Rongbao Zhao

Identification of an Intestinal Folate Transporter and the Molecular Basis for Hereditary Folate Malabsorption

Folates are essential nutrients that are required for one-carbon biosynthetic and epigenetic processes. While folates are absorbed in the acidic milieu of the upper small intestine, the underlying absorption mechanism has not been defined. We now report the identification of a human proton-coupled, high-affinity folate transporter that recapitulates properties of folate transport and absorption in intestine and in various cell types at low pH. We demonstrate that a loss-of-function mutation in this gene is the molecular basis for hereditary folate malabsorption in a family with this disease. This transporter was previously reported to be a lower-affinity, pH-independent heme carrier protein, HCP1. However, the current study establishes that a major function of this gene product is proton-coupled folate transport required for folate homeostasis in man, and we have thus amended the name to PCFT/HCP1.

Membrane transporters and folate homeostasis: intestinal absorption and transport into systemic compartments and tissues.

Members of the family of B9 vitamins are commonly known as folates. They are derived entirely from dietary sources and are key one-carbon donors required for de novo nucleotide and methionine synthesis. These highly hydrophilic molecules use several genetically distinct and functionally diverse transport systems to enter cells: the reduced folate carrier, the proton-coupled folate transporter and the folate receptors. Each plays a unique role in mediating folate transport across epithelia and into systemic tissues. The mechanism of intestinal folate absorption was recently uncovered, revealing the genetic basis for the autosomal recessive disorder hereditary folate malabsorption, which results from loss-of-function mutations in the proton-coupled folate transporter gene. It is therefore now possible to piece together how these folate transporters contribute, both individually and collectively, to folate homeostasis in humans. This review focuses on the physiological roles of the major folate transporters, with a brief consideration of their impact on the pharmacological activities of antifolates.

Resistance to antifolates

The antifolates were the first class of antimetabolites to enter the clinics more than 50 years ago. Over the following decades, a full understanding of their mechanisms of action and chemotherapeutic potential evolved along with the mechanisms by which cells develop resistance to these drugs. These principals served as a basis for the subsequent exploration and understanding of the mechanisms of resistance to a variety of diverse antineoplastics with different cellular targets. This section describes the bases for intrinsic and acquired antifolate resistance within the context of the current understanding of the mechanisms of actions and cytotoxic determinants of these agents. This encompasses impaired drug transport into cells, augmented drug export, impaired activation of antifolates through polyglutamylation, augmented hydrolysis of antifolate polyglutamates, increased expression and mutation of target enzymes, and the augmentation of cellular tetrahydrofolate-cofactor pools in cells. This chapter also describes how these insights are being utilized to develop gene therapy approaches to protect normal bone marrow progenitor cells as a strategy to improve the efficacy of bone marrow transplantation. Finally, clinical studies are reviewed that correlate the cellular pharmacology of methotrexate with the clinical outcome in children with neoplastic diseases treated with this antifolate.

Mechanisms of Membrane Transport of Folates into Cells and Across Epithelia

Until recently, the transport of folates into cells and across epithelia has been interpreted primarily within the context of two transporters with high affinity and specificity for folates, the reduced folate carrier and the folate receptors. However, there were discrepancies between the properties of these transporters and characteristics of folate transport in many tissues, most notably the intestinal absorption of folates, in terms of pH dependency and substrate specificity. With the recent cloning of the proton-coupled folate transporter (PCFT) and the demonstration that this transporter is mutated in hereditary folate malabsorption, an autosomal recessive disorder, the molecular basis for this low-pH transport activity is now understood. This review focuses on the properties of PCFT and briefly addresses the two other folate-specific transporters along with other facilitative and ATP-binding cassette (ABC) transporters with folate transport activities. The role of these transporters in the vectorial transport of folates across epithelia is considered.

Localization of the murine reduced folate carrier as assessed by immunohistochemical analysis

The reduced folate carrier (RFC1) is a major route for the transport of folates in mammalian cells. The localization of RFC1 in murine tissues was evaluated by immunohistochemical analysis using a polyclonal antibody to the C-terminus of the carrier. There was expression of RFC1 in the brush-border membrane of the jejunum, ileum, duodenum and colon. RFC1 was localized to the basolateral membrane of the renal tubular epithelium. Carrier was detected on the plasma membrane of hepatocytes but not in bile duct epithelial cells. In the choroid plexus RFC1 was highly expressed at the apical surface. It was also expressed in axons and dendrites and on the apical membrane of cells lining the spinal canal. In spleen, RFC1 was detected only in the cells of the red pulp. These data provide insights into the role that RFC1 plays in folate delivery in a variety of tissues. In particular, the localization of carrier may elucidate the role of RFC1 in the vectorial transport of folates across epithelia. The data also indicate that in kidney tubules and choroid plexus the sites of RFC1 expression are different from what has been reported previously for the folate receptor; and while RFC1 is expressed in small intestine, folate receptor is not.

SLC19A3 encodes a second thiamine transporter ThTr2.

Recently, a new family of facilitative carriers has been cloned consisting of the reduced folate (SLC19A1) and the thiamine (SLC19A2) transporters. Despite a high level of sequence identity and similarity there is essentially no functional overlap between these carriers. The former transports folates and the latter thiamine. In this paper we describe the function of SLC19A3, another member of this transporter family most recently cloned, after transient transfection of the cDNA into HeLa cells. Uptake of [3H]thiamine, but not of methotrexate nor folic acid, was enhanced in SLC19A3 transfectants relative to vector control. Similarly, in the transfectants thiamine transport increased with an increase in pH with peak activity at pH approximately 7.5. While [3H]thiamine uptake was markedly inhibited by nonlabeled thiamine it was not inhibited by several organic cations in 100-fold excess. Hence this carrier has a high degree of specificity for vitamin B1. The data indicate that SLC19A3 has the characteristics of SLC19A2 (ThTr1) and represents a second thiamine transporter (ThTr2) in this family of facilitative carriers.

The Proton-Coupled Folate Transporter: Impact on Pemetrexed Transport and on Antifolates Activities Compared with the Reduced Folate Carrier

The reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT) are ubiquitously expressed in normal and malignant mammalian tissues and in human solid tumor cell lines. This article addresses the extent to which PCFT contributes to transport of pemetrexed and to the activities of this and other antifolates relative to RFC at physiological pH. Either RFC or PCFT cDNA was stably transfected into a transporter-null HeLa cell variant to achieve activities similar to their endogenous function in wild-type HeLa cells. PCFT and RFC produced comparable increases in pemetrexed activity in growth medium with 5-formyltetrahydrofolate. However, PCFT had little or no effect on the activities of methotrexate, N-(5-[N-(3,4-dihydro-2-methyl-4-oxyquinazolin-6-ylmethyl)-N-methyl-amino]-2-thenoyl)-l-glutamic acid (raltitrexed, Tomudex; ZD1694), or Nα-(4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-l-ornithine (PT523) in comparison with RFC irrespective of the folate growth source. PCFT, expressed at high levels in Xenopus laevis oocytes and in transporter-competent HepG2 cells, exhibited a high affinity for pemetrexed, with an influx Km value of 0.2 to 0.8 μM at pH 5.5. PCFT increased the growth inhibitory activity of pemetrexed, but not that of the other antifolates in HepG2 cells grown with 5-formyltetrahydrofolate at physiological pH. These findings illustrate the unique role that PCFT plays in the transport and pharmacological activity of pemetrexed. Because of the ubiquitous expression of PCFT in human tumors, and the ability of PCFT to sustain pemetrexed activity even in the absence of RFC, tumor cells are unlikely to become resistant to pemetrexed as a result of impaired transport because of the redundancy of these genetically distinct routes.

A prominent low-pH methotrexate transport activity in human solid tumors: Contribution to the preservation of methotrexate pharmacologic activity in HeLa cells lacking the reduced folate carrier

Whereas the major folate transporter, the reduced folate carrier (RFC), has a physiological pH optimum, transport activities for folates and antifolates have been detected with low pH optima. Because the interstitial pH in solid tumors is generally acidic, the mechanisms by which antifolates are transported at low pH could be an important determinant of drug activity under these conditions. The current study quantitated the low pH methotrexate (MTX) transport activity in human solid tumor cell lines from the National Cancer Institute tumor panel and other sources. MTX influx at pH 5.5 was equal to, or greater than, influx at pH 7.4 in 29 of 32 cell lines. To assess the role of RFC in transport at low pH in one of these cell lines, a HeLa clonal line (R5) was selected for MTX resistance due to a genomic deletion of the carrier gene. MTX influx was depressed by 70% in R5 versus wild-type HeLa cells at pH 7.4. At pH 6.5, influx in these two lines was similar; as the pH was decreased to 5.5 influx increased in both cell lines. Similarly, whereas net MTX uptake over 1 h was markedly decreased in R5 cells at pH 7.4, net uptake in HeLa and R5 cells was comparable at pH 6.5. Also, as compared with MCF7 breast cancer cells, MTX uptake was markedly decreased at pH 7.4, but only minimally at pH 6.5, in the MDA-MB-231 human breast cancer cell line that lacks RFC expression. When grown with folic acid (2 μm) at pH 7.4, the IC50 for MTX was 14-fold higher in R5 as compared with wild-type HeLa cells; the difference was only 4-fold when cells when grown at pH 6.9; the IC50s were identical at this pH when the medium folate was 25 nm 5-formyltetrahydrofolate. These data demonstrate that transport activity at low pH is prevalent in human solid tumors, is RFC-independent in R5 cells and MDA-MB-231 breast cancer cells, and can preserve MTX activity in the absence of RFC at an acidic pH relevant to solid tumors in vivo.

A role for the proton-coupled folate transporter (PCFT-SLC46A1) in folate receptor-mediated endocytosis

Recently, this laboratory identified a proton-coupled folate transporter (PCFT), with optimal activity at low pH. PCFT is critical to intestinal folate absorption and transport into the central nervous system because there are loss-of-function mutations in this gene in the autosomal recessive disorder, hereditary folate malabsorption. The current study addresses the role PCFT might play in another transport pathway, folate receptor (FR)-mediated endocytosis. FRα cDNA was transfected into novel PCFT+ and PCFT– HeLa sublines. FRα was shown to bind and trap folates in vesicles but with minimal export into the cytosol in PCFT– cells. Cotransfection of FRα and PCFT resulted in enhanced folate transport into cytosol as compared with transfection of FRα alone. Probenecid did not inhibit folate binding to FR, but inhibited PCFT-mediated transport at endosomal pH, and blocked FRα-mediated transport into the cytosol. FRα and PCFT co-localized to the endosomal compartment. These observations (i) indicate that PCFT plays a role in FRα-mediated endocytosis by serving as a route of export of folates from acidified endosomes and (ii) provide a functional role for PCFT in tissues in which it is expressed, such as the choroid plexus, where the extracellular milieu is at neutral pH.

The molecular identity and characterization of a Proton-Coupled Folate Transporter—PCFT; biological ramifications and impact on the activity of pemetrexed—12 06 06

Membrane transport of folates is essential for the survival of all mammalian cells and transport of antifolates is an important determinant of antifolate activity. While a major focus of attention has been on transport mediated by the reduced folate carrier and folate receptors, a very prominent carrier-mediated folate transport activity has been recognized for decades with a low-pH optimum and substrate specificity distinct from that of the reduced folate carrier which operates most efficiently at neutral pH. This low-pH transporter represents the mechanism by which folates are absorbed in the small intestine and it is also widely expressed in other human tissues and solid tumors. Recently, this laboratory discovered the molecular identity of this transporter which is genetically unrelated to the reduced folate carrier. This transporter is proton-coupled, electrogenic, and manifests a substrate specificity that is similar to that of the low-pH transport activity previously described in mammalian cells. The key role this transporter plays in intestinal folate absorption has been confirmed by the demonstration of a mutation in this gene in the rare autosomal recessive disorder, hereditary folate malabsorption. This article reviews (1) the characteristics and prevalence of the low-pH folate transport activity, (2) its relationship to, and properties of, the recently identified Proton-Coupled Folate Transporter (PCFT), (3) the physiological and pharmacological roles of this transporter, particularly with respect to pemetrexed, and (4) the historical controversy, now resolved, on the mechanism of intestinal folate absorption.