Regulation Of Osteoclast Development And Bone Homeostasis By Rankl In Adipocytes

Backgrounds: Many studies have shown that obesity can decrease bone mineral density,resulting in bone loss.It is generally believed that adipocytes indirectly regulates bone metabolism through the secretion of a variety of adipokines.However,in recent years the literature and our early work suggest that adipocytes express Receptor activator of nuclear factor kappa-B ligand(RANKL),which acts to initatiate osteoclast differentiation by binding to its receptor RANK on the precursor cells of osteoclasts.This suggests that adipocyte might have the potential to directly regulate osteoclast formation and this hypothesis has recently been demonstrated by in vitro studies,but there are currently no in vivo evidences.In order to further reveal whether adipocytes directly regulate osteoclast development through RANKL,we made adipocyte specific Rankl transgenic mice and gene silencing mice,and analyzed the changes in the bone mass and the number of osteoclasts.The present study aims to unravel if the RANKL/OPG system in adipocytes mediates the interaction of adipocytes and preosteoclasts and regulates osteoclast development thus modulating the modeling and remodeling of bone.Methods:1.Rankl transgenic and Rankl gene silencing constructs were made by expressing Rankl c DNA and silencing sequence,respectively,under the 5.4-kb promoter of a P2.Then the adipocyte-specific Rankl transgenic mice and adipocyte Rankl gene silencing mice were made by microinjection,fertilized egg transplantation and genotype identification by model animal research center of Nanjing University.Total RNA of various tisssues was extracted from the transgenic mice and its negative control mice and reverse transcribed into c DNA,then PCR-amplified using specific primer to detect the distribution of Rankl transgene.The total protein of adipose tissue from transgenic mice and gene silencing mice and their respective negative control mice was extracted and subjected to Western blotting analysis to detect the RANKL protein levels.2,X-ray and Micro-CT were used to detect the bone histomorphometric parameters in the transgenic mice,gene silencing mice and their respective negative control mice.The bone mass was also evaluated by using Alcina Blue-Hematoxylin-Orange G staining on the tibial slides;The tibia paraffin sections were also stained with anti-tartaric acid phosphatase(TRAP)to detect the changes in the number of osteoclasts between the control and the mutant mice.Osteoclast formation from the bone marrow cells was studied and expression levels of osteoclast specific genes were examined in.Calcein double labeling was performed to measure the changes of bone dynamics parameters.ELISA was carried out to detect osteocalcin levels in the serum of the control and mutant mice.Results:1.Genotyping PCR confirmed the integration of the trasngene and silencing sequences in the a P2-Rankl transgenic(a P2-Rankl Tg)mice and a P2-Rankl silencing(a P2-Rankl KD)mice.RT-PCR revealed that Rankl transgene was specifically highly expressed in adipose tissue,mildly expressed in tibial tissue with bone marrow,but not expressed in liver,spleen,brain and tibial deprived of bone marrow.Compared with the negative control mice,the expression of RANKL protein in groin adipose tissue was increased in the transgenic mice,and decreased in the gene silencing mice compared with their repective negative control mice.2.Compared with the negative control mice,the histomorphometric parameters were negatively changed in 3-month old transgenic mice.In brief,the relative volume of trabecular bone(BV/TV)was reduced by 90%,the trabecular bone mineral density(BMD)was reduced by 71%,the number of trabecular bone(Tb.N)was reduced by 91%,the bone model index(SMI)was increased by 180%.In 3-month old transgenic mice,TRAP staining in the tibial paraffin sections showed a 77% increase in the number of osteoclasts and a 83% increase in osteoclast surface.In vitro osteoclast fromation from bone marrow cells was potentiated in the transgenic mice,with a 1.73-fold increase compared to the control group.Moreover,the m RNA expression of TRACP-5b,CTR and Ctsk were increased by 1.35,5.14 and 1.54-fold respectively,in the marrow cells of transgenic mice.Bone mineralization rate(MAR)was increased by 59% in 1 month old transgenic mice.Furthermore,the serum osteocalcin level was increased by 54% in 1-month old transgenic mice.These suggest that adipocte-specific Rankl transgenic mice(a P2-Rankl Tg)have increased bone marrow osteoclast formation and increased bone resorption,increased bone turnover rate,and reduced bone mass.3.Compared with the negative control mice,the histomorphometric parameters were positively changed in 3-month old Rankl silencing mice.In brief,the relative volume of trabecular bone(BV/TV)was increased by 67%,the trabecular bone mineral density(BMD)was increased by 28%,the number of trabecular bone(Tb.N)was increased by 45%,and the bone model index(SMI)was decreased by 17%.TRAP staining in the tibial paraffin sections of 3-month old silencing mice showed a 54% decrease in the number of osteoclasts and a 45% decrease in osteoclast surface.In vitro osteoclast fromation from bone marrow cells was attenuated in the transgenic mice,with a 40% decrease compared to the control group.Moreover,the m RNA expression of TRACP-5b,CTR and Ctsk were increased by 70%,49% and 63%,respectively,in the marrow cells of the Rankl silencing mice.Bone mineralization rate(MAR)was decreased by 49% in 1 month old silencing mice.Furthermore,the serum osteocalcin level was decreased by 31% in 1-month old silencing mice.These suggest that adipocte-specific Rankl silencing mice have decreased bone marrow osteoclast formation and reduced bone resorption,decreased bone turnover rate,and increased bone mass.Conclusion:1.Adipocyte Rankl transgenic mice and gene silencing mice were successfully made.2.Adipocyte Rankl transgenic mice(aP2-Rankl Tg)have increased bone marrow osteoclast formation,increased bone resorption,increased bone turnover,and reduce bone mass.3.Adipocyte Rankl gene silencing mice have decreased bone marrow osteoclast formation,decreased bone resorption,reduced bone turnover,and increased bone mass.4.Adipocytes can directly regulate osteoclast formation by expressing RANKL,which may confer a novel mechanism for the bone loss due to obesity.