The Plasma Membrane Lactate Transporter MCT4, but Not MCT1, Is Up-regulated by Hypoxia through a HIF-1α-dependent Mechanism

Abstract

The monocarboxylate transporter MCT4 mediates lactic acid efflux from most tissues that are dependent on glycolysis for their ATP production. Here we demonstrate that expression of MCT4 mRNA and protein was increased >3-fold by a 48-h exposure to 1% O 2 , whereas MCT1 expression was not increased. The effect was mimicked by CoCl 2 (50 M), suggesting transcriptional regulation by hypoxia-inducible factor 1␣ (HIF-1␣). The predicted promoters for human MCT1, MCT2, and MCT4 were cloned into the pGL3 vector and shown to be active (luciferase luminescence) under basal conditions. Only the MCT4 promoter was activated (>2-fold) by hypoxia. No response was found in cells lacking HIF-1␣. Four potential hypoxiaresponse elements were identified, but deletion analysis implicated only two in the hypoxia response. These were just upstream from the transcription start site and also found in the mouse MCT4 promoter. Mutation of site 2 totally abolished the hypoxic response, whereas mutation of site 1 only reduced the response. Gel-shift analysis demonstrated that nuclear extracts of hypoxic but not normoxic HeLa cells contained two transcription factors that bound to DNA probes containing these hypoxia-response elements. The major shifted band was abolished by mutation of site 2, and supershift analysis confirmed that HIF-1␣ bound to this site. Binding of the second factor was abolished by mutation of site 1. We conclude that MCT4, like other glycolytic enzymes, is up-regulated by hypoxia through a HIF-1␣-mediated mechanism. This adaptive response allows the increased lactic acid produced during hypoxia to be rapidly lost from the cell.The transport of lactic acid across the plasma membrane is of fundamental importance for all mammalian cells. Glycolytic cells (e.g. white muscle fibers and all cells under hypoxic conditions), must rapidly export lactic acid; other tissues import lactic acid to fuel respiration (brain, heart, and red muscle) or gluconeogenesis (liver and kidney) (1-3). Transport is mediated by a family of proton-linked monocarboxylate transporters (MCT(s)) 2 that are also responsible for the transport of other metabolically important monocarboxylates including the ketone bodies. The MCT family has 14 members (3), 6 of which have been functionally characterized. Of these, only MCT1-MCT4 catalyze proton-coupled transport of lactate (1, 4 -7). MCT1 is expressed in most cells (1, 3), whereas MCT4 is expressed strongly only in glycolytic tissues (e.g. white muscle) that must export large amounts of lactic acid (2, 8). In rats, MCT2 is found in tissues requiring high affinity uptake of lactate or pyruvate such as liver and kidney (both gluconeogenic) and neurons (lactate and pyruvate oxidation). However, MCT2 is absent in most human tissues, whereas MCT3 expression is largely restricted to the retinal pigment epithelium (see Refs. 1 and 3). MCT1, MCT3, and MCT4 require ancillary glycoproteins, either gp70 (Embigin) or, more often CD147 (Basigin), for their expression at the plasma membrane (9 -10). MCT2 does not associ...

Glucose deprivation increases monocarboxylate transporter 1 (MCT1) expression and MCT1-dependent tumor cell migration

“…Interestingly, heterocomplexes of CD147 with MCT4 (primarily involved in monocarboxylate export) 4,5 have been previously reported to colocalize with b1-integrin at the leading edge lamellipodia of migrating cells, 45 supporting a role of MCTs in migration. Here, we documented that MCT1-CD147 complexes that accumulate in glucose-deprived cells are present among other locations in such protrusions ( Figure 5 and Supplementary Figure 5) and regulate the migratory phenotype ( Figure 6).…”

“…Furthermore, additional controls would be involved under hypoxia, [40][41][42] explaining why hypoxia can fail to induce or can even repress MCT1 expression in vivo 3,43 whereas it stimulates MCT1 expression in vitro 44 (Figure 2a), and why we detected increased MCT1 expression at early time points (Figure 2a, 24 h) whereas others did not find MCT1 induction upon a longer exposure to hypoxia (48 h). 5 Modulating ROS can indeed impact the response of MCT1 to hypoxia (Supplementary Figure 4A).…”

“…It primarily depends on the expression and activity of monocarboxylate transporter 1 (MCT1), a passive lactate-proton symporter facilitating lactic acid uptake by oxidative TCs (whereas MCT4 channels lactic acid out of glycolytic TCs). 2,4,5 Since lactate shuttling is coupled with one proton, the use of MCT1 to fuel OXPHOS with lactate opposes extracellular acidification. A second contribution of lactate to the metabolic optimization of the tumor microenvironment is referred to as 'lactate signaling'.…”

Interfering with pH regulation in tumours as a therapeutic strategy

“…MCTs (mainly MCT1) regulate the entry and exit of lactate from tumour cells 86 , and their inhibition favours the switch from lactatefuelled respiration to glycolysis, which consecutively kills hypoxic tumour cells via glucose starvation 6 . Indeed, MCTs are overexpressed in many tumours, with MCT4 also being induced by hypoxia through HIF1 signalling, although this remains a controversial issue 87 . MCT inhibitors might therefore have utility as alternative antitumour agents, and various classes of such inhibitors -including DIDS, compound 25 and 26, (FIG.…”

Hypoxia and cancer

“…Despite a low pHe, the intracellular pH (pHi) of tumour cells is maintained at a relatively normal pH or even slightly more alkaline pH, which is reported to result from HIF-mediated up-regulation and activation of a number of membrane located transporters, exchanges, pumps and ecto-enzymes that are implicated in pH homeostasis. Among these are the growth factor activatable and amiloride-sensitive Na + /H + Exchanger (NHE-1) [32][33][34] and the H + /lactate cotransporter (monocarboxylate transporter, MCT1 and MCT4) [35]. In addition, one of the most highly HIF-induced proteins, carbonic anhydrase IX (CA IX), an enzyme that catalyzes the reversible conversion of CO 2 to carbonic acid (Fig.…”