Qingnan Li

EPO Promotes Bone Repair through Enhanced Cartilaginous Callus Formation and Angiogenesis

Erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling is involved in the development and regeneration of several non-hematopoietic tissues including the skeleton. EPO is identified as a downstream target of the hypoxia inducible factor-α (HIF-α) pathway. It is shown that EPO exerts a positive role in bone repair, however, the underlying cellular and molecular mechanisms remain unclear. In the present study we show that EPO and EPOR are expressed in the proliferating, pre-hypertrophic and hypertrophic zone of the developing mouse growth plates as well as in the cartilaginous callus of the healing bone. The proliferation rate of chondrocytes is increased under EPO treatment, while this effect is decreased following siRNA mediated knockdown of EPOR in chondrocytes. EPO treatment increases biosynthesis of proteoglycan, accompanied by up-regulation of chondrogenic marker genes including SOX9, SOX5, SOX6, collagen type 2, and aggrecan. The effects are inhibited by knockdown of EPOR. Blockage of the endogenous EPO in chondrocytes also impaired the chondrogenic differentiation. In addition, EPO promotes metatarsal endothelial sprouting in vitro. This coincides with the in vivo data that local delivery of EPO increases vascularity at the mid-stage of bone healing (day 14). In a mouse femoral fracture model, EPO promotes cartilaginous callus formation at days 7 and 14, and enhances bone healing at day 28 indexed by improved X-ray score and micro-CT analysis of microstructure of new bone regenerates, which results in improved biomechanical properties. Our results indicate that EPO enhances chondrogenic and angiogenic responses during bone repair. EPOs function on chondrocyte proliferation and differentiation is at least partially mediated by its receptor EPOR. EPO may serve as a therapeutic agent to facilitate skeletal regeneration.

The epithelial sodium channel is involved in dexamethasone-induced osteoblast differentiation and mineralization

PurposeGlucocorticoids (GCs) can facilitate bone formation, but prolonged GCs exposure in vivo can lead to osteoporosis. The mechanisms underlying these reciprocal effects have not been elucidated.MethodsThe epithelial sodium channel (ENaC) is a possible regulator of osteoblast proliferation and differentiation, so we examined whether ENaC was involved in mediating the effects of dexamethasone (Dex) on osteoblast.ResultExpression of the functional α-ENaC subunit was upregulated by 10−8M and 10−6M Dex, but decreased by 10−4M Dex. Furthermore, Dex had similar dose-dependent effects on the expression of three osteogenic genes, Cbfa1, OPN, and OC, with low concentrations enhancing expression and higher concentrations suppressing expression. The effects of Dex on osteoblast proliferation, differentiation, and mineralization were examined in the presence and absence of the ENaC specific antagonist amiloride. Dex at 10−8M and 10−6M markedly increased osteoblast proliferation, alkaline phosphatase activity (an index of differentiation), and calcium nodule formation, while 10−4M had no effect or suppressed all these responses. Amiloride blocked the Dex-induced, osteoblast differentiation and mineralization but had no effect on osteoblast differentiation and mineralization when applied alone. But such changes did not show in osteoblast proliferation. However, the Dex-induced α-ENaC expression was not blocked by RU486, a GC receptor antagonist.ConclusionThese results suggest that changes in ENaC activity may involved in Dex-induced differentiation and mineralization of osteoblast. But the Dex-induced effect on ENaC did not mediated by the GC genomic mechanism in osteoblast at this study.

Epithelial sodium channel enhanced osteogenesis via cGMP/PKGII/ENaC signaling in rat osteoblast

Abstract The amiloride-sensitive epithelial sodium channel (ENaC) is a major contributor to intracellular sodium homeostasis. In addition to epithelial cells, osteoblasts (Obs) express functional ENaCs. Moreover, a correlation between bone Na content and bone disease has been reported, suggesting that ENaC-mediated Na+ regulation may influence osteogenesis. Obs were isolated and cultured by enzyme digestion. Cell proliferation and differentiation were evaluated by WST-8 assay kit and AKP assay kit respectively. PKGII expression was silenced by siRNA. The mRNA expression was investigated by semi-quantitative PCR and the protein expression was determined by Western-blot. The cell-permeable cGMP analog 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) increased α-ENaC channel expression in primary rat Obs as indicated by RT-PCR. In addition, 8-pCPT-cGMP stimulation enhanced expression of the mRNA encoding cGMP-dependent protein kinases II (PKGII). The cGMP analog also promoted osteoblast proliferation, differentiation and induced the expression of several osteogenic genes, including core binding factor al, osteocalcin, alkaline phosphatase, collagen type I, and osteopontin. Furthermore, the expression of α-ENaC, the main functional subunit of ENaC, was reduced when a small interfering RNA specific for PKGII was introduced into Obs. Treatment with 8-pCPT-cGMP in cells transfected with the siRNA for PKGII partially reversed downregulated α-ENaC mRNA expression. Our results suggest that 8-pCPT-cGMP stimulates proliferation, differentiation, and osteogenic gene expression in Obs through cGMP/PKGII-dependent regulation of ENaC channel expression. The cGMP/PKGII signaling pathway is a potential target for pharmaceutical interventions to treat metabolic bone diseases.

Estrogen regulates the expression and activity of epithelial sodium channel in mouse osteoblasts.

Estrogen plays an important role in bone metabolism and only high dose can stimulate osteoblast bone formation. However, the underlying mechanisms have not been elucidated. The epithelial sodium channel (ENaC) is a key pathway for sodium transport in epithelia, vascular endothelium, and other tissues; although the expressions of α and γ ENaC mRNA were found in osteoblasts, the regulation of ENaC by estrogen in osteoblasts has not been studied. Our recent data confirmed the ENaC expression in mouse primary osteoblasts by immunocytofluorescence, RT-PCR, western blot, and patch clamp. Furthermore, we found estrogen (10(-5)M) increased the expression of α and γ ENaC subunits at both the mRNA and protein levels in osteoblasts. On the other hand, 17β estradiol (20 nM) increased inward Na+ currents which were inhibited by amiloride. The estrogen dose used in patch clamp is much lower than those of mRNA and protein analysis, which means single cell ENaC electrophoretic mobility is much more sensitive to estrogen than the mRNA and protein production by estrogen stimulation. Our results suggest that estrogen regulates expression and function of ENaC in osteoblasts may provide a new clue that the mechanism of high dose of estrogen influence osteoblast bone formation via ENaC activity.

Baicalin positively regulates osteoclast function by activating MAPK/Mitf signalling.

Activation of osteoblasts in bone formation and osteoclasts in bone resorption is important during the bone fracture healing process. There has been a long interest in identifying and developing a natural therapy for bone fracture healing. In this study, we investigated the regulation of osteoclast differentiation by baicalin, which is a natural molecule extracted from Eucommiaulmoides (small tree native to China). It was determined that baicalin enhanced osteoclast maturation and bone resorption activity in a dose‐dependent manner. Moreover, this involves the activation of MAPK, increased Mitf nuclear translocation and up‐regulation of downstream osteoclast‐related target genes expression. The baicalin‐induced effect on osteoclast differentiation can be mimicked by specific inhibitors of p‐ERK (U0126) and the Mitf‐specific siRNA, respectively. Protein–ligand docking prediction identified that baicalin might bind to RANK, which is the upstream receptor of p‐ERK/Mitf signalling in osteoclasts. This indicated that RANK might be the binding target of baicalin. In sum, our findings revealed baicalin increased osteoclast maturation and function via p‐ERK/Mitf signalling. In addition, the results suggest that baicalin can potentially be used as a natural product for the treatment of bone fracture.

Ferulic acid induces proliferation and differentiation of rat osteoblasts in vitro through cGMP/PKGII/ENaC signaling

Abstract Ferulic acid (FA) is an active component of the traditional Chinese herb Angelica sinensis. Numerous health benefits have been attributed to FA, but few studies have investigated the effects of FA on osteoblasts (Obs). Our work studied the effects of FA on proliferation, differentiation, and mineralization of rat calvarial Obs and examined the signaling pathways involved. Cell proliferation and differentiation were evaluated by Cell Counting Kit-8 (CCK-8) and alkaline phosphatase (ALP) assay kit, respectively. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKGII) expression was silenced by small interfering RNA (siRNA). The mRNA expression was investigated by semi-quantitative PCR. FA (40–2560 μM) promoted Ob proliferation and differentiation; at 40–640 μM, FA stimulated calcified nodule formation and increased the expression of osteogenic genes encoding osteopontin and collagen-l. FA (40–2560 μM) increased cGMP levels in Obs and upregulated the expression of PKGII, EnaCα, and ENaCγ mRNAs. Downregulated ENaCα mRNA expression in Obs transfected with the siRNA for PKGII was reversed when FA was introduced into Obs. These results demonstrated that FA promoted proliferation, differentiation, and mineralization of Obs in vitro, and enhanced osteogenic genes expression partly through the cGMP-PKGII-ENaC signaling pathway.

The influence of dietary sodium on bone development in growing rats

The present study investigated the effects of dietary sodium on bone growth in young rats. Five-week-old rats were fed one of three different diets for 60 days: low sodium (NaCl, 0.32 g/kg diet), normal sodium (NaCl, 2.6 g/kg) and high sodium (NaCl, 20 g/kg). The proximal tibial metaphysis (PTM), the fifth lumbar vertebra (LV5) and the middle part of the tibia shaft (TX) were analysed by bone histomorphometry. The expression of three osteogenesis genes, Runx2, osteopontin and osteocalcin, was determined by RT-PCR in bone samples from the skull. In both the PTM and LV5, trabecular area and thickness were increased by the low-sodium diet, while the high-sodium diet decreased trabecular area in LV5. Dynamic data revealed that sodium restriction increased bone formation parameters in the PTM and LV5, but decreased bone resorption in LV5. In TX, endosteal bone formation was enhanced by the low-sodium diet and depressed by the high-sodium diet compared to the normal sodium group. But there were no statistically changes in the cortical bone area of TX. Low-sodium intake significantly enhanced the expression of all three osteogenesis genes compared to the normal sodium group, while high-sodium intake suppressed osteogenic gene expression. Our results suggest that sodium restriction in growing rats promotes bone development by influencing both bone formation and resorption.