Role of reduced folate carrier in intestinal folate uptake

Studies from our laboratory and others have characterized different aspects of the intestinal folate uptake process and have shown that the reduced folate carrier (RFC) is expressed in the gut and plays a role in the uptake process. Little, however, is known about the actual contribution of the RFC system toward total folate uptake by the enterocytes. Addressing this issue in RFC knockout mice is not possible due to the embryonic lethality of the model. In this study, we describe the use of the new approach of lentivirus-mediated short hairpin RNA (shRNA) to selectively silence the endogenous RFC of the rat-derived intestinal epithelial cells (IEC-6), an established in vitro model for folate uptake, and examined the effect of such silencing on folate uptake. First we confirmed that the initial rate of [3H]folic acid uptake by IEC-6 cells was pH dependent with a markedly higher uptake at acidic compared with alkaline pH. We also showed that the addition of unlabeled folic acid to the incubation buffer leads to a severe inhibition (∼95%) in [3H]folic acid (16 nM) uptake at buffer pH 5.5 but not at buffer pH 7.4. We then examined the effect of treating (for 72 h) IEC-6 cells with RFC-specific shRNA on the levels of RFC protein and mRNA and observed substantial reduction in the levels of both parameters (∼80 and 78%, respectively). Such a treatment was also found to lead to a severe inhibition (∼90%) in initial rate of folate uptake at buffer pH 5.5 (but not at pH 7.4); uptake of the unrelated vitamin, biotin, on the other hand, was not affected by such a treatment. These results demonstrate that the RFC system is the major (if not the only) folate uptake system that is functional in intestinal epithelial cells.

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Differentiation-dependent regulation of the intestinal folate uptake process: studies with Caco-2 cells and native mouse intestine

AJP Cell Physiology ◽

10.1152/ajpcell.00249.2008 ◽

2008 ◽

Vol 295 (3) ◽

pp. C828-C835 ◽

Cited By ~ 18

Author(s):

Veedamali S. Subramanian ◽

Jack C. Reidling ◽

Hamid M. Said

Keyword(s):

Epithelial Cells ◽

Initial Rate ◽

Intestinal Epithelial Cells ◽

Acid Uptake ◽

Reduced Folate Carrier ◽

Mrna Levels ◽

Intestinal Epithelial ◽

Uptake Process ◽

Mouse Intestine ◽

Process Studies

Differentiation of intestinal epithelial cells is accompanied by alterations in levels of expression of many genes, including those involved in nutrient uptake. Effects of differentiation of intestinal epithelial cells on the physiological and molecular parameters of the intestinal folate uptake process are not well characterized. To address this issue, we used two models, Caco-2 cells and native mouse intestine. Studies with Caco-2 cells showed a significant increase in the initial rate of carrier-mediated folic acid uptake during differentiation (i.e., as the cells transitioned from preconfluent to confluent and then to postconfluent stages). This increase was associated with an increase in the level of expression of the human reduced folate carrier (hRFC) and the human proton-coupled folate transporter (hPCFT) both at the protein and mRNA levels with differentiation; it was also associated with a significant increase in activity of the hRFC and hPCFT promoters. Studies with native mouse intestine showed a significantly higher folate uptake in villus compared with crypt cells, which was again associated with a significantly higher level of expression of the mouse RFC and PCFT at the protein and mRNA levels. Together, these studies demonstrate that the intestinal folate uptake process undergoes differentiation-dependent regulation and that this regulation is mediated via changes in the level of expression of both the RFC and PCFT. In addition, the studies suggest the possible involvement (at least in part) of a transcriptional mechanism(s) in this type of regulation of the intestinal folate uptake process.

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Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopusoocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.1.c289 ◽

1998 ◽

Vol 274 (1) ◽

pp. C289-C294 ◽

Cited By ~ 59

Author(s):

Chandira K. Kumar ◽

Toai T. Nguyen ◽

Francis B. Gonzales ◽

Hamid M. Said

Keyword(s):

Folic Acid ◽

Epithelial Cells ◽

Cdna Clone ◽

Xenopus Oocytes ◽

Intestinal Epithelial Cells ◽

Maximal Velocity ◽

Mrna Levels ◽

Acidic Ph ◽

Intestinal Epithelial ◽

Folate Transport

We recently identified a cDNA clone from mouse small intestine, which appears to be involved in folate transport when expressed in Xenopus oocytes. The open reading frame of this clone is identical to that of the reduced folate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley, and K. H. Cowan. J. Biol. Chem. 269: 17–20, 1994). The characteristics of this cDNA clone [previously referred to as intestinal folate carrier 1 (IFC-1)] expressed in Xenopus oocytes, however, were found to be different from the characteristics of folate transport in native small intestinal epithelial cells. To further study these differences, we determined the characteristics of RFC when expressed in an intestinal epithelial cell line, IEC-6, and compared the findings to its characteristics when expressed in Xenopus oocytes. RFC was stably transfected into IEC-6 cells by electroporation; its cRNA was microinjected into Xenopus oocytes. Northern blot analysis of poly(A)+RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFC was found to be fourfold higher than uptake in control sublines. This increase in folic acid and 5-MTHF uptake was inhibited by treating IEC-6/RFC cells with cholesterol-modified antisense DNA oligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity ( V max) of the uptake process [the apparent Michaelis-Menten constant ( K m) also changed (range was 0.31 to 1.56 μM), but no specific trend was seen]. In both IEC-6/RFC and control sublines, the uptake of both folic acid and 5-MTHF displayed 1) pH dependency, with a higher uptake at acidic pH 5.5 compared with pH 7.5, and 2) inhibition to the same extent by both reduced and oxidized folate derivatives. These characteristics are very similar to those seen in native intestinal epithelial cells. In contrast, RFC expressed in Xenopus oocytes showed 1) higher uptake at neutral and alkaline pH 7.5 compared with acidic pH 5.5 and 2) higher sensitivity to reduced compared with oxidized folate derivatives. Results of these studies demonstrate that the characteristics of RFC vary depending on the cell system in which it is expressed. Furthermore, the results may suggest the involvement of cell- or tissue-specific posttranslational modification(s) and/or the existence of an auxiliary protein that may account for the differences in the characteristics of the intestinal RFC when expressed in Xenopus oocytes compared with when expressed in intestinal epithelial cells.

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Intestinal Neurod1 expression impairs paneth cell differentiation and promotes enteroendocrine lineage specification

Scientific Reports ◽

10.1038/s41598-019-55292-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Hui Joyce Li ◽

Subir K. Ray ◽

Ning Pan ◽

Jody Haigh ◽

Bernd Fritzsch ◽

...

Keyword(s):

Cell Differentiation ◽

Epithelial Cells ◽

Cell Fate ◽

Intestinal Epithelial Cells ◽

Paneth Cell ◽

Substantial Reduction ◽

Ectopic Expression ◽

Cell Lineage ◽

Intestinal Epithelial ◽

Conditional Expression

AbstractTranscription factor Neurod1 is required for enteroendocrine progenitor differentiation and maturation. Several earlier studies indicated that ectopic expression of Neurod1 converted non- neuronal cells into neurons. However, the functional consequence of ectopic Neurod1 expression has not been examined in the GI tract, and it is not known whether Neurod1 can similarly switch cell fates in the intestine. We generated a mouse line that would enable us to conditionally express Neurod1 in intestinal epithelial cells at different stages of differentiation. Forced expression of Neurod1 throughout intestinal epithelium increased the number of EECs as well as the expression of EE specific transcription factors and hormones. Furthermore, we observed a substantial reduction of Paneth cell marker expression, although the expressions of enterocyte-, tuft- and goblet-cell specific markers are largely not affected. Our earlier study indicated that Neurog3+ progenitor cells give rise to not only EECs but also Goblet and Paneth cells. Here we show that the conditional expression of Neurod1 restricts Neurog3+ progenitors to adopt Paneth cell fate, and promotes more pronounced EE cell differentiation, while such effects are not seen in more differentiated Neurod1+ cells. Together, our data suggest that forced expression of Neurod1 programs intestinal epithelial cells more towards an EE cell fate at the expense of the Paneth cell lineage and the effect ceases as cells mature to EE cells.

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Polarized targeting of the human reduced folate carrier (hRFC) in intestinal epithelial cells: Xenopus laevis oocytes as a model system

Gastroenterology ◽

10.1016/s0016-5085(08)80699-3 ◽

2001 ◽

Vol 120 (5) ◽

pp. A142

Author(s):

Veedamali S. Subramanian ◽

Jonathan S. Marchant ◽

Ian Parker ◽

Hamid M. Said

Keyword(s):

Epithelial Cells ◽

Xenopus Laevis ◽

Intestinal Epithelial Cells ◽

Model System ◽

Xenopus Laevis Oocytes ◽

Reduced Folate Carrier ◽

Intestinal Epithelial

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Polarized targeting of the human reduced folate carrier (hRFC) in intestinal epithelial cells: Xenopus laevis oocytes as a model system

Gastroenterology ◽

10.1016/s0016-5085(01)80699-5 ◽

2001 ◽

Vol 120 (5) ◽

pp. A142-A142 ◽

Cited By ~ 1

Author(s):

V SUBRAMANIAN ◽

J MARCHANT ◽

I PARKER ◽

H SAID

Keyword(s):

Epithelial Cells ◽

Xenopus Laevis ◽

Intestinal Epithelial Cells ◽

Model System ◽

Xenopus Laevis Oocytes ◽

Reduced Folate Carrier ◽

Intestinal Epithelial

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Intracellular regulation of intestinal folate uptake: studies with cultured IEC-6 epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.2.c729 ◽

1997 ◽

Vol 272 (2) ◽

pp. C729-C736 ◽

Cited By ~ 39

Author(s):

H. M. Said ◽

T. Y. Ma ◽

A. Ortiz ◽

A. Tapia ◽

C. K. Valerio

Keyword(s):

Small Intestine ◽

Folic Acid ◽

Protein Kinase ◽

Epithelial Cell ◽

Intestinal Epithelial Cell ◽

Acid Uptake ◽

Maximal Velocity ◽

Intestinal Epithelial ◽

Intracellular Regulation ◽

Uptake Process

Although the mechanism of folate uptake in the small intestine has been well characterized, very little is known about the intracellular regulation of the uptake process. Using mature confluent monolayers of the intestinal epithelial cell line IEC-6 as an in vitro intestinal epithelial cell model, we have found the uptake of folic acid to be similar to that of the native small intestine in that it is 1 ) temperature, energy, and pH dependent, 2) Na+ independent, 3) inhibited by structural analogs and anion transport inhibitors, and 4) saturable as a function of substrate concentration [apparent Michaelis constant (Km) = 0.45 +/- 0.06 microM; maximal velocity (Vmax) = 3.08 +/- 0.14 pmol x mg protein(-1) x 5 min(-1)]. Furthermore, IEC-6 cells were found by Northern blot analysis to lack the expression of the membrane folate-binding protein. Pretreatment of IEC-6 monolayers with specific protein tyrosine kinase (PTK) inhibitors genistein and tyrphostin A25 caused a significant inhibition in folic acid uptake. On the other hand, their negative controls, genistin and tyrphostin A1, respectively, had no effect. The inhibitory effect of genistein was mediated through inhibition in the Vmax of the folate uptake process with no change in the apparent Km. Pretreatment of IEC-6 monolayers with compounds that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) level (e.g., dibutyryl cAMP) also resulted in a significant (though modest) inhibition in folic acid uptake; however, specific inhibitors of protein kinase A did not affect the uptake process. Specific modulators of protein kinase C and Ca2+/calmodulin-mediated pathways did not significantly affect folic acid uptake. These results demonstrate the suitability of IEC-6 monolayers as an intestinal epithelial model to study folate transport and demonstrate for the first time that uptake of folic acid is regulated by a PTK- and a cAMP-mediated pathway.

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Role of MicroRNA-423-5p in posttranscriptional regulation of the intestinal riboflavin transporter-3

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00238.2017 ◽

2017 ◽

Vol 313 (6) ◽

pp. G589-G598 ◽

Cited By ~ 7

Author(s):

Ram Lakhan ◽

Veedamali S. Subramanian ◽

Hamid M. Said

Keyword(s):

Epithelial Cells ◽

Posttranscriptional Regulation ◽

Intestinal Epithelial Cells ◽

Significant Inhibition ◽

Luciferase Reporter ◽

Intestinal Epithelial ◽

Functional Consequences ◽

Uptake Process ◽

First Time

Riboflavin (RF) is essential for normal cellular functions and health. Humans obtain RF from exogenous sources via intestinal absorption that involves a highly specific carrier-mediated process. We have recently established that the riboflavin transporter-3 (RFVT3) is vital for the normal intestinal RF uptake process and have characterized certain aspects of its transcriptional regulation. Little is known, however, about how this transporter is regulated at the posttranscriptional level. We address this issue by focusing on the role of microRNAs. Using bioinformatics, we identified two potential interacting miRNAs with the human (h) RFVT3-3′-UTR, and showed (using pmirGLO-hRFVT3-3′-UTR) that the hRFVT3-3′-UTR is, indeed, a target for miRNA effect. Of the two putative miRNAs identified, miR-423-5p was found to be highly expressed in intestinal epithelial cells and that its mimic affected luciferase reporter activity of the pmirGLO-hRFVT3-3′-UTR construct, and also led to inhibition in RF uptake by intestinal epithelial Caco-2 and HuTu-80 cells. Furthermore, cells transfected with mutated seed sequences for miR-423-5p showed an abrogation in inhibitory effect of the miR-423-5p mimic on luciferase activity. While miR-423-5p did not affect the level of expression of the hRFVT3 mRNA, it did lead to a significant inhibition in the level of expression of its protein. Similarly, miR-423-5p was found to affect the level of expression of the mouse RFVT3 in cultured intestinal enteroids. These findings demonstrate, for the first time, that the RFVT3 is a target for posttranscriptional regulation by miRNAs in intestinal epithelial cells and that this regulation has functional consequences on intestinal RF uptake. NEW & NOTEWORTHY Our findings show for the first time that RFVT3 is a target for posttranscriptional regulation by miR-423-5p in intestinal epithelial cells, and this regulation has functional consequences on intestinal riboflavin (RF) uptake process.

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