摘要
为了研究钙敏感性受体(CaSR)和活性维生素D[1,25(OH)_2D_3]在钙磷平衡和骨骼代谢中的相互作用及机制,我们建立了CaSR和25羟维生素D-1-α羟化酶[1α(OH)ase]双基因敲除[CaSR~(-/-)1α(OH)ase~(-/-)]小鼠模型,比较分析了2周龄CaSR~(-/-)1α(OH)ase~(-/-)小鼠与相应单基因敲除及野生型(WT)同窝小鼠的表型差异。
与先前的研究结果一致,约85%的CaSR~(-/-)小鼠在出生后两周内死亡,幸存至两周者体格小,体重轻,血钙明显升高,血磷降低,血甲状旁腺素(PTH)水平明显升高,甲状旁腺肥大。两周龄1α(OH)ase~(-/-)小鼠表现为轻度低钙低磷血症,血PTH升高50%,甲状旁腺中度肥大,但能存活到成年。CaSR~(-/-)1α(OH)ase~(-/-)小鼠生存期较CaSR~(-/-)小鼠延长,2周和4周龄CaSR~(-/-)1α((OH)ase~(-/-)小鼠生存率分别为91%和42%。2周龄CaSR~(-/-)1α(OH)ase~(-/-)小鼠体重较CaSR~(-/-)小鼠明显增加,血钙正常,血磷较CaSR~(-/-)和1α(OH)ase~(-/-)小鼠低,血PTH水平较CaSR~(-/-)小鼠更高。这些结果说明,1α(OH)ase基因敲除能通过纠正CaSR~(-/-)小鼠的高钙血症而延长其存活期,但由于更严重的高PTH血症致使CaSR~(-/-)1α(OH)ase~(-/-)小鼠不能存活至成年。
为了明确CaSR和1,25(OH)_2D_3在骨骼生长发育过程中的相互作用,我们通过影像学和组织学比较分析了2周龄不同基因型小鼠骨骼的表型差异。CaSR~(-/-)小鼠表现为严重的骨骼生长障碍,包括长骨短小,钙化的次级骨化中心缺如,骨密度降低,但干骺端小梁骨密度增加。1α(OH)ase~(-/-)小鼠长骨略短,钙化的骺端较小,骨密度轻度降低。CaSR~(-/-)1α(OH)ase~(-/-)小鼠长骨长度、皮质骨和小梁骨密度均较CaSR~(-/-)小鼠明显增加。这些结果说明1α(OH)ase基因敲除能够部分改变CaSR缺失引起的严重的骨骼生长发育。
为了明确CaSR和1,25(OH)_2D_3在软骨内成骨中的相互作用,我们通过组织学和免疫组织化学的方法分析比较了2周龄不同基因型小鼠生长板的表型差异。CaSR~(-/-)小鼠表现为次级骨化中心形成延迟,增殖细胞核抗原(PCNA)阳性软骨细胞减少,软骨细胞肥大带明显增宽。1α(OH)ase~(-/-)小鼠生长板和肥大带宽度较WT小鼠稍有增宽。在2周龄CaSR~(-/-)1α(OH)ase~(-/-)小鼠,次级骨化中心已开始形成,肥大软骨细胞中已开始出现血管和成骨细胞。生长板和肥大带宽度较CaSR~(-/-)小鼠明显变窄,而PCNA阳性软骨细胞则较CaSR~(-/-)小鼠明显增加。鉴于甲状旁腺素相关蛋白(PTHrP)在软骨细胞增殖和分化中起有重要作用,我们通过PTHrP免疫组织化学染色发现PTHrP阳性细胞在CaSR~(-/-)小鼠明显减少,而在CaSR~(-/-)1α(OH)ase~(-/-)小鼠则较CaSR~(-/-)小鼠有所增加,但没有达到WT小鼠的水平。这些结果说明,1α(OH)ase基因敲除能够通过上调PTHrP,增加软骨细胞的增殖而起到部分纠正CaSR缺失引起的软骨内成骨的严重障碍。
为了明确CaSR和1,25(OH)_2D_3在骨形成和骨吸收中的相互作用,我们通过组织学,组织化学和免疫组织化学及图像分析检测了两周龄不同基因型小鼠骨组织的表型差异。与WT小鼠相比,成骨细胞数、小梁骨容量和骨钙素(OCN)阳性面积在CaSR~(-/-)小鼠均明显增加;在1α(OH)ase~(-/-)小鼠则有所减少,而在CaSR~(-/-)1α(OH)ase~(-/-)小鼠增加更为明显。抗酒石酸酸性磷酸酶(TRAP)阳性破骨细胞数在CaSR~(-/-)小鼠增加,在1α(OH)ase~(-/-)小鼠减少;而在CaSR~(-/-)1α(OH)ase~(-/-)小鼠与WT小鼠相似。NF-κB受体活化素配体(receptoractivatior of NF-κB ligand,RANKL)阳性细胞和面积变化与成骨细胞的变化一致。这些结果说明,CaSR缺失引起的成骨细胞骨形成增加是由于PTH过代偿作用的结果。
本研究进一步阐述了CaSR和1,25(OH)_2D_3在调节钙磷平衡,甲状旁腺细胞生长和PTH产生以及软骨内成骨和成骨细胞骨形成中的相互作用的机制,为后续研究和临床相关疾病的诊治提供了实验和理论依据。
To assess mechanism of interaction between calcium sensing receptor(CaSR)and active vitamin D[1,25(OH)_2D_3]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)_2D_3 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)_2D_3 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)_2D_3 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)_2D_3 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.
引文
1. Jian, W.X., Long, J.R., Li, M.X., Liu, X.H., and Deng, H. W. 2005. Genetic determination of variation and covariation of bone mineral density at the hip and spine in a Chinese population. J Bone Miner Metab 23:181-185.
2. Ricci, T.A., Chowdhury, H.A., Heymsfield, S.B., Stahl, T., Pierson, R.N., Jr., and Shapses, S.A. 1998. Calcium supplementation suppresses bone turnover during weight reduction in postmenopausal women. J Bone Miner Res 13:1045-1050.
3. Rubinacci, A., Divieti, P., Polo, R.M., Zampino, M., Resmini, G., and Tenni, R. 1996. Effect of an oral calcium load on urinary markers of collagen breakdown. J Endocrinol Invest 19:719-726.
4. Lorget, F., Kamel, S., Mentaverri, R., Wattel, A., Naassila, M, Maamer, M, and Brazier, M. 2000. High extracellular calcium concentrations directly stimulate osteoclast apoptosis. Biochem Biophys Res Commun 268:899-903.
5. Dvorak, M.M., and Riccardi, D. 2004. Ca2+ as an extracellular signal in bone. Cell Calcium 35:249-255.
6. Yamaguchi, T., Chattopadhyay, N., Kifor, O., and Brown, E.M. 1998. Extracellular calcium (Ca2+(o))-sensing receptor in a murine bone marrow-derived stromal cell line (ST2): potential mediator of the actions of Ca2+(o) on the function of ST2 cells. Endocrinology 139:3561-3568.
7. Brown, E.M., Gamba, G., Riccardi, D., Lombardi, M, Butters, R., Kifor, O., Sun, A., Hediger, M.A., Lytton, J., and Hebert, S.C. 1993. Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid. Nature 366:575-580.
8. Shoback, D., and Chang, W. 2001. Starvation amidst plenty-rickets and hypercalcemia in calcium receptor knockout mice. Endocrinology 142:3733-3735.
9. Pollak, M.R., Brown, E.M., Chou, Y.H., Hebert, S.C., Marx, S.J., Steinmann, B., Levi, T, Seidman, C.E., and Seidman, J.G 1993. Mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 75:1297-1303.
10. Brown, E.M., and MacLeod, R.J. 2001. Extracellular calcium sensing and extracellular calcium signaling. Physiol. Rev. 81:239-297.
11. Chang, W, Tu, C., Pratt, S., Chen, T.H., and Shoback, D. 2002. Extracellular Ca(2+)-sensing receptors modulate matrix production and mineralization in chondrogenic RCJ3.1C5.18 cells. Endocrinology 143:1467-1474.
12. House, M.G., Kohlmeier, L., Chattopadhyay, N., Kifor, O., Yamaguchi, T., Leboff, M.S., Glowacki, J., and Brown, E.M. 1997. Expression of an extracellular calcium-sensing receptor in human and mouse bone marrow cells. J Bone Miner Res 12:1959-1970.
13. Ho, C., Conner, D.A., Pollak, M.R., Ladd, D.J., Kifor, O., Warren, H.B., Brown, E.M., Seidman, J.G, and Seidman, C.E. 1995. A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nat Genet 11:389-394.
14. Gamer, S.C., Pi, M., Tu, Q., and Quarles, L.D. 2001. Rickets in cation-sensing receptor-deficient mice: an unexpected skeletal phenotype. Endocrinology 142:3996-4005.
15. Bouillon, R., Okamura, W.H., and Norman, A.W. 1995. Structure-function relationships in the vitamin D endocrine system. Endocr Rev 16:200-257.
16. Valdivielso, J.M., and Fernandez, E. 2006. Vitamin D receptor polymorphisms and diseases. Clin Chim Acta 371:1-12.
17. Dardenne, O., Prud'homme, J., Arabian, A., Glorieux, F.H., and St-Arnaud, R. 2001. Targeted inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets. Endocrinology 142:3135-3141.
18. Panda, D.K., Miao, D., Tremblay, M.L, Sirois J., Farookhi, R., Hendy, G.N., and Goltzman, D. 2001. Targeted ablation of the 25-hydroxyvitamin D 1alpha -hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction. Proc Natl Acad Sci U S A 98:7498-7503.
19. Kos, C.H., Karaplis, A.C., Peng, J.B., Hediger, M.A., Goltzman, D., Mohammad, K.S., Guise, T.A., and Pollak, M.R. 2003. The calcium-sensing receptor is required for normal calcium homeostasis independent of parathyroid hormone. J Clin Invest 111:1021 -1028.
20. Tu, Q., Pi, M, Karsenty, G., Simpson, L., Liu, S., and Quarles, L.D. 2003. Rescue of the skeletal phenotype in CasR-deficient mice by transfer onto the Gcm2 null background. J Clin Invest 111:1029-1037.
21. Xue, Y., Karaplis, A.C., Hendy, G.N., Goltzman, D., and Miao, D. 2005. Genetic models show that parathyroid hormone and 1,25-dihydroxyvitamin D3 play distinct and synergistic roles in postnatal mineral ion homeostasis and skeletal development. Hum Mol Genet 14:1515-1528.
22. Krebs, L.J., and Arnold, A. 2002. Molecular basis of hyperparathyroidism and potential targets for drug development. Curr Drug Targets Immune Endocr Metabol Disord 2:167-179.
23. Panda, D.K., Miao, D., Bolivar, I, Li, J., Huo, R., Hendy, G.N., and Goltzman, D. 2004. Inactivation of the 25-hydroxyvitamin D lalpha-hydroxylase and vitamin D receptor demonstrates independent and interdependent effects of calcium and vitamin D on skeletal and mineral homeostasis. J Biol Chem 279:16754-16766.
24. Karaplis, A.C., Luz, A., Glowacki, J., Branson, R.T., Tybulewicz, V.L., Kronenberg, H.M., and Mulligan, R.C. 1994. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev 8:277-289. Write to the Help Desk NCBI | NLM | NIH Department of Health & Human Services Privacy Statement | Freedom of Information Act | Disclaimer.
25. Miao, D., He, B., Karaplis, A.C., and Goltzman, D. 2002. Parathyroid hormone is essential for normal fetal bone formation. J Clin Invest 109:1173-1182.
26. Ahlstrom, M., Pekkinen, M., Riehle, U., and Lamberg-Allardt, C. 2008. Extracellular calcium regulates parathyroid hormone-related peptide expression in osteoblasts and osteoblast progenitor cells. Bone 42:483-490.
27. Sanders, J.L., Chattopadhyay, N., Kifor, O., Yamaguchi, T., Butters, R.R., and Brown, E.M. 2000. Extracellular calcium-sensing receptor expression and its potential role in regulating parathyroid hormone-related peptide secretion in human breast cancer cell lines. Endocrinology 141:4357-4364.
28. Shen, X., Mula, R.V., Li, J., Weigel, N.L., and Falzon, M. 2007. PTHrP contributes to the anti-proliferative and integrin alpha6beta4-regulating effects of 1,25-dihydroxyvitamin D(3). Steroids 72:930-938.
29. Tovar Sepulveda, V.A., and Falzon, M. 2003. Prostate cancer cell type-specific regulation of the human PTHrP gene via a negative VDRE. Mol Cell Endocrinol 204:51-64.
30. Dvorak, M.M., Siddiqua, A., Ward, D.T, Carter, D.H., Dallas, S.L., Nemeth, E.F., and Riccardi, D. 2004. Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones. Proc Natl Acad Sci U S A 101:5140-5145.
31. Dvorak, M.M., Tu, C., Elalieh, H., Chen, T., Liu, B., Kream, B.E., Bikle, D.D., Chang, W., and Shoback, D.M. 2007. Conditional Ablation of the Osteoblast Calcium-Sensing Receptor Causes Abnormalities in Skeletal Development and Mineralization. J Bone Miner Res 22 (Suppl 1):M223.
32. Miao, D., He, B., Lanske, B., Bai, X.Y., Tong, X.K., Hendy, G.N., Goltzman, D., and Karaplis, A.C. 2004. Skeletal abnormalities in Pth-null mice are influenced by dietary calcium. Endocrinology 145:2046-2053.
1.Edwards SL.Maintaining calcium balance:physiology and implications.Nuts Times 2005;101(19):58-61.
2.Petri M.Systemic lupus erythematosus:women's health issues.Bull Rheum Dis 2000;49(8):1-3.
3.王惠芬.钙、锌对免疫细胞凋落的影响.国外医学·医学地理分册 1994;15(3):118.
4.Brown EM,Gamba G,Riccardi D,et al.Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid.Nature 1993;366(6455):575-80.
5.Brown EM,MacLeod RJ.Extracellular calcium sensing and extracellular calcium signaling.Physiol.Rev.2001;81:239-297.
6.Orendac M,Kozich V,Zeman J,et al.[Clinical picture of homocystinuria with cystathionine beta-synthase deficiency in 19 Czech and Slovak patients].Cas Lek Cesk 2000;139(16):500-7.
7.Brenza HL,Kimmel-Jehan C,Jehan F,et al.Parathyroid hormone activation of the 25-hydroxyvitamin D3-lalpha-hydroxylase gene promoter.Proc Natl Acad Sci U S A 1998;95(4):1387-91.
8.Lands L.Fat,sugar and drugs on the French Riviera.GMHC Treat Issues 1999;13(3):9-11.
9.Cantley LK,Russell J,Lettieri D,Sherwood LM.1,25-Dihydroxyvitamin D3 suppresses parathyroid hormone secretion from bovine parathyroid cells in tissue culture.Endocrinology 1985;117(5):2114-9.
10.Chan YL,McKay C,Dye E,Slatopolsky E.The effect of 1,25 dihydroxycholecalciferol on parathyroid hormone secretion by monolayer cultures of bovine parathyroid cells.Calcif Tissue Int 1986;38(1):27-32.
11.Kato S,Yoshizazawa T,Kitanaka S,Murayama A,Takeyama K.Molecular genetics of vitamin D-dependent hereditary rickets.Horm Res 2002;57(3-4):73-8.
12.Oleson(周敬德译)E.钙和维生素D摄入量对人类患结肠癌危险的影响.国外医学·医学地理分册 1986;7(3):122.
13.Nayler WG,Panagiotopoulos S,Elz JS,Daly MJ.Calcium-mediated damage during post-ischaemic reperfusion.J Mol Cell Cardiol 1988;20 Suppl 2:41-54.