Biochemical and Biophysical Research Communications
Transcriptional regulation of PCFT by KLF4, HNF4α, CDX2 and C/EBPα: Implication in its site-specific expression in the small intestine
Highlights
► We examined transcription factors that may regulate PCFT expression in the intestine. ► PCFT promoter activity is basically induced by KLF4. ► KLF4-induced PCFT promoter activity is enhanced by HNF4α synergistically. ► CDX2 and C/EBPα suppress PCFT promoter activity induced by KLF4 and HNF4α.
Introduction
Proton-coupled folate transporter (PCFT) has been recently identified as the transporter responsible for the intestinal uptake of folates and analogs that include antifolate drugs such as methotrexate [1]. Its characteristics, which feature specific affinity for reduced folates as well as folate and optimum operation driven by H+ at acidic pH, are in agreement with those of the folate transport system that had long been hypothesized to be present in the small intestine [1], [2], [3], [4], [5]. Furthermore, the initial study on PCFT by Qiu et al. indicated that a loss of its function due to a homozygous mutation in its gene is responsible for hereditary folate malabsorption, underscoring its pivotal role in folate absorption [1]. It is also notable that PCFT is expressed abundantly in the upper and middle parts of the small intestine, but not at all in the lower part [3], [4], and this unique expression profile is in excellent agreement with that of the activity of folate uptake, as we demonstrated in a study using rats [4]. Elucidating the mechanism regulating the unique expression profile of PCFT in the small intestine should be of interest and help understanding its potential alteration, which could have an impact on the absorption of antifolate drugs as well as folates.
Studies on PCFT have been expanding rapidly since its identification. They include those on its transcriptional regulation, which have identified Yin Yang 1 (YY1), activator protein 1 (AP1), AP2, nuclear respiratory factor 1 (NRF1) and vitamin D receptor (VDR) as transcription factors potentially involved in that [6], [7], [8]. However, all those studies are for transcriptional regulation in the cell lines of HeLa and Caco-2, and those identified transcription factors, which are present ubiquitously in various organs, are unlikely to be able to account for the specifically high expression of PCFT in the small intestine and its unique expression profile in that organ. We, therefore, attempted in the present study to identify transcription factors that could account for such a specific and unique expression profile of PCFT, assuming the involvement of those which are more specifically or abundantly expressed in the small intestine, and to elucidate the transcriptional regulation mechanism. We also examined the status of DNA methylation, because it has been reported to be an important epigenetic factor that determines the expression of PCFT in various cell lines [9], [10].
Section snippets
Materials
Rabbit polyclonal antibody for Krüppel-like factor 4 (KLF4), rabbit polyclonal antibody for CCAAT/enhancer-binding protein α (C/EBPα) and rabbit polyclonal antibody for caudal-type homeobox transcription factor 2 (CDX2) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), rabbit polyclonal antibody for hepatocyte nuclear factor 4α (HNF4α) was from Abnova (Taipei, ROC), mouse monoclonal anti-β-actin was from Sigma–Aldrich (St. Louis, MO, USA) and goat anti-rabbit IgG (Peroxidase
Activation of PCFT promoter by KLF4
As the first step to identify transcription factors involved in the transcriptional regulation of the expression of human PCFT in the small intestine, luciferase reporter assays were performed with the reporter construct containing −1695/+96 segment of the 5′-flanking region of the human PCFT gene in HEK293 cells, assessing changes in luciferase activity induced by cotransfection of plasmids for the expression of several transcription factors, which are known to be specifically present in the
Discussion
The present study has indicated that KLF4 can be a major transcription factor that activates the promoter of the human PCFT gene. KLF4, which belongs to the family of zinc-finger transcription factors, is also known as gut-enriched Krüppel-like factor (GKLF) and highly expressed in terminally differentiated epithelial cells at the villi of the small intestine [13]. It has also been reported that KLF4 is involved as a transcriptional activator in the intestine-specific expression of various
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