Study On The Mechanism Of Connexin 43 Hemichannels Protect Bone Loss During Estrogen Deficiency

Bone tissue continuously undergoes remodeling through bone formation and bone resorption.The osteocytes,orchestrating osteoblast and osteoclast functions,comprising about 95%of bone cells,are embedded within the bone matrix and communicate with neighboring osteocytes and cells on the bone surface(such as osteoblast and osteoclast)through an extensive network of long dendritic processes.Gap junctions are intercellular channels that provide direct cell-cell communication between adjacent cells.Connexin 43(Cx43)forms gap junctions that mediate osteocyte coupling and are critical for maintaining proper bone physiology,including survival and differentiation of osteoblasts,and skeletal development.Hemichannels(HCs),which abundantly located on the cell surface of osteocytes,are extensively involved in the communication between osteocytes and their extracellular environment.Also HCs play an important roles in autocrine/paracrine signaling,cell survival,and mechanotransduction.Cell-based studies suggested that hemichannels are responsive to mechanical stimulation,resulting in the opening of these channels.Connexin-based gap junctions and HCs allow the passage of small molecules(MW<1000Da),such as essential metabolites and second messengers,such as PGE2,ADP,and ATP.These released extracellular factors are known to regulate bone metabolism.In recently years,the importance of Cx43 in bone formation and remodeling,and responses to mechanical loading and parathyroid hormone has been illustrated using osteoblast and osteocyte specific conditional Cx43 knockout(cKO)mouse models.However,these gene cKO models could not elucidate the specific role of connexin channels given that Cx43 forms both gap junction channels and HCs.We generated two transgenic mouse models driven by DMP1 promoter with the expression of Cx43 mutants predominantly in osteocytes:(1)R76W,with dominant-negative effects on gap junction channels;(2)△130-136,with dominant negative effects on both gap junction channels and HCs.These two transgenic mouse models allow us to dissect differential functions of gap junctions vs HCs formed by Cx43.We showed that impairment of Cx43 HCs negatively affected bone formation,remodeling and osteocyte viability.The skeletal phenotype of A130-136 mice was significantly different than the WT and R76W,including increased BMD,enlarged midshaft cortical bone,greater osteocyte apoptosis,and poorer material properties.In previous experiments,we used male mice.In order to detect whether there is gender difference in the effect of Cx43 HCs on bone,we selected female mice of the same age to repeat parts of the experiments.Our findings suggest that effect of Cx43 HCs on bone in female mice was similar to male mice.Osteoporosis and osteopenia,are common among postmenopausal women and feature a high risk of fragility fracture.Decreased estrogen is closely associated with an increase in osteoclast-mediated bone resorption and a decrease in osteoblast-mediated bone formation,which contribute to bone loss during aging.Furthermore,estrogen deficiency,oxidative stress(ROS),micro-damage,and aging influence the viability of osteocytes,which are required for mediating the efficient bone remodeling,quality maintenance,and damage repair processes of bone.Bilateral ovariectomy in female mouse is a mature animal models,which can be successfully established to simulate osteoporosis induced by estrogen deficiency.After castration,the body is in a low estrogen micro-environment.Thus,the process of bone turnover is accelerated,bone metabolism is strengthened.Meanwhile,osteocytes are active,leading to loss of bone mass and disruption of normal physiological structure,which can easily lead to osteoporosis.To study the specific role of osteocyte Cx43 HCs in estrogen-deficient bone tissue,we conducted variectomies on WT and two transgenic mouse strains,R76W and A130-136,and then determined bone structure,bone histomorphometry,osteocyte viability,and bone markers of these ovariectomized(OVX)mouse lines.By using various histochemical and bone histomorphometry approaches in cortical and trabecular bones,we show that Cx43 HCs play an important role in osteocyte viability,bone remodeling,and OS-related lipid peroxidation under mouse models of estrogen deficiency induced osteoporosis.SECTION ⅠConnexin 43 Channels Are Essential for Normal Bone Structure and Osteocyte ViabilityObjectiveTo determine whether Cx43 hemichannels influence on maintaining BMD,cortical bone structure,osteocyte viability,and osteoclast numbers and activity in female mice.Methods1.BMD measurement.The BMD was measured in all mice using adual-energy X-ray absorptiometry(DXA)scanner.The BMD of the spine,femur,and a total bone measurement were acquired.2.Histology,immunohistochemistry,and histomorphometric analysis.Femur and tibia of mice were embedded in paraffin and 5-μm-thick sections were cut onto glass slides and stained with hematoxylin and eosin(H&E)to quantify the numbers of empty and total osteocytic lacunae.The In Situ Cell Death Detection Kit,TMR red was used for detection and quantification of bone cell apoptosis given broken DNAs were labeled with TUNEL reaction mixture.Tissue sections were tartrate-resistant acid phosphatase(TRAP)stained using Leucocyte Acid Phosphatase Staining Kit to determine osteoclast number(N.Oc),osteoblast number(N.Ob),and bone surface(BS)in the trabecular bone and along the endocortical surface from the metaphysis to the upper midshaft.3.Isolation of RNA and protein samples from bone tissues.In order to quantification of biomarkers of bone formation and resorption.Real-time PCR was performed to detect the mRNA expression of OPG and RANKL in bone using the ABI 7900 PCR device and SYBR Green.Results1.Altered skeletal phenotypes in Cx43 transgenic mice.A significant increase of BMD was observed in the total body,spine,and femur measurements of △130-136 mice,compared to WT and R76W.BMD values of R76W mice were similar to WT.2.Histological analysis showed an increase in the numbers of empty lacunae in cortical bones of △130-136 mice,but the presence of empty lacunae in WT and R76W mice is minimal.An increase of TUNEL labeling in osteocytes in A130-136 compared to WT and R76W mice.The number of osteoclasts was increased in △130-136 mice,although the osteoblast numbers were not significantly altered.3.The mRNA levels of RANKL levels were significantly increased in bone tissues of △130-136 mice as compared to controls,meanwhile the ratio of RANKL/OPG mRNA of A130-136 mice was close significantly increased.Conclusions1.Transgenic models helps define the specific function and mechanism of osteocytic gap junction channels and hemichannels in osteocyte viability and the function of osteocytes in sustaining bone structure,and remodeling.2.There is not gender difference in the effect of Cx43 HCs on bone.3.The effect of Cx43 HCs on bone in female mice was similar to male mice.Cx43 HCs are important in maintaining BMD,cortical bone structure,osteocyte viability,and osteoclast numbers and activity.SECTION IIConnexin 43 Hemichannels Protect Bone Loss During Estrogen DeficiencyObjectiveTo study the specific role of osteocyte Cx43 HCs in estrogen-deficient bone tissue.Methods1.Cell culture.MLO-Y4 cells were cultured to determine the effects of estrogen deficiency on Cx43 expression in osteocytes.2.Dye uptake assay.The activities of Cx43 HCs in response to estrogen withdrawal were further studied using ethidium bromide(EtBr)dye uptake assay.3.Animal models and surgery procedure.We used a 17-week-old WT and transgenic female mice ovariectomy or sham operations(as controls)were performed to model postmenopausal osteoporosis.4.Isolation of RNA from bone tissues and RT-PCR.Real-time PCR was performed with total RNA isolated from humerus to detect the messenger RNA expression of SOD2.5.BMD and Mico-CT analysis.The BMD of the spine,femur,and a total bone measurement were acquired.The structural properties of cortical and trabecular bones were evaluated using a desktop micro-computed tomography imagingsystem.After thresholding,the bone volume/total volume(BV/TV),trabecular thickness(Tb.Th),trabecular spacing(Tb.Sp),and trabecular number(Tb.N)were quantified.Cortical bone analyses included the diaphyseal total area(Tt.Ar),bone area(Ct.Ar),and the area moment of inertia(MMI).6.Mechanical testing of femur.To test bone material and biomechanical properties using "Three-point bending" tests.7.Bone histology and histomorphometry.TUNEL staining was used for detection and quantification of bone cell under apoptosis.To measure the number of osteoblasts and osteoclasts,we used modified Masson’s trichrome and TRAP Staining.8.Immunohistochemistry.We used anti-4-HNE and Cx43 mmunohistochemistry staining to assess the OS-induced lipid peroxidation and Cx43 expression.9.Calcein and Alizarin labeling,and dynamic bone histomorphometry.Mice were injected of Calcein and Alizarin.The rate of bone formation was measured by dynamic histomorphometry via double labeling assay.Results1.Estrogen deficiency reduced Cx43 expression and HCs function.2.Acceleration of bone loss and alteration of bone phenotypes with the impairment of osteocytic Cx43 HCs in OVX mice.Increased bone loss and altered structure of vertebral trabecular bones in A130-136 mice after ovariectomy.Minimal alterations of cortical bone properties in △130-136 mice after ovariectomy.3.Increased deterioration of bone material properties in △130-136 mice after ovariectomy.4.Increased cortical osteocyte apoptosis and empty lacunae in OVX △130-136 mice.5.Attenuation of ovariectomy-induced osteoclast activation with impairment of HCs.Reduction of Oc.S changes in trabecular and cortical bones of △130-136 mice after ovariectomy.6.Minimal changes of bone dynamic histomorphometry parameters in ovariectomized A130-136 transgenic mice compared with WT and R76W groups.7.Increased oxidative stress level in △130-136 mice after ovariectomy.ConclusionsLoss of Cx43 HCs in osteocytes arguments the catabolic effect of ovariectomy on bone tissue and bone remodeling with the decrease of both bone formation and bone resorption.Cx43 HCs is likely to play a protective role against bone loss due to postmenopausal estrogen-deficiency and aging.