The Human Monocarboxylate Transporter MCT1: Gene Structure and Regulation

M. A. Cuff and S. P. Shirazi-Beechey

Epithelial Function and Development Group
Department of Veterinary Preclinical Sciences
The University of Liverpool
Brownlow Hill and Crown Street
L69 7ZJ Liverpool, United Kingdom

To the Editor: We read with concern the recent paper by Hadjiagapiou et al. (4) that examines the regulation of the monocarboxylate transporter isoform 1 (MCT1) gene promoter in Caco-2 cells and would like to make a number of comments with regard to 1) errors in their data and 2) misquotation and misrepresentation of prior publications by others in this important study area.

MCT1 is the prototype of a family of proteins that play an important role in the transport of monocarboxylates across the cell membrane in a variety of cell types (5). MCT1 is particularly prominent in the colonic epithelium where it functions to transport short-chain fatty acids (most notably, butyrate) from the lumen across the colonocyte luminal membrane (7, 8). We demonstrated several years ago that MCT1 abundance is markedly downregulated during the transition from normality to malignancy in the colon and, more recently, that its expression is required for butyrate to exert many of its effects in cultured colonic epithelial cells (1, 6). Not surprisingly, the regulation of MCT1 expression in the intestine is an important area of study. We have previously reported that human colonic MCT1 expression is responsive to its substrate, butyrate, and that this regulation involves the dual control of transcription and mRNA stability (2). We have also determined the site of transcription initiation, isolated the MCT1 gene promoter, and demonstrated the MCT1 gene structure to comprise five exons interrupted by four introns. The first of these introns occurs in the 5’-untranslated region (UTR) encoding DNA, spans greater than 26 kb, and accounts for more than one-half of the entire MCT1 transcription unit (3).

Given that the isolation of the human MCT1 gene promoter was first published in full more than three years ago (3) and the sequence submitted to the European Molecular Biology Laboratories database (Accession no. AJ438944), it is surprising that the first part of the paper by Hadjiagapiou et al. focuses on repetition of this isolation. This aside, a major concern is the inaccurate description by Hadjiagapiou et al. of where the MCT1 promoter DNA sequence is located in relation to the genome. The authors specifically and repeatedly state that the transcriptional start site is located only 281 bp upstream from the translational start site (i.e., the MCT1 coding region) and this is reinforced and illustrated by the contiguous DNA sequence shown in their Fig. 1. We want make it clear that these data are incorrect and ignore the large (26 kb) intron that interrupts the MCT1 5'-UTR (3). The presence of this intron is clearly indicated by a database blast of the MCT1 5'-RACE sequence, which shows that the sequence of the MCT1 message and that of the MCT1 genomic DNA diverge 45 bp upstream of the MCT1 coding region and only reconverge ~26 kb upstream in the genomic DNA. Moreover, this arrangement is described prominently in our prior publication detailing the first isolation of the MCT1 gene promoter and determination of the gene structure of human MCT1 (3). It is also confirmed by the latest human genome-sequencing assembly. Accordingly, it is unfortunate that Hadjiagapiou et al. appear to have overlooked this and report that the MCT1 promoter lies >26 kb downstream from where it is actually located in the genome. We feel that this is misleading and will of course confuse future study of the regulation of MCT1 expression.

We also want to make readers aware of a number of factual errors that Hadjiagapiou et al. report regarding our previously published data of the isolation and characterization of the MCT1 gene promoter. First, Hadjiagapiou et al. state that the MCT1 gene promoter was isolated from AA/C2 cells. We assume Hadjiagapiou et al. meant to refer to AA/C1 colonic epithelial cells; however, this too is incorrect because the MCT1 promoter was in fact isolated by our screening of a chromosome 1-specific genomic DNA cosmid library derived directly from human tissue (3). Second, Hadjiagapiou et al. report that our determination of the MCT1 transcription initiation site employed total RNA from AA/C1 cells, and they use this to rationalize their assignment of the transcriptional start site to an alternative downstream location in Caco-2 cells. This is also incorrect. Our determination of the MCT1 transcription initiation site employed total RNA derived directly from human colonic tissue and not AA/C1 cells. Once again, this is clearly detailed in the paper they refer to (3).

We hope the above information and comments will serve to clarify the scientific record, reduce any confusion for those interested in the regulation of MCT1 expression, and so facilitate advancing future research in this area.

REFERENCES

  1. Cuff M, Dyer J, Jones M, and Shirazi-Beechey SP. The human colonic monocarboxylate transporter isoform 1: its potential importance to colonic tissue homeostasis. Gastroenterology 128: 676–686, 2005.[CrossRef][ISI][Medline]
  2. Cuff MA, Lambert DW, and Shirazi-Beechey SP. Substrate-induced regulation of the human colonic monocarboxylate transporter, MCT1. J Physiol 539: 361–371, 2002.[Abstract/Free Full Text]
  3. Cuff MA, and Shirazi-Beechey SP. The human monocarboxylate transporter, MCT1: genomic organization and promoter analysis. Biochem Biophys Res Commun 292: 1048–1056, 2002.[CrossRef][ISI][Medline]
  4. Hadjiagapiou C, borthakur A, dahdal R, Gill R, Malakooti J, Ramaswamy K, and Dudeja PK. Role of USF1 and USF2 as potential repressor proteins for human intestinal monocarboxylate transporter 1 promoter. Am J Physiol Gastrointest Liver Physiol 228: G1118–G1126, 2005.[CrossRef]
  5. Halestrap AP and Price NT. The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation. Biochem J 343: 281–299, 1999.[CrossRef][ISI][Medline]
  6. Lambert DW, Wood IS, Ellis A, and Shirazi-Beechey SP. Molecular changes in the expression of human colonic nutrient transporters during the transition from normality to malignancy. Br J Cancer 86: 1262–1269, 2002.[CrossRef][ISI][Medline]
  7. Ritzhaupt A, Ellis A, Hosie KB, and Shirazi-Beechey SP. The characterization of butyrate transport across pig and human colonic luminal membrane. J Physiol 507: 819–830, 1998.[Abstract/Free Full Text]
  8. Ritzhaupt A, Wood IS, Ellis A, Hosie KB, and Shirazi-Beechey SP. Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport L-lactate as well as butyrate. J Physiol 513: 719–732, 1998.[Abstract/Free Full Text]

 

REPLY

Pradeep K Dudeja, Jaleh Malakooti, and K. Ramaswamy

Section of Digestive Diseases and Nutrition
Department of Medicine
University of Illinois at Chicago
Chicago, Illinois 60612

To the Editor: We would like to address the points raised in the letter of Drs. Cuff and Shirazi-Beechey and comments such as "repeating their work," "errors in the data," and "misquotation and misrepresentations of prior publications." To set the record straight and to bring to your attention all the facts pertaining to the human MCT1 promoter cloning, we would like to provide a chronological update of our work. We would also like to assure you and Drs. Cuff and Shirazi-Beechey that the promoter data given in this manuscript is not simply repeating their work after their publication of 2002 (4); rather, we had initiated the cloning work much earlier than their group. Furthermore, what are termed as "errors in our data" are actually just the standard way of presenting the data. Finally, what is termed as "misquotation and misrepresentation of prior publications" happen to be relatively minor, honest omissions, for which we truly apologize.

We are certainly aware of and recognize the importance of this field of study focused at elucidating the cellular and molecular mechanisms of human intestinal short-chain fatty acid (SCFA) absorption and their regulation. Our laboratories had made significant contributions in this important area of research and have been instrumental in establishing the techniques for the purification of the human intestinal plasma membranes (10, 12, 16) and establishing the existence of carrier-mediated mechanisms of SCFA transport across the human ileal luminal and colonic antipodal plasma membranes (11, 13, 17). As early as in 1996 (9), we were interested in investigating MCT1 as a potential candidate gene involved in intestinal butyrate uptake; therefore, we submitted an abstract to the American Gastroenterological Association (AGA) in December 1996 and first presented our studies of MCT1 expression in the human tissues including the intestine at the annual AGA meeting in May 1997. Since then, several publications and presentations from our laboratories have demonstrated the role of MCT1 in human intestinal SCFA absorption as well as its regulation both at the membrane level as well as at the transcriptional level. Some of these important findings include the regulation of butyrate uptake by protein kinase C (1), modulation of butyrate transport in response to infection by enteropathogenic Eschericia coli involving changes in surface expression of MCT1 (2), regulation of butyrate transport by serotonin (6) and somatostatin (15) involving alterations in MCT1 levels at the luminal membranes, expression of various MCT isoforms along the length of the human intestine (5, 9), MCT1 promoter cloning (8), and regulation of the MCT1 promoter by USF1 and -2 (7), NF-{kappa}B (3), and AP2 (14). We also certainly recognize the important contributions of Dr. Shirazi-Beechey in this area of investigation. We sincerely believe that there is much more to be achieved and that there is room for several investigators to coexist even in a very competitive environment.

Chronology of MCT1 promoter cloning and justification for our presentation of the MCT1 promoter cloning data. Drs. Cuff and Shirazi-Beechey in their letter have claimed that they had published the MCT1 promoter sequence in April 2002 (4) and that the first part of our manuscript in question includes simply repeating their work. However, it should be obvious from the chronology presented below that our studies of MCT1 promoter cloning were initiated and submitted for presentation/publication earlier than their publication. Our studies of the human MCT1 promoter cloning were also published in April 2002 as an abstract (8). These data were presented in May of 2002 at the Annual AGA conference, and this particular abstract was submitted to AGA (December 2001) much before even Dr. Shirazi-Beechey submitted her manuscript for publication in Biochem Biophys Res Commun (March 2002). I would like you and the readers to know that the MCT1 promoter cloning began in our laboratories in 1999. This is evident from our submission of the partial sequence of the promoter to GenBank on February 29, 2000 (Accession #AF239919). It should be noted that Drs. Cuff and Shirazi-Beechey completely omitted referring to this MCT1 promoter sequence deposited in GenBank in their manuscript (4). It took us about three years until we were able to obtain the full-length promoter sequence. Drs. Cuff and Shirazi-Beechey have claimed that there are errors in our reporting of the data, because we report that the transcription initiation site is 281 bp upstream of the translation initiation site, and we have ignored the presence of a 26-kb intron in the 5'-UTR. Of course we were aware of the presence of the intron. The sequence of human chromosome 1 (AL158844 [GenBank] ; April 2001) was available well before the publication of their article. As we have cited in our manuscript, the primer used to obtain the promoter sequence was based on the nucleotide sequence of MCT1 genomic DNA (AL158844 [GenBank] ; April, 2001) upstream from our reported cDNA (AF239919 [GenBank] ); therefore, we were aware of the presence of an intron that disrupts the 5'-UTR. However, because the focus of our studies (7) was not the characterization of the genomic organization of the MCT1 gene, we did not refer to any of the MCT1 introns. Whenever one talks about the location of the transcription initiation site, it is the standard practice not to include the intron sequences interrupting the 5'-UTR in the genomic DNA (in fact, the UTR is defined as a part of an mRNA molecule not coding for a protein), and that is exactly what was done in our manuscript that is being termed as "errors in the data." We have simply stated that the transcription initiation site is 281 nt upstream from the translation start site on the nucleotide sequence that we have reported. We can cite numerous manuscripts published in outstanding journals where the intron sequences are not included in describing the transcription initiation site. So in our view, it is a very unfortunate and serious comment to call a standard way of presentation of data as "errors in the data." The reason for our presentation of the first two figures in the above-mentioned manuscript pertaining to MCT1 promoter cloning was for us to show how we obtained the MCT1 promoter for our MCT1 regulation studies by USF1 and -2. The regulation by USFs was the main focus of the concerned manuscript, as is also evident from its title. Although our group and Dr. Shirazi-Beechey’s group cloned the promoter almost parallel to each other, but they were able to publish it before us, we went several steps ahead to examine the regulation of MCT1 promoter, and that is why the overall promoter cloning part was only a small portion of this manuscript. This was another reason that we did not deposit our full promoter sequence in the GenBank. Also in the manuscript, we never claimed that this was the first ever cloning of the MCT1 promoter by us; rather we made several references to the MCT1 promoter cloning by Dr. Shirazi-Beechey. Of course we misquoted the source of their genomic DNA as well as RNA to be the human colonic AA/C1 cells, which were used by them for promoter expression studies. This was an honest omission on our part, and we truly apologize for that. If needed, we would be happy to put an addendum to correct the records.

We believe that our responses to Dr. Shirazi-Beechey’s letter and concerns are important to set the record straight for the scientific community working in this area of research and should clarify confusions, if any, created by the concerned letter with respect to the "errors in the data."

REFERENCES

  1. Alrefai WA, Tyagi S, Gill RK, Saksena S, Hadjiagapiou C, Mansour F, Ramaswamy K, and Dudeja PK. Regulation of butyrate uptake in Caco-2 cells by phorbol 12-myristate 13 acetate, PMA. Am J Physiol Gastrointest Liver Physiol 286: G197–G203, 2004.[Abstract/Free Full Text]
  2. Borthakur A, Gill RK, Hodges K, Ramaswamy K, Hecht G, and Dudeja PK. Enteropathogenic Eschericia coli (EPEC) inhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level. Am J Physiol Gastrointest Liver Physiol. In press; 10.1152/ajpgi.00302.2005.
  3. Borthakur A, Saksena S, Gill R, Alrefai WA, Ramaswamy K, and Dudeja PK. Regulation of human intestinal monocarboxylate transporter 1 promoter by butyrate (Abstract). Gastroenterology 128: A45, 2005.[CrossRef]
  4. Cuff MA and Shirazi-Beechey SP. The human monocarboxylate transporter, MCT1: genomic organization and promoter analysis. Biochem Biophys Res Commun 292: 1048–1056, 2002.[CrossRef][ISI][Medline]
  5. Gill R, Hadjiagapiou C, Alrefai WA, Saksena S, Carrol R, Goldstein J, Ramaswamy K, and Dudeja PK. Expression and membrane localization of MCT isoforms along the length of the human intestine. Am J Physiol Cell Physiol, May 2005; doi:10.1152/ajpcell.00112.2005.
  6. Gill R, Saksena S, Alrefai WA, Tyagi S, Ramaswamy K, and Dudeja PK. Serotonin inhibits MCT1 activity by retrieval of the tansporter from the apical membranes in Caco-2 cells (Abstract). Gastroenterology 126: A145, 2004.[CrossRef]
  7. Hadjiagapiou C, Borthakur A, Dohdal RY, Gill RK, Malakooti J, Ramaswamy K, and Dudeja PK. The role of upstream stimulatory factors 1 and 2 (USF1 and USF2) in regulation of human monocarboxylate transporter 1 (MCT1) promoter. Am J Physiol Gastrointest Liver Physiol 288: G1118–G1126, 2005.[Abstract/Free Full Text]
  8. Hadjiagapiou C, Dahdal RY, Ramaswamy K, and Dudeja PK. Molecular cloning of the human monocarboxylate transporter 1 (MCT1) promoter (Abstract). Gastroenterology 122: A536, 2002.
  9. Hadjiagapiou C, Hausman A, Dudeja PK, Layden TJ, Davidson NO, and Ramaswamy K. Distribution of monocarboxylate transporter 1 (MCT 1) in human tissues (Abstract). Gastroenterology 111: A367, 1997.
  10. Harig JM, Dudeja PK, Knaup SM, Shoshara J, Ramaswamy K., and Brasitus TA. Apical plasma membrane vesicles formed from organ donor colon demonstrate Na+ and H+ conductances and Na+/H+ exchange. Biochem Biophys Res Commun 167: 438–443, 1990.[CrossRef][ISI][Medline]
  11. Harig JM, Ng EK, Dudeja PK, Brasitus TA, and Ramaswamy K. Transport of n-butyrate into human colonic luminal membrane vesicles. Am J Physiol Gastrointest Liver Physiol 271: G415–G422, 1996.[Abstract/Free Full Text]
  12. Harig JM, Rajendran VM, Barry JA, and Ramaswamy K. D-Glucose and L-leucine transport by human intestinal brush-border membrane vesicles. Am J Physiol Gastrointest Liver Physiol 256: G618–G623, 1989.[Abstract/Free Full Text]
  13. Harig JM, Soergel KH, Barry JA, and Ramaswamy K. Transport of propionate by human ileal brush-border membrane vesicles. Am J Physiol Gastrointest Liver Physiol 260: G776–G782, 1991.[Abstract/Free Full Text]
  14. Saksena S, Gill RK, Tyagi S, Alrefai WA, Malakooti J, Ramaswamy K, and Dudeja PK. Regulation of MCT1 promoter by AP2 transcription factor in Caco-2 cells (Abstract). Gastroenterology 128: A367, 2005.
  15. Theegala S, Tyagi S, Gill R, Alrefai WA, Dwivedi A, Ramaswamy K, and Dudeja PK. Somatostatin upregulates butyrate uptake in Caco-2 cells via recycling of monocarboxylate transporter 1 (MCT1)(Abstract). Gastroenterology 128: A368, 2005.
  16. Tyagi S, Joshi V, Alrefai WA, Gill RK, Ramaswamy K, and Dudeja PK. Evidence for a Na+-H+ exchange across human colonic basolateral plasma membranes purified from organ donor colons. Dig Dis Sci 45: 2282–2289, 2000.[CrossRef][ISI][Medline]
  17. Tyagi S, Venugopalakrishnan J, Ramaswamy K, and Dudeja PK. Mechanism of n-butyrate uptake in the human proximal colonic basolateral membranes. Am J Physiol Gastrointest Liver Physiol 282: G676–G682, 2002.[Abstract/Free Full Text]




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