Intestinal alkaline phosphatase gene expression is activated by ZBP-89.
Intestinal alkaline phosphatase (IAP) is an enterocyte differentiation marker that functions to limit fat absorption. Zinc finger binding protein-89 (ZBP-89) is a Kruppel-type transcription factor that appears to promote a differentiated phenotype in the intestinal epithelium. The purpose of this study was to investigate the regulation of IAP gene expression by ZBP-89. RT-PCR, quantitative real-time RT-PCR, Western blot analyses, and reporter assays were used to determine the regulation of IAP by ZBP-89 in HT-29 and Caco-2 colon cancer cells. ZBP-89 knockdown was achieved by specific short interfering (si)RNA. EMSA and chromatin immunoprecipitation (ChIP) were performed to examine the binding of ZBP-89 to the IAP promoter. The results of RT-PCR, quantitative real-time PCR, and Western blot analyses showed that ZBP-89 was expressed at low levels in Caco-2 and HT-29 cells, whereas IAP was minimally expressed and absent in these cells, respectively. Transfection with ZBP-89 expression plamid increased IAP mRNA and protein levels in both cell lines, whereas knockdown of endogenous ZBP-89 by siRNA reduced basal levels of IAP gene expression in Caco-2 cells. IAP-luciferase reporter assays, EMSA, and ChIP established that ZBP-89 activated the IAP gene through a response element (ZBP-89 response element: 5'-CCTCCTCCC-3') located between -1018 and -1010 bp upstream of the AUG start codon. We conclude that ZBP-89 is a direct transcriptional activator of the enterocyte differentiation marker IAP. These findings are consistent with the role that this transcription factor is thought to play as a tumor suppressor and suggests its possible function in the physiology of fat absorption.
Malo
Madhu S
MS
Gastrointestinal Unit and Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, 02114, USA.
Mozumder
Moushumi
M
Zhang
Xiao Bo
XB
Biswas
Shaluk
S
Chen
Alexander
A
Bai
Long-Chuan
LC
Merchant
Juanita L
JL
Hodin
Richard A
RA
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D018938:Caco-2 Cells; D004268:DNA-Binding Proteins / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D019073:HT29 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D014157:Transcription Factors / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology*
D004268:DNA-Binding Proteins; D014157:Transcription Factors; C110951:ZNF148 protein, human; D000469:Alkaline Phosphatase