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ARS Home » Northeast Area » Boston, Massachusetts » Jean Mayer Human Nutrition Research Center On Aging » Research » Publications at this Location » Publication #112689
Title: DEVELOPMENT OF AN ASSAY FOR THE HUMAN CALBINDIN D9K PROTEIN AND DEMONSTRATION OF VITAMIN D-DEPENDENT CALBINDIN D9K EXPRESSION IN CACO-2 CELLS, A HUMAN INTESTINAL CELL LINE

Author
item WOOD, RICHARD - HNRCA-TUFTS
item TCHACK, LAURIE - HNRCA-TUFTS

Submitted to: Journal of Bone and Mineral Research
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2000
Publication Date: 9/1/2000
Citation: WOOD, R.J., TCHACK, L. DEVELOPMENT OF AN ASSAY FOR THE HUMAN CALBINDIN D9K PROTEIN AND DEMONSTRATION OF VITAMIN D-DEPENDENT CALBINDIN D9K EXPRESSION IN CACO-2 CELLS, A HUMAN INTESTINAL CELL LINE. JOURNAL OF BONE AND MINERAL RESEARCH. 2000;15(1):s323.

Interpretive Summary:

Technical Abstract: Calbindin D9K is a vitamin D-responsive calcium binding protein believed to play an important role in the transfer of calcium across the mammalian enterocyte. We have expressed the open reading frame of human calbindin D9K in E. coli as a fusion protein with a C-terminal histidine tag to facilitate purification using immobilized metal affinity chromatography. The purified protein was used as an antigen in rabbits to develop a polyclonal antibody to human calbindin D9K, which was subsequently found to cross react on Western blots with an appropriately sized vitamin D-responsive protein from Caco-2 cells, a human intestinal cell line. An ELISA assay was then developed to measure calbindin D9K and determine the responsiveness of calbindin D to 1,25-dihydroxyvitamin D treatment. We have previously reported that Caco-2 cells express increased human calbindin 9K mRNA in response to treatment with 1,25-dihydroxyvitamin D. In the current study, we have shown that Caco-2 cells express detectable amounts of calbindin D9K under basal cell culture conditions. Moreover, treatment with 100 nmol/l 1,25-dihydroxyvitamin D for 48h, a dose of 1,25-dihydroxyvitamin D that increases calcium transport in Caco-2 cells, resulted in a significant (p< 0.004, paired t-test) 125% increase in calbindin D from 71 ng calbindin D/mg pro to 160 ng calbindin/mg pro. These findings in Caco-2 cells are consistent with previous observations of 1,25-dihydroxyvitamin D-associated increases in calbindin D mRNA in human intestine and the notion that calbindin D9K may mediate vitamin D-induced calcium absorption.