Mechanism Of Interaction Between Calcium Sensing Receptor And Active Vitamin D On Calcium And Phosphate Homeostasis And On Skeletal Metabolism

To assess mechanism of interaction between calcium sensing receptor(CaSR)and active vitamin D[1,25(OH)₂D₃]on calcium and phosphate homeostasis and on skeletal metabolism,we created a CaSR and 25-hydroxyvitamin D-1-hydroxylase[1(OH)ase]double gene knockout[CaSR^-/-1α(OH)ase^-/-]animal model and compared their phenotypes with those of each single gene knockout and wild-type animals at 2-weeks of ages.Our results confirmed that around 85%CaSR^-/-mice died at within 2 weeks after birth.Size of body and body weight were reduced markedly,serum calcium levels were raised,serum phosphorus levels were decreased and serum parathyroid hormone(PTH)levels were raised significantly,the size of parathyroid glands was enlarged in 2-week-old CaSR^-/-mice compared to their WT littermates.1α(OH)ase^-/-mice are viable until adult.Serum calcium and phosphorus levels were reduced,serum PTH levels were raised to 150%of WT mice and the size of parathyroid glands was enlarged moderately in 2-week-old 1α(OH)ase^-/-mice compared to their WT littermates. CaSR^-/-1α(OH)ase^-/-mice were survival longer than CaSR^-/-mice,their survival rate reached 91%and 42%at 2 and 4 weeks of ages, respectively,and their serum calcium levels were normalized.Size of body and body weight were increased,serum phosphorus levels were reduced,and PTH levels were raised and the size of parathyroid glands was enlarged in 2-week-old CaSR^-/-1α(OH)ase^-/-mice compared to age-matched CaSR^-/-littermates.These results indicate that deletion of 1α(OH)ase gene in CaSR^-/-mice makes animal survival longer due to rescued hypercalcemia,however,CaSR^-/-1α(OH)ase^-/-mice cannot survival until adult due to more severe hyperparathyroidism.To assess the interaction between CaSR and 1,25(OH)₂D₃ on skeletal growth and development,the skeletal phenotypes of mice with different genotypes were analyzed at 2-weeks of ages by radiography,micro-CT and histology.CaSR^-/-mice displayed severe skeletal growth retardation including shorter long bones without calcified secondary ossification and reduced bone mineral density (BMD),however,BMD was increased in trabecular bone at the metaphyseal region.The length of long bones was slight shorter, calcified epiphysis was smaller and BMD was reduced in 1α(OH)ase^-/- mice compared to their WT littermates.The length of long bones and BMD in cortical and trabecular bone were increased significantly in CaSR^-/-1α(OH)ase^-/-mice compared to CaSR^-/-littermates.These results indicate that the deletion of 1α(OH)ase gene in CaSR^-/-mice can rescue partially severe skeletal growth retardation. To assess the interaction between CaSR and 1,25(OH)₂D₃ on endochondral bone formation,the phenotypes of growth plates in mice with different genotypes were analyzed at 2-weeks of ages by histology and immunohistochemical staining for proliferating cell nuclear antigen(PCNA and parathyroid hormone related peptide (PTHrP).The formation of the secondary ossification was delayed, PCNA positive chodrocytes were reduced and the hypertrophic zone of growth plates was enlarged in CaSR^-/-mice.The thickness of growth plates and hypertrophic zone were slight enlarged in 1α(OH)ase^-/-mice. The secondary ossification appeared at 2 weeks of ages with vessels and osteoblasts among hypertrophic chondrocytes in CaSR^-/-1α(OH)ase^-/-mice.The thickness of growth plates and hypertrophic zone were reduced and PCNA positive chondrocytes were increased significantly in CaSR^-/-1α(OH)ase^-/-mice compared to CaSR^-/- littermates.In view of the fact,PTHrP plays an important role in the proliferation and differentiation of chondrocytes,we examined the expression of PTHrP in chondrocytes by immunostaining.Results showed that PTHrP positive chondrocytes were reduced markedly in CaSR^-/-mice compared to their WT littermates and were increased in CaSR^-/-1α(OH)ase^/-mice compared to CaSR^-/-littermates although they did not reached to WT levels.Consequently,the deletion of 1α(OH)ase gene in CaSR^-/-mice rescued partially severe skeletal growth retardation may mediated through the up-regulation of PTHrP which stimulates the proliferation of chodrocytes.To assess the interaction between CaSR and 1,25(OH)₂D₃ on osteoblastic bone formation and osteoclastic bone resorption,the phenotypes of bone tissues in mice with different genotypes were analyzed at 2-weeks of ages by histology,histochemical staining, immunostaining and image analysis.Osteoblast number,trabecular bone volume and osteocalcin positive areas were increased significantly in CaSR^-/-mice and were decreased in 1α(OH)ase^-/-mice compared to their WT littermates,whereas these parameters were further increased in CaSR^-/-1α(OH)ase^-/-mice even compared to CaSR^-/-littermates.The number of TRAP positive osteoclasts were increased significantly in CaSR^-/-mice and were decreased in 1α(OH)ase^-/-mice,was normalized in CaSR^-/-1α(OH)ase^-/-mice.Alterations of RANKL positive osteoblasts and positive areas were consistent with those of osteoblasts. Our findings imply that increased osteoblastic bone formation in CaSR deficient mice results from over compensation of PTH.This study further expound the mechanism of interaction between CaSR andl,25(OH)₂D₃ on calcium and phosphate homeostasis,the cell growth of parathyroid glands and PTH production,endochodral and osteoblastic bone formation.It provides experimental and theoretical evidence for subsequent research and the diagnosis and treatment of related diseases.