DOI QR코드

DOI QR Code

Concentrations of Calcium-binding Protein and Bone Gla-protein in Culture Medium and CaBP mRNA Expression in Osteoblasts of Broiler Chickens

  • Guo, Xiaoyu (College of Animal Science, Inner Mongolia Agricultural University) ;
  • Yan, Sumei (College of Animal Science, Inner Mongolia Agricultural University) ;
  • Shi, Binlin (College of Animal Science, Inner Mongolia Agricultural University) ;
  • Feng, Yongmiao (Huhhot Vocational College)
  • Received : 2010.02.17
  • Accepted : 2010.07.09
  • Published : 2011.02.01

Abstract

This study was conducted to determine the effects of excess vitamin A on alkaline phosphatase (ALP) activity, contents of calcium-binding protein (CaBP), bone gla-protein (BGP) in culture medium and CaBP mRNA expression in chicken osteoblasts in vitro. Osteoblastic cells in the tibia from 1-day-old Arbor Acre broiler chickens were isolated using enzyme digestion. The subconfluenced cells were divided into eight treatments with six replicates in each treatment and cultured in a medium containing either vehicle or different levels of vitamin A (0, 0.2, 0.6, 1.0, 2.0, 5.0, 10.0 and $20.0\;{\mu}g$/ml), and the control received an equivalent volume of ethanol. The incubation lasted 48 h. The results showed that vitamin A down-regulated ALP activity in the culture medium as well as CaBP mRNA expression of osteoblasts in a linear dose-dependent manner (p = 0.124 and p<0.10, respectively), and suppressed the contents of BGP and CaBP in the culture medium in a quadratic dose-dependent manner (p<0.05 and p<0.10, respectively) with increasing addition of vitamin A. The addition of 0-$0.2\;{\mu}g$/ml vitamin A to the culture medium increased ALP activity, BGP and CaBP contents as well as CaBP mRNA expression compared with other groups, but positive effects of vitamin A tended to be suppressed when vitamin A was increased to $1.0\;{\mu}g$/ml, and adverse effects occurred when vitamin A was increased to 10.0-$20.0\;{\mu}g$/ml. These results implied that there was a threshold level of vitamin A inclusion beyond which inhibitory effects occurred, and the mechanism by which overdose of vitamin A reduced bone growth in chickens was probably reduced osteoblastic cell activity, and inhibited expression of CaBP mRNA and CaBP secretion.

Keywords

Vitamin A;Broiler Chicken;Calcium-binding Protein;mRNA Expression;Osteoblast;Bone Gla-protein

References

  1. Anderson, J. J. 2002. Over supplementation of vitamin A and osteoporotic fracture in the elderly: To supplement or not to supplement with vitamin A. J. Bone Miner. Res. 17(8):1359-1362. https://doi.org/10.1359/jbmr.2002.17.8.1359
  2. Balmain, N. 1991. Calbindin-D9k. A vitamin-D-dependent, calcium-binding protein in mineralized tissues. Clin. Orthop. Relat. Res. 265:265-276.
  3. Balm, N. and B. F. Cai. 1991. Calcium-binding protein: Calbindin-D9K. J. Foreign Medical Sciences: Section of Orthopaedics. 12(4):223-226.
  4. Chen, Y. J., S. M. Yan, H. Y. Li, P. Cao and Z. X. Xu. 2008. Effect of vitamin A and vitamin E on growth performance and immune functions in broilers. Chinese Journal of Inner Mongolia Agricultural University 29(1):14-19.
  5. Feng, Y. M., S. M. Yan, H. Y. Li and L. H. Fang. 2007. Effect of dietary vitamin A on CaBP-m RNA expressions in tissues of broilers. Chinese J. Animal Husbandry and Veterinary Medicine. 39:7-9.
  6. Fell, H. B. and E. Mellanby. 1952. The effect of hypervitaminosis A on embryonic limb-bones cultivated in vitro. J. Physiol. 116:320-349. https://doi.org/10.1113/jphysiol.1952.sp004708
  7. Hough, S., L. V. Avioli, H. Muir, D. Gelderblom, G. Jenkins, H. Kurasi, E. Slatopolsky, M. A. Bergfeld and S. L. Teitelbaum. 1988. Effects of hypervitaminosis A on the bone and mineral metabolism of the rat. Endocrinology 122:2933-2939. https://doi.org/10.1210/endo-122-6-2933
  8. Harada, H., R. Miki, S. Masushige and S. Kato. 1995. Gene expression of retinoic acid receptors, retinoid-X receptors, and cellular retinol-binding protein I in bone and its regulation by vitamin A. Endocrinology 136:5329-5335. https://doi.org/10.1210/en.136.12.5329
  9. Linse, S., E. Thulin, L. K. Gifford, D. Radzewsky, J. Hagan, R. R. Wilk and K. S. Akerfeldt. 1997. Domain organization of calbindin D28k as determined from the association of six synthetic EF-hand fragments. Protein Sci. 6(11):2385-2396.
  10. Melhus, H., K. Michaelsson, A. Kindmark, R. Bergstrom, L. Holmberg, H. Mallmin, A. Wolk and S. Ljunghall. 1998. Excessive dietary intake of vitamin A is associated with reduced bone mineral density and increased risk for hip fracture. J. Ann. Intern Med. 129:770-778. https://doi.org/10.7326/0003-4819-129-10-199811150-00003
  11. Magnusson, P., L. Larsson, M. Magnusson, M. W. J. Davie and C. A. Sharp. 1999. Isoforms of bone alkaline phosphatase: Characterization and origin in human trabecular and cortical bone. J. Bone Miner. Res. 14:1926-1933. https://doi.org/10.1359/jbmr.1999.14.11.1926
  12. Mellanby, E. 1947. Vitamin A and bone growth: the reversibility of vitamin A deficiency changes. J. Physiol. 105:382-399. https://doi.org/10.1113/jphysiol.1947.sp004178
  13. Naveh-Many, T., R. Marx, E. Keshet, J. W. Pike and J. Silver. 1990. Regulation of 1,25-dihydroxyvitamin D3 receptor gene expression by 1,25-dihydroxyvitamin D3 in the parathyroid in vivo. J. Clin. Invest. 86(6):1968-1975. https://doi.org/10.1172/JCI114931
  14. Owen, T. A., M. Aronow, V. Shalhoub, L. M. Barone, L. Wilming, M. S. Tassinari, M. B. Kennedy, S. Pockwinse, J. B. Lian, G. S. Stein, A. Michael and S. Victoria. 1990. Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationship in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracelluar matrix. J. Cell Physiol. 143:420-430. https://doi.org/10.1002/jcp.1041430304
  15. Park, C. K., Y. Ishimi, M. Ohmura, M. Yamaguchi and S. Ikegami. 1996. Vitamin A and carotenoids stimulate differentiation of mouse osteoblastic cells. J. Nutr. Sci. Vitaminol. (Tokyo). Jun; 43(3):281-296.
  16. Rohde, C. M. and H. DeLuca. 2003. Bone resorption activity of all-trans retinoic acid is independent of vitamin D in rats. J. Nutr. 133:777-783.
  17. Raisz, L. G. 1965. Bone resorption in tissue culture. Factors influencing the response to parathyroid hormone. J. Clin. Invest. 44:103-116. https://doi.org/10.1172/JCI105117
  18. Risteli, L. and J. Risteli. 1993. Biochemical markers of bone metabolism. Ann. Med. 25:385-393. https://doi.org/10.3109/07853899309147301
  19. Schmittgen, T. D. and B. A. Zakrajsek. 2000. Effect of experimental treatment on house-keeping gene expression: validation by real-time, quantitative RT-PCR. Biochem. Biophys. Methods 46(1-2):69-81. https://doi.org/10.1016/S0165-022X(00)00129-9
  20. Thomasset, M., J. M. Dupret, A. Brehier and C. Perret. 1990. Calbindin-D9K (CaBP9K) gene: a model for studying the genomic actions of cacitriol and calcium in mammals. Adv. Exp. Med. Biol. 269:35-36. https://doi.org/10.1007/978-1-4684-5754-4_5
  21. Whiting, S. J. and B. Lemke. 1999. Excess retinol intake may explain the high incidence of osteoporosis in northern Europe. Nutr. Rev. 57(6):192-195.
  22. Wada, S. and S. Kamiya. 2006. Bone and bone related biochemical examinations. Bone and collagen related metabolites. Osteocalcin(OC). Clin. Calcium 16(6):1017-1021.
  23. Wang, H. F. 2001. Spectrum of a graph of osteocyte and the technique of osteocyt cultivation in vitro. M. Shanghai: shanghai scientific and technical publishers. 61-63.
  24. Wolke, R. E., H. D. Eaton, S. W. Nielsen and C. F. Helmboldt. 1969. Qualitative and quantitative osteoblastic activity in chronic porcine hypervitaminosis A. J. Pathol. 97:677-686. https://doi.org/10.1002/path.1710970413
  25. Yan, S. M., Y. M. Feng, H. Q. Zhang and B. L. Shi. 2007. Effects of vitamin A and vitamin D on metabolism of calcium and phosphorous in broilers. Chin. J. Anim. Nutr. 19(3):218-224.
  26. Zhi. H. Y., E. Li and J. Zhang. 2001. Effect of all trans retinoid acid on newborn rat calvarial osteoblastic cells in vitro. J. Acta Nutrimenta Sinica. 2:167-169.