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Cloning of the monocarboxylate transporter isoform MCT2 from rat testis provides evidence that expression in tissues is species-specific and may involve post-transcriptional regulation.

ABSTRACT: The cDNA for the monocarboxylate transporter MCT2 from rat testis has been cloned and sequenced. The derived protein sequence shows 82% identity with that from hamster. Rat MCT2 has a relative insertion of five amino acids in the N-terminal sequence preceding the first predicted transmembrane segment. MCT2 appears to be less highly conserved between species than MCT1. Using Northern blotting of RNA from rat and mouse tissues, MCT2 message was demonstrated to be abundant in the testis where a smaller, less abundant MCT2 transcript was also present. Low levels of a slightly different-sized transcript were found in rat and mouse liver, and mouse kidney. In hamster, only one-size transcript was detected at relatively high abundance in all the tissues examined. Antibodies were raised against a peptide derived from the extreme C-terminus of rat MCT2, and Western blotting with these detected MCT2 in membrane fractions prepared from rat testis, liver and brain but not those from heart or skeletal muscle. In hamster, MCT2 was detected in liver, heart and testis but not in brain [Garcia, Brown, Pathak, and Goldstein (1995) J. Biol. Chem. 270, 1843-1849]. For both rat MCT1 and MCT2 there were marked differences between the relative abundance of their respective messages and the amount of protein in membrane fractions from different tissues. This suggests that expression of both of these transporters in different tissues may be species-specific and regulated post-transcriptionally. The different-sized MCT2 transcripts may arise from alternative splicing. Starvation of rats for up to 48 h did not lead to any change in MCT1 or MCT2 expression in the liver, as determined by either Northern or Western blotting.

SUBMITTER: Jackson VN

PROVIDER: S-EPMC1218450 | biostudies-other | 1997 Jun

REPOSITORIES: biostudies-other

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Project description:Monocarboxylate transporter 8 (MCT8) deficiency or the Allan-Herndon-Dudley Syndrome (AHDS) is an X-linked psychomotor disability syndrome with around 320 clinical cases described worldwide. SLC16A2 gene mutations, encoding the thyroid hormone (TH) transporter MCT8, result in intellectual disability due to impaired TH uptake in the developing brain. MCT8 deficiency is a multi-organ affecting disease with a predominant neuronal cell-based pathology, with the glial component inadequately investigated. However, deficiency in myelin, a key component of white matter (WM) enabling fast nerve conduction, is a TH-dependent hallmark of the disease. Nevertheless, analysis of the myelin status in AHDS patients has led to conflicting interpretations. The majority of individual case studies reported delayed myelination, that was restored later in life. In contrast, post-mortem studies and high-resolution MRIs detected WM (micro-) abnormalities throughout adolescence, suggesting permanent hypomyelination. Thus, interpretations vary depending on methodology to investigate WM microstructure. Further, it is unknown whether the mutation within the MCT8 is linked to the severity of the myelin deficiency. Consequently, terminology is inconsistent among reports, and AHDS is occasionally misdiagnosed as another WM disorder. The evolutionary conserved TH signaling pathway that promotes the generation of myelinating oligodendrocytes enabled deciphering how the lack of MCT8 might affect myelinogenesis. Linking patient findings on myelination to those obtained from models of MCT8 deficiency revealed underlying pathophysiological mechanisms, but knowledge gaps remain, notably how myelination progresses both spatially and temporally in MCT8 deficiency. This limits predicting how myelin integrity might benefit therapeutically, and when to initiate. A recurrent observation in clinical trials is the absence of neurological improvement. Testing MCT8-independent thyromimetics in models, and evaluating treatments used in other demyelinating diseases, despite different etiologies, is crucial to propose new therapeutic strategies combatting this devastating disease.

Project description:Hypoxic regions are frequent in glioblastoma (GBM), the most common type of malignant adult brain tumor, and increased levels of tumor hypoxia have been associated with worse clinical outcomes. To unmask genes important in hypoxia, we treated GBM neurospheres in hypoxia and identified monocarboxylate transporter-4 (MCT4) as one of the most upregulated genes. To investigate the clinical importance of MCT4 in GBM, we examined clinical outcomes and found that MCT4 overexpression is associated with shorter patient survival. Consistent with this, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM, and MCT4 downregulation correlated with the less aggressive G-CIMP (Glioma CpG Methylator Phenotype) subset of GBM. Immunohistochemical analysis of tissue microarrays confirmed that MCT4 protein levels were increased in high-grade as compared with lower-grade astrocytomas, further suggesting that MCT4 is a clinically relevant target. To test the requirement for MCT4 in vitro, we transduced neurospheres with lentiviruses encoding short-hairpin RNAs (shRNAs) against MCT4, resulting in growth inhibition of 50-80% under hypoxia in two lines. MCT4 knockdown was associated with a decreased percentage of cells expressing the stem-cell marker CD133 and increased apoptotic fraction. We also found that flow-sorted CD133-positive cells had almost sixfold higher MCT4 levels than CD133-negative cells, suggesting that the stem-like population might have a greater requirement for MCT4. Most importantly, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo. Interestingly, whereas MCT4 is a well-characterized lactate exporter, we found that both intracellular and extracellular lactate levels did not change following MCT4 silencing, suggesting a novel lactate export-independent mechanism for growth inhibition in GBMs. To identify this potential mechanism, we performed microarray analysis on control and shMCT4-expressing neurospheres and found a dramatic reduction in the expression of multiple Hypoxia-Inducible Factor (HIF)-regulated genes following MCT4 knockdown. The overall reduction in HIF transcriptional response was further validated using a hypoxia response element (HRE)-dependent green-fluorescent protein (GFP) reporter line.

Dataset Information

Cloning of the monocarboxylate transporter isoform MCT2 from rat testis provides evidence that expression in tissues is species-specific and may involve post-transcriptional regulation.

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