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Hemoglobin Is Expressed by Alveolar Epithelial Cells

2006; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês

10.1074/jbc.m509314200

1083-351X

Danforth A. Newton, K. Murali Krishna Rao, Richard A. Dluhy, John E. Baatz,

Erythrocyte Function and Pathophysiology

Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the α- and β-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both α- and β-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung. Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the α- and β-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both α- and β-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung. The concept of hemoglobin as solely a carrier protein of O2 and CO2 in erythroid cells has undergone revision in recent years. Heme-binding proteins with the globin structural motif are found throughout the natural world, in both prokaryotes and eukaryotes (1Hardison R.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5675-5679Crossref PubMed Scopus (290) Google Scholar, 2Vinogradov S.N. Hoogewijs D. Bailly X. Arredondo-Peter R. Guertin M. Gough J. Dewilde S. Moens L. Vanfleteren J.R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11385-11389Crossref PubMed Scopus (130) Google Scholar). Many of these proteins are involved in oxygen storage or transport, but all discovered thus far also function in binding products of nitric oxide (NO) 2The abbreviations used are: NO, nitric oxide; AE1, erythrocyte anion exchanger; ATI, alveolar type I cell; ATII, alveolar type II cell; FBS, fetal bovine serum; HbA, hemoglobin A heterotetramer; HBA, α-globin; HBB, β-globin; HBQ, θ-globin; MEL, mouse erythroleukemia cell line; qPCR, quantitative real-time PCR; RT, reverse transcription. metabolism. Vertebrate hemoglobin itself has been clearly shown to not only scavenge NO and its metabolic derivatives, but also to transport and generate NO, releasing the gas in tissues to dilate blood vessels (3Cosby K. Partovi K.S. Crawford J.H. Patel R.P. Reiter C.D. Martyr S. Yang B.K. Waclawiw M.A. Zalos G. Xu X. Huang K.T. Shields H. Kim-Shapiro D.B. Schechter A.N. Cannon 3rd, R.O. Gladwin M.T. Nat. Med. 2003; 9: 1498-1505Crossref PubMed Scopus (1438) Google Scholar, 4Huang Z. Shiva S. Kim-Shapiro D.B. Patel R.P. Ringwood L.A. Irby C.E. Huang K.T. Ho C. Hogg N. Schechter A.N. Gladwin M.T. J. Clin. Investig. 2005; 115: 2099-2107Crossref PubMed Scopus (429) Google Scholar). In fact, an emerging concept on the primordial function of hemoglobin-like proteins is that they protect cells against oxidative and nitrosative stress (5Crawford M.J. Goldberg D.E. J. Biol. Chem. 1998; 273: 12543-12547Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 6Gross S.S. Lane P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9967-9969Crossref PubMed Scopus (92) Google Scholar). Despite this widespread distribution of hemoglobin-like proteins (including myoglobin, neuroglobin, and cytoglobin), it has long been thought that hemoglobin itself is expressed only in cells of erythroid lineage in adult vertebrates. This notion may also be revised in the coming years, as the expression of non-erythroid hemoglobins has now been reported in activated macrophages from adult mice and cells of the lens (7Liu L. Zeng M. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6643-6647Crossref PubMed Scopus (123) Google Scholar, 8Wride M.A. Mansergh F.C. Adams S. Everitt R. Minnema S.E. Rancourt D.E. Evans M.J. Mol. Vis. 2003; 9: 360-396PubMed Google Scholar). Here we demonstrate that some types of respiratory epithelial cells of human, rat, or mouse origin, including alveolar type II (ATII) epithelial cells (also called type II pneumocytes) and Clara cells, also express hemoglobin. In vivo, these cells are the primary producers of pulmonary surfactant, essential for normal lung function as well as innate immunity (9Mallory Jr., G.B. Pediatr. Respir. Rev. 2001; 2: 151-158PubMed Google Scholar, 10Wright J.R. J. Clin. Investig. 2003; 111: 1453-1455Crossref PubMed Scopus (114) Google Scholar, 11Poynter S.E. LeVine A.M. Crit. Care Clin. 2003; 19: 459-472Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Also, ATII are stem cells that undergo significant phenotypic changes to terminally differentiate into alveolar type I (ATI) cells, which comprise much of the surface area of the alveolar epithelium and function in gas exchange (12Bishop A.E. Cell Prolif. 2004; 37: 89-96Crossref PubMed Scopus (96) Google Scholar, 13Williams M.C. Annu. Rev. Physiol. 2003; 65: 669-695Crossref PubMed Scopus (216) Google Scholar). Interestingly, although the exact details have yet to be elucidated, there is recent evidence that both ATI and ATII cells can be derived from hematopoietic stem cells, an intriguing discovery that may corroborate with our observation concerning hemoglobin expression (12Bishop A.E. Cell Prolif. 2004; 37: 89-96Crossref PubMed Scopus (96) Google Scholar, 13Williams M.C. Annu. Rev. Physiol. 2003; 65: 669-695Crossref PubMed Scopus (216) Google Scholar, 14Kotton D.N. Ma B.Y. Cardoso W.V. Sanderson E.A. Summer R.S. Williams M.C. Fine A. Development. 2001; 128: 5181-5188Crossref PubMed Google Scholar, 15Krause D.S. Theise N.D. Collector M.I. Henegariu O. Hwang S. Gardner R. Neutzel S. Sharkis S.J. Cell. 2001; 105: 369-377Abstract Full Text Full Text PDF PubMed Scopus (2482) Google Scholar). The hemoglobin genes found in mammals arose by duplication before mammalian divergence and are similarly arranged in both primates and rodents (16Hardison R. Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical Management.in: Steinberg M.H. Forget B.G. Higgs D.R. Nagel R.L. Cambridge University Press, Cambridge, UK2001: 95-116Google Scholar, 17Hardison R.C. Chui D.H. Riemer C. Giardine B. Lehvaslaiho H. Wajcman H. Miller W. Hemoglobin. 2001; 25: 183-193Crossref PubMed Scopus (29) Google Scholar). α and β genetic loci are found on separate chromosomes and each includes numerous genes that are active at various stages of development. In erythroid cells, the expression of these genes is tightly coordinated to produce functional hemoglobin heterotetramers consisting of two α-globin-like and two β-globin-like chains. The genes of the α globin cluster in human (with rodent orthologs) include: α HBA-1 and -2 (Hba-1 and -2), ζ HBZ (Hba-x), and θ HBQ (Hbq). The β-globin cluster includes: β HBB (Hbb-b1 and b2 major/minor), δ HBD (rodent pseudogene), γ HBG-1 and -2 (Hbb-bh1/z), and ∈ HBE (Hbb-y). Each locus also includes several pseudogenes (17Hardison R.C. Chui D.H. Riemer C. Giardine B. Lehvaslaiho H. Wajcman H. Miller W. Hemoglobin. 2001; 25: 183-193Crossref PubMed Scopus (29) Google Scholar). Adult hemoglobin protein found in erythrocytes primarily consists of HbA, an α2β2 tetramer with four bound heme prosthetic groups. Here we demonstrate that adult forms of hemoglobin mRNAs and polypeptides are indeed expressed by human and rodent pulmonary epithelial cells. Our novel finding may have enormous implications in the physiology and pathology of the lung because of the many defined roles inherent to the structures of the hemoglobin molecule and its derived peptides, including gas exchange, NO metabolism, blood pressure regulation, and protection against oxidative and nitrosative stress. Cell Culture—The human ATII adenocarcinoma cell line A549 (18Lieber M. Smith B. Szakal A. Nelson-Rees W. Todaro G. Int. J. Cancer. 1976; 17: 62-70Crossref PubMed Scopus (1027) Google Scholar) and Clara-like adenocarcinoma cell line H441 (19Brower M. Carney D.N. Oie H.K. Gazdar A.F. Minna J.D. Cancer Res. 1986; 46: 798-806PubMed Google Scholar) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Hyclone, Provo, UT). The mouse transformed ATII cell line MLE-15 (20Wikenheiser K.A. Vorbroker D.K. Rice W.R. Clark J.C. Bachurski C.J. Oie H.K. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11029-11033Crossref PubMed Scopus (292) Google Scholar) was cultured in HITES medium (RPMI 1640 with 5 μg/ml insulin, 10 μg/ml transferrin, 30 nm sodium selenite, 10 nm hydrocortisone, 10 nm β-estradiol, and 10 mm HEPES) supplemented with 2% FBS. The human melanoma cell line FO-1 (21Glovanella B.C. Stehlin J.S. Santamaria C. Yim S.O. Morgan A.C. Williams Jr., L.J. Leibovitz A. Fialkow P.J. Mumford D.M. J. Natl. Cancer Inst. 1976; 56: 1131-1142Crossref PubMed Scopus (75) Google Scholar) and mouse fibroblast cell line NIH-3T3 (22Jainchill J.L. Aaronson S.A. Todaro G.J. J. Virol. 1969; 4: 549-553Crossref PubMed Google Scholar) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS. The transformed murine erythroleukemia cell line MEL (23Volloch V. Schweitzer B. Rits S. Exp. Cell Res. 1987; 173: 38-48Crossref PubMed Scopus (9) Google Scholar) was cultured in Iscove's modified Dulbecco's medium supplemented with 20% FBS and 55 μm 2-mercaptoethanol. MEL cells were induced to differentiate by 48 h culture with 6 units/ml epoetin (recombinant erythropoietin, Amgen, Thousand Oaks, CA) and 3 mm hexamethylene bisacetamide (Sigma). Primary ATII cells were obtained as previously described from Sprague-Dawley rats (24Rao K.M. Porter D.W. Meighan T. Castranova V. Environ. Health Perspect. 2004; 112: 1679-1686Crossref PubMed Scopus (58) Google Scholar) or C57B/6 mice (25Rice W.R. Conkright J.J. Na C.L. Ikegami M. Shannon J.M. Weaver T.E. Am. J. Physiol. 2002; 283: L256-L264Crossref PubMed Scopus (142) Google Scholar) and cultured on Matrigel (BD Biosciences) in Dulbecco's modified Eagle's medium, 10% FBS containing 10 ng/ml keratinocyte growth factor (Peprotech, Rocky Hill, NJ) to maintain the ATII phenotype. Except where noted, all culture media and additives were obtained from Invitrogen. Cell lines were obtained from the American Type Culture Collection (Manassas, VA), except FO-1, kindly provided by Dr. Sebastiano Gattoni-Celli, Medical University of South Carolina, and MEL, kindly provided by Dr. Clark Brown, Medical University of South Carolina. Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 in air. PCR and Quantitative PCR—RNA was purified from all cell types using a QuickPrep Total RNA kit, and cDNA was generated from 3 μg of purified total RNA and an oligo(dT) primer using a First Strand Synthesis kit (both kits from Amersham Biosciences). Conventional RT-PCR was performed using Taq DNA polymerase (New England BioLabs, Beverly, MA) for 35 cycles (60 °C annealing temperature). Reaction products were analyzed by electrophoresis on 4-12% polyacrylamide Tris borate-EDTA buffer gels (Novex, Invitrogen) followed by ethidium bromide staining. Results of experiments were verified by repetition of RT-PCR with RNA extracted from different aliquots of cells (at least three independent reactions performed per template/primer combination). Quantitative real-time PCR (qPCR) and data analyses were performed using the MyiQ thermal cycler (Bio-Rad) (50 cycles, 62 °C annealing/extension). Sequence-specific internal probes dual-labeled with 6-carboxyfluorescein/carboxytetramethylrhodamine (TaqMan probes) were employed to measure fluorescence because of accumulating amplicons (Table 1). For relative quantification in qPCR, a mathematical model was used that incorporated the effects of the efficiency of amplification for each primer pair over a 104 range of template dilutions (26Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25597) Google Scholar). qPCR were run in triplicate for each sample, and three independent experiments were performed. The results were expressed as mean ± S.D. To verify the synthesis of predicted amplicons, qPCR products were analyzed by electrophoresis on polyacrylamide gels followed by ethidium bromide staining.TABLE 1Oligonucleotide primers and probes used for PCR in these studiesPrimer pair or probeOligonucleotide sequenceaF, forward/sense; or R, reverse/antisense primers.GenBank™HBA, humanF: 5′-ATGTTCCTGTCCTTCCCCACCACCAAG-3′BC050661R: 5′-GCTTAACGGTATTTGGAGGTCAGCACG-3′HBB, humanF: 5′-GTGAACGTGGATGAAGTTGGTGGTGAG-3′NM_000518R: 5′-TTGGACAGCAAGAAAGCGAGCTTAGTG-3′HBQ, humanF: 5′-TACACGACAGAGGCCCTGGAAAGGACC-3′BC056686R: 5′-AGTTCAGCGGTACTCGGAAACCAGCGC-3′HBA, mouse/ratF: 5′-CTCTCTGGGGAAGACAAAAGCAAC-3′BC043020R: 5′-GGTGGCTAGCCAAGGTCACCAGCA-3′HBB, mouseF: 5′-CACAACCCCAGAAACAGACA-3′NM_008220R: 5′-CTGACAGATGCTCTCTTGGG-3′HBB, ratF: 5′-TGAACCCTGATGATGTTGGTGGCGAGG-3′NM_033234R: 5′-AAGACAAGAGCAGGAAAAGAGGTTTAG-3′HBQ, mouse/ratF: 5′-TACACGACCGAGGCCTTGGAGAGGAC-3′XM_220266R: 5′-AGTTCAGCGATCCTTGGAGACCAGTGC-3′AE1, humanF: 5′-TGGACCTGCTGCTGGTAGTAG-3′NM_000342R: 5′-ATCTGGATGCCCGTGAATAAG-3′AE1, mouse/ratF: 5′-TGGCTGCTGTCATCTTCATCTAC-3′NM_011403R: 5′-TTTGGGCTTCATCACAACAGG-3′SPB, humanF: 5′-GGACATCGTCCACATCCTTAACAAGATG-3′BC032785R: 5′-ATTGCTGCTCGGAGAGATCCTGTGTGTG-3′SPB, mouse/ratF: 5′-GTGCCAAGAGTGTGAGGATATTGTCCACCTCC-3′NM_147779R: 5′-GGACACAGCCACAGCCAGCACACCCTTG-3′ARF, human/mouse/ratF: 5′-GCCAGTGTCCTTCCACCTGTC-3′M36340R: 5′-GCCTCGTTCACACGCTCTCTG-3′HBB, human qPCRF: 5′-ACTCCTGAGGAGAAGTCTGCCGTTAC-3′NM_000518R: 5′-TTGTCACAGTGCAGCTCACTCAGTGTG-3′HBA, mouse qPCRF: 5′-ATGTTTGCTAGCTTCCCCACCACCAAG-3′BC043020R: 5′-GGTGGCTAGCCAAGGTCACCAGCA-3′HBB, mouse qPCRF: 5′-TGATGCTGAGAAGGCTGCTGTCTCTG-3′NM_008220R: 5′-GTGCCCTTGAGGCTGTCCAAGTGA-3′ACTB, human qPCRF: 5′-GGGAAATCGTGCGTGACATTAAG-3′X00351R: 5′-TGTGTTGGCGTACAGGTCTTTG-3′ACTB, mouse qPCRF: 5′-GGGAAATCGTGCGTGACATCAAAG-3′NM_007393R: 5′-TGTGTTGGCATAGAGGTCTTTAC-3′HBA probe qPCR5′-CAACTTCAAGCTCCTGAGCCACTGCCTGCTGGTG-3′BC043020HBB probe qPCR5′-CCCTGGGCAGGCTGCTGGTTGTCTACCCTTG-3′NM_008220ACTB probe qPCR5′-CCAGCCTTCCTTCTTGGGTATGGAATCCTGTGGCA-3′NM_007393a F, forward/sense; or R, reverse/antisense primers. Open table in a new tab Oligonucleotide primer pairs were designed for hemoglobin gene transcripts from the α- and β-globin clusters and are shown in Table 1. Some primer pairs were later redesigned for compatibility with qPCR analysis. All oligonucleotide primers and probes were synthesized by Integrated DNA Technologies (Coralville, IA). DNA Sequencing—Select PCR amplicons were gel purified and subjected to DNA sequencing by the Medical University of South Carolina Biotechnology Facility using the same primer pairs used for amplification. Resulting sequences were analyzed using the NCBI BLAST engine. Western Blotting—Cells were washed four times in phosphate-buffered saline and lysed in 2% SDS. Whole cell lysates were subjected to SDS-PAGE (NuPAGE gels, Invitrogen), blotted onto nitrocellulose, and probed with polyclonal antibodies to α- or β-globin (Santa Cruz Biotechnology, Santa Cruz, CA) or pan hemoglobin (Bethyl Laboratories, Montgomery, TX) according to the manufacturers instructions. Horseradish peroxidase-conjugated secondary antibodies and SuperSignal West Dura chemiluminescence kit (both from Pierce Biotechnology) were used for visual detection with a FluorChem 8900 Imager (Alpha Innotech, San Leandro, CA). Purified human hemoglobin tetramer (HbA, Sigma) and α-globin purified from bovine pulmonary surfactant (27Baatz J.E. Zou Y. Cox J.T. Wang Z. Notter R.H. Protein Expr. Purif. 2001; 23: 180-190Crossref PubMed Scopus (23) Google Scholar) (identity confirmed by N-terminal protein sequencing) were used as standards. Protein Sequencing—Whole cell lysate from rat ATII primary cells was subjected to SDS-PAGE, silver-stained (28Acierno P.M. Newton D.A. Brown E.A. Maes L.A. Baatz J.E. Gattoni-Celli S. J. Transl. Med. 2003; 1: 3Crossref PubMed Scopus (37) Google Scholar), and excised gel slices in the 16-20-kDa range were submitted to the Taplin Biological Mass Spectrometry Facility (Harvard Medical School, Boston, MA). Peptides were generated by trypsin digestion before tandem mass spectrometry sequencing. Hemoglobin mRNA Is Expressed by Pulmonary Epithelial Cells—Conventional RT-PCR was used to measure the expression of genes from the α- and β-globin clusters in several human and rodent cell lines and primary cell cultures. Oligonucleotide primer pairs specific for various globin gene transcripts were meticulously designed to amplify individual sequences among the closely homologous globin genes while distinguishing between cDNA and genomic DNA amplicons and avoiding common allelic variations (Table 1). The expression of several globin genes was easily detectable by RT-PCR analyses of several types of pulmonary epithelial cells (Fig. 1). Specifically, PCR products corresponding to α-globin (HBA) and β-globin (HBB), the primary globin mRNAs expressed by erythroid precursor cells in adult mammals, were found in human cell lines A549 (ATII-like adenocarcinoma) and H441 (Clara cell-like adenocarcinoma), mouse cell line MLE-15 (transformed ATII cells), and ATII primary cells isolated from normal rat and mouse lungs (day 3 cultures). Interestingly, the human A549 and H441 cell lines also express the mRNA of θ-globin (HBQ), an α homolog that is the least-characterized globin gene (thought to be a pseudogene in rodents until recently) (29Hsu S.L. Marks J. Shaw J.P. Tam M. Higgs D.R. Shen C.C. Shen C.K. Nature. 1988; 331: 94-96Crossref PubMed Scopus (38) Google Scholar). The usage of ATII primary cells from normal rat and mouse lungs, although difficult to maintain in culture, was important because it has been reported that transformation with herpes or Rous sarcoma viruses can activate α-globin transcription in some cell lines (30Cheung P. Panning B. Smiley J.R. J. Virol. 1997; 71: 1784-1793Crossref PubMed Google Scholar, 31Groudine M. Weintraub H. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 4464-4468Crossref PubMed Scopus (61) Google Scholar), and certain patterns of aberrant gene expression are hallmarks of most tumor cell lines. As controls for cell phenotype and purity (especially for the primary cultures), Fig. 1 also shows that these cells expressed genes characteristic of pulmonary epithelial cells (e.g. surfactant protein B), but not other erythroid-specific genes, such as erythrocyte anion exchanger (AE1, band 3 protein) (32Passow H. Rev. Physiol. Biochem. Pharmacol. 1986; 103: 61-203PubMed Google Scholar). In addition to microscopic examination of ATII preparations, the absence of a PCR product generated by using primers specific for the AE1 gene transcript (very abundant in erythroid precursors) strongly suggests that globin gene expression in these ATII primary cultures was not the result of erythroid cell contamination. All tested cell lines expressed the ubiquitous housekeeping gene ADP-ribosylation factor 1 (33Bobak D.A. Nightingale M.S. Murtagh J.J. Price S.R. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6101-6105Crossref PubMed Scopus (82) Google Scholar). Because of these findings, additional control reactions for RT-PCR analyses were also performed (Fig. 2). The human melanoma cell line FO-1 and mouse fibroblast cell line NIH-3T3 did not express transcripts from globin genes, surfactant protein B, or AE1. Amplification of cDNA prepared from human blood and the mouse erythroid cell line MEL (induced to differentiate in cell culture) clearly demonstrated the presence of HBA, HBB, HBQ, and AE1 transcripts. The unlikely contamination of cell culture medium (including 10% FBS) with globin gene nucleic acid template was analyzed by adding to the PCR both complete medium and medium "cDNA" (cDNA synthesis reaction was performed with undetectable product of RNA extraction of culture medium; no nucleic acid was actually detected spectrophotometrically). No PCR products were detected in cell culture medium (further confirmed by combining A549 template cDNA with the medium preparation to eliminate the possibility of false negatives because of PCR polymerase inhibition). All tested cells expressed ADP-ribosylation factor 1 transcript, demonstrating the integrity of the RNA preparations. Hemoglobin Gene Expression Was Verified by DNA Sequencing—Select PCR products, including those from mouse ATII primary cells, were subjected to DNA sequencing to verify that they were indeed the result of specific amplification of globin gene transcripts. The sequences obtained were compared with those in GenBank using the NCBI BLAST engine and shown to be essentially perfect matches with the corresponding globin mRNA (Table 2).TABLE 2DNA sequencing of RT-PCR productsSequenced PCR productGenBank™ matchA549, HBA primersHomo sapiens hemoglobin, α 1 and 2 (HBA), mRNAaHuman HBA1 and HBA2 mRNAs share identical coding region sequences as flanked by these primers. (example: 100% nucleotide match to bases 120-449 of GenBank BC032122)A549, HBB primersH. sapiens hemoglobin, β (HBB), mRNA (example: 100% nucleotide match to bases 109-515 of GenBank NM_000518)A549, HBQ primersH. sapiens hemoglobin, θ 1 (HBQ), mRNA (example: 100% nucleotide match to bases 95-454 of GenBank BC056686)H441, HBA primersH. sapiens hemoglobin, α 1 and 2 (HBA), mRNAaHuman HBA1 and HBA2 mRNAs share identical coding region sequences as flanked by these primers. (example: 100% nucleotide match to bases 120-454 of GenBank BC032122)H441, HBB primersH. sapiens hemoglobin, β (HBB), mRNA (example: 100% nucleotide match to bases 109-510 of GenBank NM_000518)H441, HBQ primersH. sapiens hemoglobin, θ 1 (HBQ), mRNA (example: 100% nucleotide match to bases 95-454 of GenBank BC056686)MLE, HBA primersMus musculus hemoglobin α, adult chain 1, mRNA (example: 100% nucleotide match to bases 22-356 of GenBank BC043020)MLE, HBB primersM. musculus hemoglobin, β major chain (Hbb-b1), mRNA (example: 99% nucleotide match to bases 36-551 of GenBank XM_489729)MEL, HBQ primersM. musculus hemoglobin, θ 1 (Hbq1), mRNA (example: 100% nucleotide match to bases 123-460 of GenBank NM_175000)Mouse ATII,bMouse primary ATII cells. HBA primersM. musculus hemoglobin α, adult chain 1, mRNA (example: 100% nucleotide match to bases 22-352 of GenBank BC043020)Mouse ATII, HBB primersM. musculus hemoglobin, β major chain (Hbb-b1), mRNA (example: 100% nucleotide match to bases 35-551 of GenBank XM_489729)a Human HBA1 and HBA2 mRNAs share identical coding region sequences as flanked by these primers.b Mouse primary ATII cells. Open table in a new tab Along with the absence of RT-PCR products obtained with tissuespecific primers like surfactant protein B and AE1, the species specificity of these globin amplicons confirmed by DNA sequencing further eliminates the unlikely possibility of PCR cross-contamination (e.g. from mouse MEL cells or bovine serum in culture medium). In addition, because of the design of the oligonucleotide primers, every PCR product shown in Figs. 1 and 2 and Table 2 would contain intron sequences if derived from amplification of contaminating genomic DNA. It is also worth noting that the results in Fig. 2 and Table 2 clearly demonstrate that the mouse erythroid cell line MEL expresses θ-globin, a poorly defined globin homolog, especially in rodents. Relative Levels of Globin Gene Expression Are Different Among Cell Types—Using real time qPCR, the relative levels of HBA and HBB gene expression were compared between mouse pulmonary epithelial cells and induced MEL erythroid cells (Table 3). For this analysis, quality control was performed by determining amplification efficiency for each primer pair and by normalizing globin expression to β-actin expression to correct for differences in starting cDNA template concentrations (26Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25597) Google Scholar). As expected, the erythroid cells clearly expressed much higher levels of both HBA and HBB mRNAs than did MLE-15 and ATII primary cells. In fact, HBA expression in MEL cells was slightly greater than β-actin expression, certainly making it one of the most abundant cellular mRNAs. Still, globin gene expression was easily detected in both mouse ATII cell types, and the amplification detection thresholds decreased in a dose-dependent manner over a 105-fold template dilution curve (data not shown). All three cells types expressed more HBA than HBB mRNA, with ratios of ∼9:1, 10:1, and 27:1 for MEL, primary ATII, and MLE-15 cells, respectively.TABLE 3Quantitative analysis of globin gene expressionRatiosNormalized globin mRNA expression-foldComparison of globin gene expression in different mouse cellsMEL HBA:ATII HBA35.8 ± 4.4MEL HBA:MLE-15 HBA147 ± 9.3MEL HBB:ATII HBB40 ± 4.7MEL HBB:MLE-15 HBB440 ± 16.3ATII HBA:MLE-15 HBA4.1 ± 1.1ATII HBB:MLE-15 HBB11.0 ± 2.2Relative expression of different globin genesMEL HBA:HBB9.0 ± 2.8ATII HBA:HBB10.1 ± 2.6MLE-15 HBA:HBB26.9 ± 3.9 Open table in a new tab qPCR was also performed with human A549 and H441 cells, and although not precisely compared with mouse cells because of the necessity for making even greater assumptions for reference gene normalization and internal probe efficiency, it was clear that globin gene expression in these human cell lines was closer to that of the mouse pulmonary epithelial cells rather than erythroid cells. Again, amplification detection thresholds for globin genes in A549 and H441 decreased in a dose-dependent manner over a template dilution curve, and more HBA than HBB was expressed in each cell line (data not shown). Hemoglobin Protein Is Expressed by Pulmonary Epithelial Cells—Immunoblotting was performed to determine whether hemoglobin polypeptide subunits are expressed by lung epithelial cells (Fig. 3). Using polyclonal antibodies that demonstrated species cross-reactivity, α- and β-globin were detected in lysates from human A549 and H441 cells, mouse MLE-15 cells, and rat primary ATII cells (mouse primary ATII cells not available for testing). Interestingly, the hemoglobin from pulmonary epithelial cells migrated more slowly than that from erythroid sources, including that from MEL cells. This observation is yet to be explained, but is consistent with that seen in previously reported non-erythroid hemoglobin expression (7Liu L. Zeng M. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6643-6647Crossref PubMed Scopus (123) Google Scholar). Not surprisingly, much higher levels of globin subunits were detected in induced MEL cells compared with that in pulmonary epithelial cells (∼25-fold less total MEL protein was loaded to obtain roughly comparable levels of detection). Most likely because of the extremely stable conformation of the hemoglobin molecule, multiple forms of the protein, including aggregates larger than the heterotetramer, were commonly detected by immunostaining even after detergent and heat denaturation. For example, the α-globin dimers shown in Fig. 3 were consistently the most abundant forms seen in SDS-PAGE analyses of A549 and H441 cell lysates (in fact, monomers were occasionally difficult to detect even with MEL lysates). Because of this variability, the use of purified hemoglobin tetramer for comparison in immunoblotting greatly assisted in the interpretation of the protein expression data. Although all cells were thoroughly washed before lysis, culture medium containing 10% FBS was also tested to eliminate the possibility of contamination with bovine hemoglobin. No immunostaining was detected with these antibodies, which clearly would recognize bovine α-globin as demonstrated with a purified standard. When the staining intensities of these antibodies were normalized (assuming the HbA heterotetramer standard contained equimolar amounts of α-