Characterization of a Novel Airway Epithelial Cell-specific Short Chain Alcohol Dehydrogenase/Reductase Gene Whose Expression Is Up-regulated by Retinoids and Is Involved in the Metabolism of Retinol

Abstract

Multiple retinoic acid responsive cDNAs were isolated from a high density cDNA microarray membrane, which was developed from a cDNA library of human tracheobronchial epithelial cells. Five selected cDNA clones encoded the sequence of the same novel gene. The predicted open reading frame of the novel gene encoded a protein of 319 amino acids. The deduced amino acid sequence contains four motifs that are conserved in the short-chain alcohol dehydrogenase/reductase (SDR) family of proteins. The novel gene shows the greatest homology to a group of dehydrogenases that can oxidize retinol (retinol dehydrogenases). The mRNA of the novel gene was found in trachea, colon, tongue, and esophagus. In situhybridization of airway tissue sections demonstrated epithelial cell-specific gene expression, especially in the ciliated cell type. Both all-trans-retinoic acid and 9-cis-retinoic acid were able to elevate the expression of the novel gene in primary human tracheobronchial epithelial cells in vitro. This elevation coincided with an enhanced retinol metabolism in these cultures. COS cells transfected with an expression construct of the novel gene were also elevated in the metabolism of retinol. The results suggested that the novel gene represents a new member of the SDR family that may play a critical role in retinol metabolism in airway epithelia as well as in other epithelia of colon, tongue, and esophagus. Multiple retinoic acid responsive cDNAs were isolated from a high density cDNA microarray membrane, which was developed from a cDNA library of human tracheobronchial epithelial cells. Five selected cDNA clones encoded the sequence of the same novel gene. The predicted open reading frame of the novel gene encoded a protein of 319 amino acids. The deduced amino acid sequence contains four motifs that are conserved in the short-chain alcohol dehydrogenase/reductase (SDR) family of proteins. The novel gene shows the greatest homology to a group of dehydrogenases that can oxidize retinol (retinol dehydrogenases). The mRNA of the novel gene was found in trachea, colon, tongue, and esophagus. In situhybridization of airway tissue sections demonstrated epithelial cell-specific gene expression, especially in the ciliated cell type. Both all-trans-retinoic acid and 9-cis-retinoic acid were able to elevate the expression of the novel gene in primary human tracheobronchial epithelial cells in vitro. This elevation coincided with an enhanced retinol metabolism in these cultures. COS cells transfected with an expression construct of the novel gene were also elevated in the metabolism of retinol. The results suggested that the novel gene represents a new member of the SDR family that may play a critical role in retinol metabolism in airway epithelia as well as in other epithelia of colon, tongue, and esophagus. retinoic acid retinoic acid receptor retinoid X receptor retinol dehydrogenase SDR, short chain (alcohol) dehydrogenase/reductase TBE, cell-specific human retinol dehydrogenase polymerase chain reaction untranslated region high performance liquid chromatography base pair(s) Vitamin A (retinol) and its metabolites (retinoids) are essential to the development and maintenance of the airway epithelial phenotype (1Huang T.H. Ann D.K. Zhang Y.J. Chang A.T. Crabb J.W. Wu R. Am. J. Respir. Cell Mol. Biol. 1994; 10: 192-201Crossref PubMed Scopus (26) Google Scholar, 2Jetten A.M. Rearick J.I. Smits H.L. Biochem. Soc Trans. 1986; 14: 930-933Crossref PubMed Scopus (33) Google Scholar, 3Wu, R. (ed). (1997) in Growth and Differentiation of Tracheobronchial Epithelial Cells. Lung Growth and Development (McDonald, J. A., ed) pp. 211-241,Marcel Dekker, Inc., New YorkGoogle Scholar, 4Jetten A.M. Nervi C. Vollberg T.M. J. Natl. Cancer Inst. Monogr. 1992; 13: 93-100PubMed Google Scholar). Epithelial tissues, including the airway epithelia, are vitamin A target tissues that require retinoids (5Wolbach S.B. Howe P.R. J. Exp. Med. 1925; 42: 753-781Crossref PubMed Scopus (1094) Google Scholar, 6Fell H.B. Mellanby E. J. Physiol. 1953; 119: 470-488Crossref PubMed Scopus (185) Google Scholar, 7De Luca L.M. Roop D. Huang F.L. Acta Vitamin. Enzymol. 1985; 7 (suppl.): 13-20PubMed Google Scholar). Vitamin A metabolites, such as all-trans-retinoic acid (RA)1 and 9-cis-RA, are important regulators for gene transcription as the ligands for various transcriptional factors of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families (for review, see Ref. 8Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2605) Google Scholar). Extensive progress has been made in determining the specificity of these RA metabolites with the interactions with these RARs and RXRs (8Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2605) Google Scholar, 9Allenby G. Bocquel M.T. Saunders M. Kazmer S. Speck J. Rosenberger M. Lovey A. Kastner P. Grippo J.F. Chambon P. Levin A.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 30-34Crossref PubMed Scopus (671) Google Scholar). As compared with these RA metabolites, retinol is not as potent as those RAs in terms of the interactions with these receptors and in terms of transcriptional regulation (10Connor M.J. Smit M.H. Biochem. Pharmacol. 1987; 36: 919-924Crossref PubMed Scopus (39) Google Scholar, 11Kurlandsky S.B. Xiao J.H. Duell E.A. Voorhees J.J. Fisher G.J. J. Biol. Chem. 1994; 269: 32821-32827Abstract Full Text PDF PubMed Google Scholar). Thus, it was suggested that retinol has to be metabolized to various RAs to exert its biological activity (11Kurlandsky S.B. Xiao J.H. Duell E.A. Voorhees J.J. Fisher G.J. J. Biol. Chem. 1994; 269: 32821-32827Abstract Full Text PDF PubMed Google Scholar). However, the mechanism by which vitamin A and its metabolites exert this activity to regulate airway development and the maintenance of mucociliary functions in epithelium is unknown. As lipid soluble compounds, retinoids can readily cross the membrane into any cell in the body from the plasma circulation. The major form of retinoid in plasma circulation is all-trans-retinol at about 1.5–2.0 μm (12Ribaya-Mercado J.D. Mazariegos M. Tang G. Romero-Abal M.E. Mena I. Solomons N.W. Russell R.M. Am. J. Clin. Nutr. 1999; 69: 278-284Crossref PubMed Scopus (39) Google Scholar, 13Booth S.L. Tucker K.L. McKeown N.M. Davidson K.W. Dallal G.E. Sadowski J.A. J. Nutr. 1997; 127: 587-592Crossref PubMed Scopus (88) Google Scholar). However, the level of RA in the plasma is around 4–14 nm (14De Leenheer A.P. Lambert W.E. Claeys I. J. Lipid Res. 1982; 23: 1362-1367Abstract Full Text PDF PubMed Google Scholar, 15Eckhoff C. Nau H. J. Lipid Res. 1990; 31: 1445-1454Abstract Full Text PDF PubMed Google Scholar), which may be insufficient to supply the needed cellular RA level for its biological activity in vitamin A target tissues and cells. In addition, in some vitamin A target tissues and cells, such as skin and keratinocytes (16Jurukovski V. Markova N.G. Karaman-Jurukovska N. Randolph R.K. Su J. Napoli J.L. Simon M. Mol. Genet. Metab. 1999; 67: 62-73Crossref PubMed Scopus (51) Google Scholar), the access to blood vessels can be quite limited. Thus, there is a need in these vitamin A target cells for an efficient machinery to transport and to metabolize plasma retinol into various RA metabolites. The production of RA from retinol can take place within the cell if the cell contains enzymes that can sequentially oxidize retinol to retinaldehyde and retinaldehyde to RA. The reversible oxidation of retinol to retinaldehyde has been suggested to be the rate-limiting step in the metabolism of retinol to retinoic acid and therefore the step most likely to be tightly regulated by the cell (17Chai X. Boerman M.H. Zhai Y. Napoli J.L. J. Biol. Chem. 1995; 270: 3900-3904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 18Napoli J.L. Posch K.P. Fiorella P.D. Boerman M.H. Biomed. Pharmacother. 1991; 45: 131-143Crossref PubMed Scopus (102) Google Scholar). The second step, the oxidation of retinaldehyde to retinoic acid, seems to be irreversible (reviewed in Ref. 19Duester G. Biochemistry. 1996; 35: 12221-12227Crossref PubMed Scopus (234) Google Scholar). Consistent with the first step in this model of retinol metabolism in vitamin A target cells, there have been reports of multiple enzymes with retinol dehydrogenase activities that are isoform-specific or cell type-specific. These enzymes include (but are not limited to) RODH-I (17Chai X. Boerman M.H. Zhai Y. Napoli J.L. J. Biol. Chem. 1995; 270: 3900-3904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), RODH-II (20Chai X. Zhai Y. Popescu G. Napoli J.L. J. Biol. Chem. 1995; 270: 28408-28412Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), RODH-III (21Chai X. Zhai Y. Napoli J.L. Gene ( Amst. ). 1996; 169: 219-222Crossref PubMed Scopus (63) Google Scholar), RDH-4 (22Gough W.H. VanOoteghem S. Sint T. Kedishvili N.Y. J. Biol. Chem. 1998; 273: 19778-19785Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), hRDH-E (16Jurukovski V. Markova N.G. Karaman-Jurukovska N. Randolph R.K. Su J. Napoli J.L. Simon M. Mol. Genet. Metab. 1999; 67: 62-73Crossref PubMed Scopus (51) Google Scholar), 9- and 11-cRDH (23Mertz J.R. Shang E. Piantedosi R. Wei S. Wolgemuth D.J. Blaner W.S. J. Biol. Chem. 1997; 272: 11744-11749Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 24Gamble M.V. Shang E. Zott R.P. Mertz J.R. Wolgemuth D.J. Blaner W.S. J. Lipid Res. 1999; 40: 2279-2292Abstract Full Text Full Text PDF PubMed Google Scholar, 25Gamble M.V. Mata N.L. Tsin A.T. Mertz J.R. Blaner W.S. Biochim. Biophys. Acta. 2000; 1476: 3-8Crossref PubMed Scopus (32) Google Scholar), RDH-5 (26Simon A. Hellman U. Wernstedt C. Eriksson U. J. Biol. Chem. 1995; 270: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 27Driessen C.A. J.J. Sci. 1995; 36: Google Scholar, A. J. S. Eriksson U. 1996; 36: PubMed Scopus Google Scholar, J. X. Eriksson U. Napoli J.L. Biochem. J. 1999; PubMed Scopus Google Scholar), X. Zhai Y. Napoli J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), and J. X. Napoli J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). of these enzymes are of the short chain (alcohol) dehydrogenase/reductase (SDR) gene in the alcohol dehydrogenase and families have been in the oxidation of retinaldehyde to retinoic acid, which is the second step in the metabolism of retinol to retinoic are reports of dehydrogenases that this step T. P. A.M. Biochem. Cell Biol. 1998; PubMed Scopus Google Scholar, I. G. Genet. 1999; PubMed Google Scholar, P. Chambon P. P. 1997; PubMed Scopus Google Scholar, P. X. Napoli J.L. Biochim. Biophys. Acta. 1997; PubMed Scopus Google Scholar, P. X. Napoli J.L. Gene ( Amst. ). 1997; PubMed Scopus Google Scholar, R. Biol. 2000; PubMed Scopus Google Scholar, D. P. P. J. Biochem. 1996; PubMed Scopus Google Scholar). at of these retinaldehyde dehydrogenases is in epithelial cells in with a role in the of retinaldehyde to retinoic acid in these cells. However, the critical that retinol to retinaldehyde is in airway epithelial cells. In this the and of a novel airway epithelial cell-specific SDR gene from a cDNA library from primary human tracheobronchial epithelial cells, a a high density cDNA microarray Five selected RA responsive cDNA clones encoded the same novel gene. The of the novel cDNA sequence a protein that contains amino acid motifs that are conserved in the SDR family of the SDR the novel gene is most to the retinol that this novel gene represents a new member of the SDR family that may play a critical role in retinoid metabolism in airway airway tissues were from the of at with The for human tissues has been and by the cells were isolated from these tissues by a and in a as Wu R. J. 1991; 13: Scopus (39) Google Scholar). cells were in tissue or with or in or with The was from that in Ref. Wu R. J. 1991; 13: Scopus (39) Google the the and in a of This airway epithelial cells to and mucociliary in especially the and cells were in the for 7 and the were in the and liquid for the in and were for 7 to and RA at nm or at various was as in the various of in was isolated from cells by a step acid P. N. Biochem. 1987; PubMed Scopus Google Scholar). COS cells were in cDNA clones were from primary human cells that been for an in the the and such an in the human cells into a mucociliary epithelium that in The cDNA clones were a the cDNA developed a high density microarray membrane membrane, This cDNA high density microarray membrane was with cDNA and from and of primary human cells, as Wu R. Huang M.H. Chang Wu 1998; PubMed Scopus Google Scholar). a of or clones were selected for These the of to the of the in these to for of to as to the of a novel in some in situhybridization to cell gene was at the sequence was with Inc., and with the homology to in was by the or at the for The was by the with the sequence with of the same cDNAs that the novel gene are in the with the at the of the multiple any of these cDNAs as with and a of the cDNA this was to the region of the novel cDNA was in frame to a in the This was by a of the The was the of the novel the was a The was with and into which been with the same The was by of were a and to a was to the membrane by The from the novel cDNA was with to a activity of with a The were in at for a of by in the same at for were with for at and with for at the second were for if in and for various at were to for various the novel expression in tissues, of from various tissues were and was as tissues were from at the of The sequence of the cDNA was by from human the with nm an and the chain to the A from the region of the gene was in with the the same and the from the library were to to the The were with or to and The were in with and to the Inc., to and was as J.L. J. Google Scholar). A was Inc., deduced amino acid sequence of The was to multiple to its and were as (1Huang T.H. Ann D.K. Zhang Y.J. Chang A.T. Crabb J.W. Wu R. Am. J. Respir. Cell Mol. Biol. 1994; 10: 192-201Crossref PubMed Scopus (26) Google Scholar). The specificity of the was by and cells were as T.H. J.A. Wu R. PubMed Scopus Google Scholar, Y.J. L.M. J.H. 1982; 31: Full Text PDF PubMed Scopus Google Scholar, R. Wu J. Physiol. 1986; 127: PubMed Scopus (39) Google Scholar). protein were by the of the protein were to to PubMed Scopus Google Scholar). were or to the with a at was a Inc., and the primary and cells were in or with some of the cells were for and protein The cells were with μm retinol or the of the for COS cells were transfected with or the from group was for protein The COS cells were to retinol or an of the for cells were in by into of Cell were at Cell were in a The were to cell A of was for protein by the was to the were by of of of by of and were at for the was to an and in the The was in of alcohol and for the and of and were a cell were for retinoids an of alcohol The was of were by at these the has a of for RA of at a of for retinol are retinoids are as The retinoid was from to the of the and from the of are from The of is an of retinol dehydrogenase activity the of dehydrogenase activity is which is to RA. and retinoids were made in and by and J.A. The and New Scholar). The of retinoid was to retinoid that were by of the the The of the retinoids was by determining the of isolated of the and were of retinoids of cDNA clones were selected as clones for Y. P. M. M. J. M. R. Y. P. C. C. M. and R. in that the of clones were regulated by the was a and the other were to be by of the encoded novel cDNA Five of the novel and and cDNA at the of a novel gene. A of these clones The cDNA sequence of this novel gene was by The cDNA contains a a predicted open reading frame of which is the and a The of the open reading frame a of 319 amino with a predicted of selected cDNA clones have the same and the clones in the of the a transcriptional These that the clones are cDNAs from a the of the cDNA of the various of the novel cDNA sequence as homology at the level to of the SDR gene A of the with the cDNA sequence an to clones as retinol dehydrogenase and and retinol dehydrogenase and other the has been for these of the with the clones that the clones were short in sequence at the of the novel gene are in the of of the which may In to the homology to clones the cDNA also homology to multiple of the SDR This various dehydrogenases and as well as the SDR J. X. Napoli J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google of various and isoform-specific retinol A of the deduced amino acid sequence of the gene the of four which are conserved in of SDR gene family M. H. Exp. Med. Biol. 1995; PubMed Scopus (26) Google Scholar). These motifs are the amino acid at at at and at amino acid the a of various SDR gene family was developed The in the the and include for which there is a of its a This that the gene is a novel member of the SDR gene the the novel gene was to the SDR J. X. Napoli J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google and other SDR this the gene encoded in the clones as a cell-specific dehydrogenase gene gene expression, from tissues were demonstrated the of in trachea, colon, tongue, and In these tissues, a was This was in to the Ref. M. R. R. J.A. A of in to and The of the tissues and cell gene was sections from airway and As in the epithelial cells with the to the gene. The was to the ciliated cell This was in the airway epithelium including that of the region not was in the region not The with demonstrated gene expression in human cells was regulated by As in was elevated in of the of the of the suggested that the elevation was most in the demonstrated a of in by and and in elevation the RA were for to elevate at A demonstrated that the by was an that be A to the deduced amino acid was The of the to the protein was a in which a protein was in COS cells. The of that is to transfected cells The also this The is not by and it is of with the not The was to and human tissue sections the tissue was most of the ciliated cells, there was for other epithelial cell such as cells or the cells the These the The of the of the not the These are with tissue of or sections with not any not the of this found the metabolism of all-trans-retinol in primary human was by an as the of retinaldehyde by the retinoid were to retinaldehyde in any of cell In at other the retinaldehyde in the metabolism of retinol to retinoic acid was not by (10Connor M.J. Smit M.H. Biochem. Pharmacol. 1987; 36: 919-924Crossref PubMed Scopus (39) Google Scholar). retinaldehyde is to and of its Biochem. PubMed Scopus Google Scholar, Biochim. Biophys. Acta. PubMed Scopus Google Scholar). therefore retinoic acid as the of the as the of the reversible oxidation of retinol to the which is to RA. As in the RAs as the metabolites of all-trans-retinol in primary human be this the metabolism of all-trans-retinol in primary human cells was and compared in that were in or As have demonstrated of cells with the of and 7 of from and with demonstrated that the of protein are also elevated 7 the of of RA retinol of retinoic acid cells 7 As in and were elevated in that been in and with The level of the same in the all-trans-retinol The level of also the same in in the of retinol These results suggested that human cells were able to metabolize retinol that elevated expression of the retinol dehydrogenase gene the role of in retinol a in COS cells, not and not to a of can and that COS cells transfected with the construct COS cells transfected with the not A As in COS cells the to metabolize retinol with the expression have a airway epithelial cell-specific short chain alcohol dehydrogenase with a role in the metabolism of from of human cells. The gene was found as of demonstrated that the expression of this gene be elevated by various This elevation in a and expression of this gene is and cell type-specific. Both and in demonstrated that the expression of this gene in airway tissue at the airway especially in the ciliated cell type. This gene is also in colon, and in sections demonstrated the expression of this gene at the epithelial cell In addition, the gene was to the SDR gene family and to retinol dehydrogenase and have also demonstrated a the metabolism of retinol in and the expression of this gene These results that the found gene is a major in the regulation of all-trans-retinol metabolism in various epithelial tissues, including that of the are of to the role of this new gene in the metabolism of retinol. of is the of motifs that are of SDR gene family The first at amino acid is a as the A. P.D. The and New that is important in The sequence at amino acid has been suggested as the of dehydrogenase/reductase the functions of the at amino acid and at amino acid are these motifs are quite conserved in the SDR gene In addition, the demonstrated that the gene is well into the SDR gene family with a to various SDR such as the dehydrogenase and multiple retinol of to the role of this gene in retinol metabolism is from a have a of the metabolism of retinol in with the expression of this gene to the expression of the gene in also an enhanced retinol metabolism in this However, retinoic acid is to the expression of in human a gene such as alcohol dehydrogenase also have been and have been for retinol metabolism the Thus, the may not be to the that functions as a retinol the role of this gene in the retinol transfected COS cells with an expression construct of COS cells the expression of the gene the that were COS cells were able to metabolize all-trans-retinol into RA. The with the to the retinol These results that this found gene is a major in the metabolism of retinol in with protein to the of this new gene. high homology to SDR gene family there are some this gene and the of this gene of the SDR gene family have a conserved amino acid sequence J. X. Napoli J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), gene not this of the SDR gene family have homology to other to any of homology is at the amino acid is that a new of retinol dehydrogenase that is from the SDR gene family This is by the in which that its of the Vitamin A is as an important vitamin that the and the development of airway were reports that a important role in the regulation of airway epithelial cell A.M. P. J.H. P. Biochem. J. 1999; PubMed Google Scholar). In addition, RARs and RXRs have for various RAs (8Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2605) Google Scholar, 9Allenby G. Bocquel M.T. Saunders M. Kazmer S. Speck J. Rosenberger M. Lovey A. Kastner P. Grippo J.F. Chambon P. Levin A.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 30-34Crossref PubMed Scopus (671) Google Scholar). the of the metabolites by airway epithelial cells be critical to the development of airway tissues and cell In this may play a critical role in the regulation of the cellular retinol from not this gene has a for a of retinol. In demonstrated that all-trans-retinol was metabolized into and by cells with expression of this gene. protein and a the specificity for to be of retinol tissues in various tissues and in human skin Napoli and to that RA is in in a of vitamin A target these RA activities are from those of alcohol dehydrogenase activities J.L. Posch K.P. Fiorella P.D. Boerman M.H. Biomed. Pharmacother. 1991; 45: 131-143Crossref PubMed Scopus (102) Google Scholar). the epithelia of the a retinol dehydrogenase to for the A. A. Eriksson U. J. Cell Sci. 1999; PubMed Google Scholar, A. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). and cells need retinol dehydrogenases of critical role in retinoid metabolism S. Piantedosi R. J. A. S.L. Blaner W.S. J. Lipid Res. 2000; Full Text Full Text PDF PubMed Google Scholar). The that multiple retinol dehydrogenases are in the (17Chai X. Boerman M.H. Zhai Y. Napoli J.L. J. Biol. Chem. 1995; 270: 3900-3904Abstract Full Text Full Text PDF PubMed Scopus (131) Google W.H. VanOoteghem S. Sint T. Kedishvili N.Y. J. Biol. Chem. 1998; 273: 19778-19785Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, J. M. Napoli J.L. 1999; PubMed Scopus Google Scholar, M.H. Napoli J.L. Biochemistry. 1995; PubMed Scopus Google a need for this that also a if not role in the retinoid metabolism in the In have a novel retinol dehydrogenase in the airway is in the of retinoic that this gene a critical role in the retinoid metabolism in airway epithelia by the first step in the to the of retinoic acid in Russell and Chang for the