Correlations between neuropeptides and inflammatory cytokines in SF were evaluated by partial correlation analysis. The proinflammatory effects of SP and CGRP on synoviocytes obtained from patients with moderate DDH were investigated in vitro by real-time PCR and ELISA. The mechanisms of those effects were evaluated by Western blot analysis and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) DNA binding assay.ResultsSignificantly increased levels of neuropeptides and inflammatory cytokines were observed in synovium and SF from patients in the severe DDH group compared with the moderate DDH and control groups. In moderate DDH samples, SP in SF correlated with tumor necrosis factor (TNF)-α, and CGRP in SF correlated with TNF-α and interleukin (IL)-10. In the severe DDH group, SP in SF correlated with interleukin (IL)-1β, TNF-α and IL-10. CGRP in SF correlated with TNF-α. Additionally, SP might have had obvious proinflammatory effects on synoviocytes through the activation of NF-κB.ConclusionsThe upregulation of SP and CGRP in synovium and SF might participate in the inflammatory process of arthritis in DDH. The activation of the NF-κB pathway seems indispensable in the proinflammatory effect of SP on synoviocytes. This original discovery may indicate a potential clinical drug target and the development of innovative therapies for DDH.

Hypoxia facilitates the survival of nucleus pulposus cells in serum deprivation by down-regulating excessive autophagy through restricting ROS generation

Nucleus pulposus (NP) cells reside in a hypoxic environment in vivo, while the mechanisms of how NP cells maintain survival under hypoxia are not clear. Autophagy is an important physiological response to hypoxia and implicated in the survival regulation in most types of cells. This study was designed to investigate the role of autophagy in the survival of NP cells under hypoxia. We found that appropriate autophagy activity was beneficial to the survival of NP cells in serum deprivation, while excessive autophagy led to death of the NP cells. Hypoxia facilitated the survival of NP cells in serum deprivation by down-regulating excessive autophagy. Hypoxia down-regulated the autophagy activity of NP cells through restricting the production of reactive oxygen species (ROS) and inactivating the AMPK/mTOR signaling pathway, and possibly through a pathway involving HIF-1α. We believed that understanding the autophagy response of NP cells to hypoxia and its role in cell survival had important clinical significance in the prevention and treatment of degenerative discogenic diseases.

LGR4 acts as a key receptor for R-spondin 2 to promote osteogenesis through Wnt signaling pathway

R-spondin proteins are identified as secreted agonists of the canonical Wnt/β-catenin signaling pathway, and leucine-rich repeat-containing G-protein-coupled receptors (LGR) are recognized as R-spondin receptors. The potential role of R-spondin 2 (Rspo2) and LGR4 in mediating osteogenesis remains poorly understood. In our in vitro experiments, we found that Rspo2 could promote osteogenesis through activating the Wnt signaling pathway in MC3T3-E1 cells. However, this effect of Rsop2 disappeared in the cells with functional disruption of LGR4. Meanwhile, Rspo2 significantly inhibited osteoclastogenesis and this effect of Rspo2 was dependent on the presence of osteoblasts with normal function of LGR4. In our in vivo experiments, we found that application of exogenous Rspo2 rescued the bone loss and improved the microarchitecture of bone in OVX mice. Rspo2 could be a positive regulator of bone metabolism through activating the canonical Wnt/β-catenin signaling, and LGR4 acted as a key receptor for Rspo2 to promote osteogenesis.

Evidence of the Role of R-Spondin 1 and Its Receptor Lgr4 in the Transmission of Mechanical Stimuli to Biological Signals for Bone Formation

The bone can adjust its mass and architecture to mechanical stimuli via a series of molecular cascades, which have been not yet fully elucidated. Emerging evidence indicated that R-spondins (Rspos), a family of secreted agonists of the Wnt/β-catenin signaling pathway, had important roles in osteoblastic differentiation and bone formation. However, the role of Rspo proteins in mechanical loading-influenced bone metabolism has never been investigated. In this study, we found that Rspo1 was a mechanosensitive protein for bone formation. Continuous cyclic mechanical stretch (CMS) upregulated the expression of Rspo1 in mouse bone marrow mesenchymal stem cells (BMSCs), while the expression of Rspo1 in BMSCs in vivo was downregulated in the bones of a mechanical unloading mouse model (tail suspension (TS)). On the other hand, Rspo1 could promote osteogenesis of BMSCs under CMS through activating the Wnt/β-catenin signaling pathway and could rescue the bone loss induced by mechanical unloading in the TS mice. Specifically, our results suggested that Rspo1 and its receptor of leucine-rich repeat containing G-protein-coupled receptor 4 (Lgr4) should be a novel molecular signal in the transmission of mechanical stimuli to biological signal in the bone, and this signal should be in the upstream of Wnt/β-catenin signaling for bone formation. Rspo1/Lgr4 could be a new potential target for the prevention and treatment of disuse osteoporosis in the future.

Blockade of substance P receptor attenuates osteoporotic pain, but not bone loss, in ovariectomized mice.

Objective The aim of this study was to investigate the effect of a substance P (SP) receptor (NK1 receptor [NK1-R]) antagonist on hyperalgesia and bone metabolism in ovariectomized mice. Methods Thirty-six 9-week-old mice were subjected to either bilateral ovariectomy or sham surgery. Three weeks after the operation, the mice were treated with either a single-dose injection or 2-week repeated daily administration of L-703606, an NK1-R antagonist. Behavioral tests were performed for pain assessment; tibiae and the third lumbar vertebrae were dissected and assessed for microarchitectural or biomechanical properties. The expressions of SP and NK1-R in the dorsal root ganglia and spinal cord were also evaluated. Results Both single-dose injection and 2-week repeated injections of L-703606 led to a significant increase in nociceptive threshold in ovariectomized mice. However, the antihyperalgesic effect faded at 2 hours and almost disappeared at 5 hours after a single-dose injection. With the 14-day repeated treatment of ovariectomized mice, the effect was not detectable at 24 hours after the first injection but was obvious at 24 hours after 1-week and 2-week administrations and still existed at 48 hours after the last injection. Ovariectomized mice at the hyperalgesic state had enhanced SP immunoreactivity in the dorsal root ganglia and up-regulated SP and NK1-R expressions in the spinal cord. However, no significant change in serum SP level was detected. Two-week treatment with L-703606 could down-regulate these expressions but failed to salvage the deteriorated trabecular microstructure and reduced compressive strength in ovariectomized mice. Conclusions Estrogen deficiency–induced hyperalgesia is achieved through up-regulation of SP and NK1-R expressions. Blockade of SP receptor can alleviate pain but cannot ameliorate bone loss. NK1-R antagonist is not recommended for the treatment of estrogen deficiency osteoporosis.

Sympathetic neuron can promote osteoblast differentiation through BMP signaling pathway.

Communication between sympathetic neurons and osteoblasts through the adrenergic receptor pathway has already been reported. To investigate whether the sympathetic neurons have a direct effect on osteoblast differentiation, an in vitro Transwell coculture system was established in which osteoblasts were cocultured with sympathetic neurons with no cell-to-cell contact. The expression of osteogenesis-related genes was upregulated in osteoblasts cocultured with sympathetic neurons. Meanwhile, bone morphogenetic protein (BMP) mRNA and protein expressions were detected in sympathetic neurons, and BMP secretion from sympathetic neurons was also confirmed. However, transfection with BMP-2 and/or BMP-6 siRNA in sympathetic neurons caused a down-regulation of osteogenesis-related genes in the cocultured osteoblasts. Sympathetic neurons promoted osteoblast differentiation through BMP signaling pathway, implying that the integrity of sympathetic neurons was important for optimal bone formation and remodeling.

Evidence for an Important Role of Smad-7 in Intervertebral Disc Degeneration.

Smad-7 inhibited the transforming growth factor beta (TGF-β)-induced proteoglycan synthesis in chondrocytes and completely antagonized the effect of TGF-β on the proliferation of the cells. The aim of this study was to evaluate the contribution of Smad-7 to the pathophysiology of disc degeneration by determining the expression of Smad-7 in the degenerative intervertebral discs and its effect on the extracellular matrix metabolism of disc cells. Instability of the lumbar spine produced by imbalanced dynamic and static forces was used to induce intervertebral disc degeneration in rats. The expression of Smad-7 was assessed by the immunohistochemical method. Disc cell apoptosis was detected by in situ TUNEL staining. The effect of Smad-7 overexpression on the matrix metabolism of disc cells was analyzed in vitro by real-time polymerase chain reaction (PCR) and Western blotting. Finally, intradiscal injection of the Smad-7 overexpression lentivirus was performed to evaluate the in vivo effect of Smad-7 on disc degeneration. Radiographic and histomorphological examinations showed that lumbar disc degeneration became more and more severe in the rats with induced instability. Immunohistochemical observation demonstrated increasing protein expression of Smad-7 in the degenerative discs. A significantly positive correlation was found between Smad-7 expression and the degree of disc degeneration and between Smad-7 expression and disc cell apoptosis. Overexpression of Smad-7 in disc cells inhibited the expression of TGF-β1, collagen type-I, collagen type-II, and aggrecan and promoted the expression of MMP-13, but did not change the expression of ADAMTS-5. The in vivo findings illustrated that intradiscal injection of lentivirus vector with Smad-7 overexpression accelerated the progress of disc degeneration. In conclusion, Smad-7 was highly expressed in the degenerative discs. Overexpression of Smad-7 weakened the protective role of TGF-β and accelerated the progress of disc degeneration. Interference on Smad-7 might be a potential therapeutic method for the prevention and treatment of degenerative disc diseases.