Identification of vitamin D-stimulated alkaline phosphatase in IEC-6 cells, a rat small intestine crypt cell line.
The IEC-6 cell line was derived from newborn rat small intestinal crypts and maintains characteristics of nontransformed crypt epithelial cells. We have reported that alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated in IEC-6 cells by 1,25-dihydroxycholecalciferol (1,25(OH)2D3). Using reverse transcription polymerase chain reaction, we determined that the ALP in IEC-6 cells is the liver/bone/kidney ALP (LALP) isoenzyme and not the mature enterocyte form. Levamisole, a specific blocker for LALP, blocked the ALP activity of IEC-6 cells. Kinetic studies showed that 1,25(OH)2D3 increases Vm values of ALP in IEC-6 cells while Km values remain the same. Northern analysis of LALP mRNA levels in IEC-6 cells indicated that 10(-7) M 1,25(OH)2D3 acts by increasing alkaline phosphatase activity at the gene expression level.
Jeng
Y J
YJ
Department of Pharmacology, University of Texas Medical Branch at Galveston 77555-1031.
Watson
C S
CS
Thomas
M L
ML
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002117:Calcitriol / Q000494:pharmacology*; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression / Q000187:drug effects; D007421:Intestine, Small / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D007978:Levamisole / Q000494:pharmacology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping
D017931:DNA Primers; D007527:Isoenzymes; D012333:RNA, Messenger; D007978:Levamisole; D000469:Alkaline Phosphatase; D002117:Calcitriol