RFC-1 Gene Expression Regulates Folate Absorption in Mouse Small Intestine

Abstract

Mediated folate compound transport inward in isolated luminal epithelial cells from mouse small intestine was delineated as pH-dependent and non-pH-dependent components on the basis of their differential sensitivity to the stilbene inhibitor, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid. pH dependence was manifested as higher maximum capacity (Vmax) for influx of l, L-5-CH3-H4folate at acidic pH compared with neutral or alkaline pH with no effect on saturability (Km). The pH-dependent component was relatively insensitive to inhibition by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid and highly saturable (Km or Ki = 2 to 4 microM) in the case of folic acid, folate coenzymes, and 4-aminofolate analogues as permeants or inhibitors. The non-pH-dependent component was highly sensitive to 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid and poorly and variably saturable (Km or Ki = 20 to >2000 microM) with respect to these folate compounds. Only the pH-dependent transport component was developmentally regulated, showing much higher maximum capacity for l,L-5-CH3-H4folate influx in mature absorptive rather than proliferative crypt cells. The increase in pH-dependent influx during maturation was associated with an increase in RFC-1 gene expression in the form of a 2.5-kilobase RNA transcript and 58-kDa brush-border membrane protein detected by folate-based affinity labeling and with anti-mouse RFC-1 peptide antibodies. The size of this protein was the same as that encoded by RFC-1 mRNA. The treatment of mature absorptive cells with either the affinity label or the anti-RFC-1 peptide antibodies inhibited influx of l, L-[3H]-5-CH3-H4folate in a concentration-dependent manner. These results strongly suggest that pH-dependent folate absorption in this tissue is regulated by RFC-1 gene expression.

Characterization of a Folate Transporter in HeLa Cells with a Low pH Optimum and High Affinity for Pemetrexed Distinct from the Reduced Folate Carrier

“…However, recent studies from this laboratory indicate that in human solid tumors, in general, there is very prominent transport activity at low pH that is usually equal to or often exceeds transport mediated by RFC at physiologic pH (9). The mechanism(s) underlying this low pH transport activity has not been clarified, although there is some evidence suggesting that in cells of intestinal origin this transport activity is related, at least in part and in some way, to RFC (11)(12)(13)(14). This study addresses the mechanism of folate transport at low pH using a HeLa cell line with prominent low pH activity in which RFC was deleted from the genome and therefore cannot contribute at all, qualitatively or quantitatively, to the transport characteristics observed.…”

Resistance to antifolates

“…The mechanism by which folates are transported in the intestine at low pH is not entirely clear. Supporting a role for RFC in this process are the observations that the protein is present at the apical brush-border membrane (Chiao et al, 1997;Wang et al, 2001), activity at low pH is enhanced by transfection of the cDNA into intestinal cells (Kumar et al, 1998;Rajgopal et al, 2001), and an antibody to RFC inhibited 5-CH 3 -THF transport into intestinal cells (Chiao et al, 1997).…”

A mouse model of hereditary folate malabsorption: deletion of the PCFT gene leads to systemic folate deficiency

“…[6][7][8] The RFC1 transporter is expressed ubiquitously, including the brush-border membrane of epithelial cells in the small intestine. 9 Although RFC1 is necessary for folate transport in erythroid cells, its involvement in intestinal folate uptake has not been confirmed. 6 Inactivation of RFC1 in mice by homologous recombination led to either embryonic lethality or defective erythropoiesis in pups born to mothers who were supplemented with 1 mg daily subcutaneous doses of folic acid.…”