Defining the transcriptional regulation of the intestinal sodium-glucose cotransporter using RNA-interference mediated gene silencing.
BACKGROUND
The sodium glucose cotransporter (SGLT1) is responsible for all active intestinal glucose uptake. Hepatocyte nuclear factors 1 alpha and beta (HNF 1 alpha and HNF 1 beta) activate the SGLT1 promoter, whereas GATA-binding protein 5 (GATA-5) and caudal-type homeobox protein 2 (CDX2) regulate transcription of other intestinal genes. We investigated SGLT1 regulation by these transcription factors using promoter studies and RNA interference.


METHODS
Chinese hamster ovary (CHO) cells were transiently cotransfected with an SGLT1-luciferase promoter construct and combinations of expression vectors for HNF 1 alpha, HNF 1 beta, CDX2, and GATA-5. Caco-2 cells were stably transfected with knockdown vectors for either HNF 1 alpha or HNF 1 beta. mRNA levels of HNF 1 alpha, HNF 1 beta, and SGLT1 were determined using quantitative polymerase chain reaction (qPCR).


RESULTS
HNF 1 alpha, GATA-5, and HNF 1 beta significantly activated the SGLT1 promoter (P < .05). Cotransfection of GATA-5 with HNF 1 alpha had an additive effect, whereas HNF 1 beta and CDX2 antagonized HNF 1 alpha and GATA-5. SGLT1 expression was significantly reduced in HNF 1 alpha or HNF 1 beta knockdowns (P < .001). HNF alpha knockdown significantly reduced HNF 1 beta expression and vice versa (P < .005).


CONCLUSIONS
HNF 1 alpha and HNF 1 beta are important transcription factors for endogenous SGLT1 expression by cultured enterocytes. GATA-5 and CDX2 also regulate SGLT1 promoter activity and show cooperativity with the HNF1s. We, therefore, propose a multifactorial model for SGLT1 regulation, with interactions between HNF1, GATA-5, and CDX2 modulating intestinal glucose absorption.
Balakrishnan
Anita
A
Department of Surgery, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Stearns
Adam T
AT
Rhoads
David B
DB
Ashley
Stanley W
SW
Tavakkolizadeh
Ali
A
D000818:Animals; D000071616:CDX2 Transcription Factor; D016466:CHO Cells; D018938:Caco-2 Cells; D006224:Cricetinae; D003412:Cricetulus; D050992:GATA5 Transcription Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D020868:Gene Silencing; D051538:Hepatocyte Nuclear Factor 1-alpha / Q000378:metabolism / Q000494:pharmacology; D051539:Hepatocyte Nuclear Factor 1-beta / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000494:pharmacology; D006801:Humans; D011401:Promoter Regions, Genetic / Q000187:drug effects; D034622:RNA Interference; D051269:Sodium-Glucose Transporter 1 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D000071616:CDX2 Transcription Factor; C491270:CDX2 protein, human; D050992:GATA5 Transcription Factor; D051538:Hepatocyte Nuclear Factor 1-alpha; D018398:Homeodomain Proteins; D051269:Sodium-Glucose Transporter 1; D051539:Hepatocyte Nuclear Factor 1-beta