HOX Genes in Human Lung
2001; Elsevier BV; Volume: 158; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64042-4
ISSN1525-2191
AutoresHeiko Golpon, Mark W. Geraci, Mark D. Moore, Heidi L. Miller, Gary J. Miller, Rubin M. Tuder, NORBERT F. VOELKEL,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoHOX genes belong to the large family of homeodomain genes that function as transcription factors. Animal studies indicate that they play an essential role in lung development. We investigated the expression pattern of HOX genes in human lung tissue by using microarray and degenerate reverse transcriptase-polymerase chain reaction survey techniques. HOX genes predominantly from the 3′ end of clusters A and B were expressed in normal human adult lung and among them HOXA5 was the most abundant, followed by HOXB2 and HOXB6. In fetal (12 weeks old) and diseased lung specimens (emphysema, primary pulmonary hypertension) additional HOX genes from clusters C and D were expressed. Using in situ hybridization, transcripts for HOXA5 were predominantly found in alveolar septal and epithelial cells, both in normal and diseased lungs. A 2.5-fold increase in HOXA5 mRNA expression was demonstrated by quantitative reverse transcriptase-polymerase chain reaction in primary pulmonary hypertension lung specimens when compared to normal lung tissue. In conclusion, we demonstrate that HOX genes are selectively expressed in the human lung. Differences in the pattern of HOX gene expression exist among fetal, adult, and diseased lung specimens. The altered pattern of HOX gene expression may contribute to the development of pulmonary diseases. HOX genes belong to the large family of homeodomain genes that function as transcription factors. Animal studies indicate that they play an essential role in lung development. We investigated the expression pattern of HOX genes in human lung tissue by using microarray and degenerate reverse transcriptase-polymerase chain reaction survey techniques. HOX genes predominantly from the 3′ end of clusters A and B were expressed in normal human adult lung and among them HOXA5 was the most abundant, followed by HOXB2 and HOXB6. In fetal (12 weeks old) and diseased lung specimens (emphysema, primary pulmonary hypertension) additional HOX genes from clusters C and D were expressed. Using in situ hybridization, transcripts for HOXA5 were predominantly found in alveolar septal and epithelial cells, both in normal and diseased lungs. A 2.5-fold increase in HOXA5 mRNA expression was demonstrated by quantitative reverse transcriptase-polymerase chain reaction in primary pulmonary hypertension lung specimens when compared to normal lung tissue. In conclusion, we demonstrate that HOX genes are selectively expressed in the human lung. Differences in the pattern of HOX gene expression exist among fetal, adult, and diseased lung specimens. The altered pattern of HOX gene expression may contribute to the development of pulmonary diseases. Homeodomain genes are a superfamily of genes encoding transcription factors, which are well known for their important functions regulating developmental processes such as body patterning and organogenesis.1Mark M Rijli FM Chambon P Homeobox genes in embryogenesis and pathogenesis.Pediatr Res. 1997; 42: 421-429Crossref PubMed Scopus (215) Google Scholar, 2Bogue CW Lou LJ Vasavada H Wilson CM Jacobs HC Expression of Hoxb genes in the developing mouse foregut and lung.Am J Respir Cell Mol Biol. 1996; 15: 163-171Crossref PubMed Scopus (59) Google Scholar, 3Krumlauf R Hox genes in vertebrate development.Cell. 1994; 78: 191-201Abstract Full Text PDF PubMed Scopus (1793) Google Scholar Homeodomain genes are widely distributed in all animal groups including mammals and eukaryotes, accounting for ∼0.1% of the vertebrate genome.4Boncinelli E Homeobox genes and disease.Curr Opin Genet Dev. 1997; 7: 331-337Crossref PubMed Scopus (85) Google Scholar The family of vertebrate homeodomain genes can be divided into two subfamilies: the clustered homeodomain genes known as HOX genes or class I homeobox genes; and the nonclustered or divergent homeodomain genes such as PAX, EMX, OTX, and MSX, named after their homologs in Drosophila (paired, empty spiracles, orthodenticle, and muscle segment homeobox genes, respectively).5Stein S Fritsch R Lemaire L Kessel M Checklist: vertebrate homeobox genes.Mech Dev. 1996; 55: 91-108Crossref PubMed Scopus (98) Google Scholar The protein products of the HOX genes are helix-turn-helix transcription factors that share a highly conserved 60-amino acid DNA-binding region, known as the homeobox. In humans, 39 HOX genes are known, which are organized into four physical clusters (HOXA, HOXB, HOXC, and HOXD). Most studies regarding HOX gene expression have focused on embryonic development and, hence, little information is available regarding their expression in adult tissues. Recently, some functions of HOX genes in adult tissues have been described. For example, deficits in group 9 paralogous genes (Hoxa-9, Hoxb-9, Hoxd-9; mouse nomenclature) resulted in abnormal cellular proliferation and differentiation in the mammary gland of adult mice.6Chen F Capecchi MR Paralogous mouse Hox genes, Hoxa9, Hoxb9, and Hoxd9, function together to control development of the mammary gland in response to pregnancy.Proc Natl Acad Sci USA. 1999; 6: 541-546Crossref Scopus (164) Google Scholar It has also been reported that the size of the prostate was decreased by inactivation of Hoxd-13.7Podlasek CA Duboule D Bushman W Male accessory sex organ morphogenesis is altered by loss of function of Hoxd-13.Dev Dyn. 1997; 208: 454-465Crossref PubMed Scopus (107) Google Scholar Moreover, abnormal homeobox gene expression has been described in a number of leukemias.8van Oostveen J Bijl J Raaphorst F Walboomers J Meijer C The role of homeobox genes in normal hematopoiesis and hematological malignancies.Leukemia. 1999; 13: 1675-1690Crossref PubMed Scopus (134) Google Scholar, 9Kawagoe H Humphries RK Blair A Sutherland HJ Hogge DE Expression of HOX genes, HOX cofactors, and MLL in phenotypically and functionally defined subpopulations of leukemic and normal human hematopoietic cells.Leukemia. 1999; 13: 687-698Crossref PubMed Scopus (184) Google Scholar HOX gene expression in leukemic cell lines seems to be lineage-restricted, and a regulatory role for HOX genes in blood cell maturation has been proposed. HOXA5 might function as an important regulator of hematopoietic cell lineage determination and maturation.10Crooks GM Fuller J Petersen D Izadi P Malik P Pattengale PK Kohn DB Gasson JC Constitutive HOXA5 expression inhibits erythropoiesis and increases myelopoiesis from human hematopoietic progenitors.Blood. 1999; 94: 519-528Crossref PubMed Google Scholar, 11Fuller JF McAdara J Yaron Y Sakaguchi M Fraser JK Gasson JC Characterization of HOX gene expression during myelopoiesis: role of HOXA5 in lineage commitment and maturation.Blood. 1999; 93: 3391-3400Crossref PubMed Google Scholar Presently, very little information is available regarding the role of HOX genes in the adult respiratory system.12Kappen C Hox genes in the lung.Am J Respir Cell Mol Biol. 1996; 15: 156-162Crossref PubMed Scopus (46) Google Scholar Apart from the description of HOX gene expression in lung cancer cell lines,13Cillo C Faiella A Cantile M Boncinelli E Homeobox genes and cancer.Exp Cell Res. 1999; 248: 1-9Crossref PubMed Scopus (156) Google Scholar, 14Tiberio C Barba P Magli MC Arvelo F Le Chevalier T Poupon MF Cillo C HOX gene expression in human small-cell lung cancers xenografted into nude mice.Int J Cancer. 1994; 58: 608-615Crossref PubMed Scopus (79) Google Scholar HOX gene expression has only been reported in cultured human bronchial fibroblasts, where retinoic acid caused an induction of HOXA5 expression.15Bernacki SH Nervi C Vollberg TM Jetten AM Homeobox 1.3 expression: induction by retinoic acid in human bronchial fibroblasts.Am J Respir Cell Mol Biol. 1992; 7: 3-9Crossref PubMed Scopus (10) Google Scholar It is also known that 16 homeobox genes are differentially expressed in embryonic and newborn mouse lungs,2Bogue CW Lou LJ Vasavada H Wilson CM Jacobs HC Expression of Hoxb genes in the developing mouse foregut and lung.Am J Respir Cell Mol Biol. 1996; 15: 163-171Crossref PubMed Scopus (59) Google Scholar, 16Bogue CW Gross I Vasavada H Dynia DW Wilson CM Jacobs HC Identification of Hox genes in newborn lung and effects of gestational age and retinoic acid on their expression.Am J Physiol. 1994; 266: L448-L454PubMed Google Scholar, 17Chinoy MR Volpe MV Cilley RE Zgleszewski SE Vosatka RJ Martin A Nielsen HC Krummel TM Growth factors and dexamethasone regulate Hoxb5 protein in cultured murine fetal lungs.Am J Physiol. 1998; 274: L610-L620PubMed Google Scholar and here again the pattern of gene expression in the developing lung is affected by retinoic acid.16Bogue CW Gross I Vasavada H Dynia DW Wilson CM Jacobs HC Identification of Hox genes in newborn lung and effects of gestational age and retinoic acid on their expression.Am J Physiol. 1994; 266: L448-L454PubMed Google Scholar, 18Cardoso WV Mitsialis SA Brody JS Williams MC Retinoic acid alters the expression of pattern-related genes in the developing rat lung.Dev Dyn. 1996; 207: 47-59Crossref PubMed Scopus (78) Google Scholar Because some mouse Hox mutants have severe disruption of lung morphogenesis, Hox genes might play an important role in branching morphogenesis, epithelial cell fate, and the differentiation of conducting airway epithelium.19Volpe MV Martin A Vosatka RJ Mazzoni CL Nielsen HC Hoxb-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate.Histochem Cell Biol. 1997; 108: 495-504Crossref PubMed Scopus (64) Google Scholar Specifically, Hoxa-1 mutants failed to initiate respiration20Lufkin T Dierich A LeMeur M Mark M Chambon P Disruption of the Hox-1.6 homeobox gene results in defects in a region corresponding to its rostral domain of expression.Cell. 1991; 66: 1105-1119Abstract Full Text PDF PubMed Scopus (636) Google Scholar and Hoxa-3 mutants die shortly after birth, likely because of pulmonary failure.21Chisaka O Capecchi MR Regionally restricted developmental defects resulting from targeted disruption of the mouse homeobox gene hox-1.5.Nature. 1991; 350: 473-479Crossref PubMed Scopus (756) Google Scholar A smaller lung was also noted for Hoxa-1/Hoxb-1 double mutants.22Rossel M Capecchi MR Mice mutant for both Hoxa1 and Hoxb1 show extensive remodeling of the hindbrain and defects in craniofacial development.Development. 1999; 126: 5027-5040Crossref PubMed Google Scholar Homozygous mutants for Hoxa-5 showed perinatal lethality because of improper tracheal and lung morphogenesis that caused tracheal occlusion and respiratory distress.23Aubin J Lemieux M Tremblay M Berard J Jeannotte L Early postnatal lethality in Hoxa-5 mutant mice is attributable to respiratory tract defects.Dev Biol. 1997; 192: 432-445Crossref PubMed Scopus (171) Google Scholar Loss of Hoxa-5 function may also impair the production of surfactant-associated proteins.23Aubin J Lemieux M Tremblay M Berard J Jeannotte L Early postnatal lethality in Hoxa-5 mutant mice is attributable to respiratory tract defects.Dev Biol. 1997; 192: 432-445Crossref PubMed Scopus (171) Google Scholar Despite the growing evidence linking homeobox genes to murine lung embryogenesis and development, information regarding the expression of HOX genes in the human adult lung is still lacking. In the present study, we surveyed HOX gene expression in normal and diseased human adult lungs. The most abundant HOX genes identified by us in normal lung, HOXA5, HOXB2, and HOXB6, were the subject of further expression studies in the normal lung and in the lungs from patients with emphysema and pulmonary hypertension. Total RNA was extracted from the lung tissues by using the Trizol reagent (Life Technologies, Inc., Gaithersburg, MD). Polyadenylated (poly A+) RNA was purified from total RNA using oligo-dT-linked Oligotex Resin (Qiagen, Valencia, CA). One microgram of poly A+ RNA was converted to double-stranded cDNA (ds-cDNA) by using Superscript Choice System (Life Technologies, Inc., Rockville, MD). An oligo-dT primer containing a T7 RNA polymerase promoter (Promega Corp., Madison, WI) was used. After second strand synthesis, the reaction mixture was extracted with phenol-chloroform-isoamyl alcohol and the ds-cDNA was recovered by ethanol precipitation. In vitro transcription, to generate biotin-labeled cRNA, was performed using a T7 Megascript kit by Ambion (Austin, TX). ds-cDNA template (1.5 μl) was transcribed in the presence of a mixture of unlabeled ATP, CTP, GTP, and UTP. In addition, biotin-labeled CTP and UTP (bio-11-CTP and bio-16-UTP) from Enzol (Farmingdale, NY) was added to the mixture. Biotin-labeled cRNA was purified using an RNeasy affinity column by Qiagen. To ensure optimal hybridization to the oligonucleotide array, the cRNA was fragmented. Fragmentation was performed by incubating the cRNA at 94°C for 35 minutes to produce cRNA fragments between 35 to 200 bases in length. The sample was then added to a hybridization solution containing 100 mmol/L MES, 1 mol/L Na+, and 20 mmol/L of ethylenediaminetetraacetic acid in the presence of 0.01% Tween 20 to a final cRNA concentration of 0.05 mg/ml. Hybridization was performed for 18 to 20 hours by incubating 200 ml of the sample to the Affymetrix gene chip (Affymetrix, Santa Clara, CA). After hybridization, the hybridization solutions were removed and the gene chips were washed and stained with streptavidin-phycoerythrin. Chips were read at a resolution of 6 μm with a Hewlett-Packard gene array scanner (Hewlett-Packard Co., Boise, ID). Detailed protocols for data analysis of Affymetrix microarrays and extensive documentation of the sensitivity and quantitative aspects of the method have been described.24Lockhart DJ Dong H Byrne MC Follettie MT Gallo MV Chee MS Mittmann M Wang C Kobayashi M Horton H Brown EL Expression monitoring by hybridization to high-density oligonucleotide arrays.Nature Biotechnol. 1996; 14: 1675-1680Crossref PubMed Scopus (2835) Google Scholar, 25Golub TR Slonim DK Tamayo P Huard C Gaasenbeek M Mesirov JP Coller H Loh ML Downing JR Caligiuri MA Bloomfield CD Lander ES Molecular classification of cancer: class discovery and class prediction by gene expression monitoring.Science. 1999; 286: 531-537Crossref PubMed Scopus (9382) Google Scholar Briefly, each gene is represented by the use of ∼20 perfectly matched and mismatched control probes. The mismatched probes act as specificity controls that allow the direct subtraction of both background and cross-hybridization signals. The number of instances in which the perfectly matched hybridization signal is larger than the mismatched signal is computed along with the average of the logarithm of the perfectly matched:mismatched ratio (after background subtraction) for each probe set. These values are used to make a matrix-based decision concerning the presence or absence of an RNA molecule. To determine the quantitative RNA abundance, the average of the differences representing perfectly matched minus mismatched for each gene-specific probe family is calculated, after discarding the maximum, the minimum, and any outliers beyond three standard deviations. All of the aforementioned calculations are functions performed by the Gene Chip Analysis Suite (Affymetrix). The Gene Chip Analysis Suite is a program that is used to analyze expression data from all Affymetrix expression arrays. This analysis software is specifically designed to determine intensity of hybridization from all features on the array. Specifically, this program determines the intensity of expression for all genes on the array. One chip with adult mouse lung (pooled sample of five FVB/N mice), one chip of human fetal lung (pooled sample of at least three lungs with a gestation age of 12 weeks), three chips with normal human adult lung tissue, three chips with emphysematous human lung tissue, and three chips with primary pulmonary hypertension (PPH) human lung tissue were hybridized and analyzed for the expression of HOX genes. To survey expression of HOX genes in normal human lung tissue, we used a degenerate RT-PCR technique.16Bogue CW Gross I Vasavada H Dynia DW Wilson CM Jacobs HC Identification of Hox genes in newborn lung and effects of gestational age and retinoic acid on their expression.Am J Physiol. 1994; 266: L448-L454PubMed Google Scholar Because the homeobox domain of most of the HOX proteins is flanked by the highly conserved ELEKEF and KIWFQN motifs, we used a set of degenerate PCR primers recognizing these motifs.26Balavoine G Telford MJ Identification of planarian homeobox sequences indicates the antiquity of most Hox/homeotic gene subclasses.Proc Natl Acad Sci USA. 1995; 92: 7227-7231Crossref PubMed Scopus (48) Google Scholar The sequence of the degenerate primers were (International Union of Pure and Applied Chemistry Code used): 5′-GCT CTA GAR YTN GAR AAR GAR TT-3′; 5′-GGA ATT CRT TYT GRA ACC ADA TYT T-3′ (for the ELEKEF and KIWFQN motifs, respectively). Total RNA from human fetal lung (pooled sample of at least three lungs with a gestation age of 12 weeks), normal human adult lung (n = 2), emphysematous lungs (n = 3), and lungs from patients with PPH (n = 3) was isolated using the RNeasy mini kit (Qiagen) and converted to ds-cDNA using the Reverse Transcription System kit (Promega Corp.). Five μl of the cDNA mixture served as a template for PCR amplification in a 25-μl reaction using 2 μmol/L each of sense and antisense degenerate oligonucleotide primers (200 μmol/L of dNTP, 4.5 mmol/L of MgCl2, 0.65 U of Taq polymerase). The reaction conditions were initial denaturation at 94°C for 4 minutes, followed by 32 cycles of 94°C for 1 minute, 40°C for 1 minute, 72°C for 1 minute, and a final extension at 72°C for 5 minutes. PCR products were separated in 2% NuSieve 3:1 agarose by gel electrophoresis. The 125-bp band containing homeobox amplicons was excised and purified with the QIAquick gel extraction kit from Qiagen. The concentration of PCR products was estimated by comparison to DNA mass standards. Approximately 20 ng of the PCR product was ligated into the pCR2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and expanded in One Shot (Invitrogen) cells. An efficient cloning reaction produced ∼200 positive (>95%) colonies on each plate, which were easily identified by white color on X-Gal-containing medium. Only colonies of efficient cloning reactions were sequenced. The cloned inserts from overnight minipreps were isolated using the QIA well 8 ultra plasmid kit (Qiagen). Twenty-seven colonies originating from human fetal lung (normal adult lung = 47, emphysema = 51, PPH = 47 sequenced colonies) were sequenced at the Cancer Center Core Lab., University of Colorado Health Sciences Center (Denver, CO), using the dideoxy sequencing method of Sanger and colleagues.27Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors.Proc Natl Acad Sci USA. 1977; 74: 5463-5467Crossref PubMed Scopus (58968) Google Scholar Sequences were compared to those in the nonredundant database of the National Center for Biotechnology Information using the BLAST search algorithm. Lung tissue samples were obtained from six patients with PPH (three males, three females; 36 ± 5.47 [SEM] years). Two of the lung specimens were obtained at autopsy—three patients were undergoing open lung biopsy for diagnostic purposes and one patient was undergoing lung transplantation. All of these patients had severely elevated pulmonary artery pressure as documented by right heart catheterization. We also obtained lungs from six patients [three males, three females; 59.17 ± 4.32 (SEM) years] with severe emphysema who were either undergoing lung transplantation, lung volume reduction surgery, or lobectomy. All six patients were chronic cigarette smokers with pulmonary obstructive changes documented by lung function studies. Normal lung tissue was obtained from six patients (three males, three females; 62.5 ± 6.28 [SEM] years) undergoing open lung biopsy for diagnostic purposes [localized inflammation (n = 1) or primary or metastatic malignancies (n = 5)]. Histologically, these tissues did not show any histological abnormalities. Total RNA from human embryonic lung tissues at 12 weeks of age (embryonic period/pseudoglandular stage) was obtained from ViroGen (Watertown, MA). Quantitative RT-PCR was performed using the SYBR Green PCR core reagents (Perkin-Elmer, Foster City, CA). Direct detection of the polymerase chain reaction product was monitored with the Gene Amp 5700 sequence detection system (ABI Prism, Perkin-Elmer) by measuring the increase in fluorescence caused by the binding of SYBR Green to ds-DNA. The primers used were HOXA5 (forward 5′-CGCCCAACCCCAGATCTA-3′; reverse, 5′-GGCCGCCTATGTTGTCATG-3′; 66-bp band product), HOXB2 (forward 5′-TCTCCCCTAGCCTACAGGGTTC-3′; reverse, 5′-GGTGAAAAAATCCAGCTCTTCCT-3′; 71-bp band product), HOXB5 (forward 5′-GAGATCGCCCACGCACTCT-3′; reverse, 5′-TTCATGCGCCGGTTCTG-3′; 68-bp band product), HOXB6 (forward 5′-GCCCCCTGCCCCAAT-3′; reverse, 5′-CCCAATCTTCTTCTATCTCCTAACTGA-3′; 67-bp band product), HOXC5 (forward 5′-CCCAGAGTCAATAAATAGTTGGACAA-3′; reverse, 5′-CCATAGTCCCTGCCACGAAT-3′; 71-bp band product). We confirmed that the primer pairs amplified the expected HOX genes by cloning and sequencing the PCR products. The absence of nonspecific amplification was confirmed either by running dissociation curves or examining PCR amplification products by agarose gel electrophoresis. To determine the optimal primer concentrations, which gave the highest levels of specific products and minimal nonspecific DNA and primer-dimer products, we performed a primer optimization as described in the manufacturer's protocol. RT and amplification were performed by using one-step RT-PCR. A 50-μl reaction mix contained 5 μl of 10× SYBR PCR buffer, 5 μl of template, 6 μl of 25 mmol/L MgCl2, 6 μl of 10 mmol/L dNTPs, 1 μl of each primer, 1 μl of RNase inhibitor, and 25 μl of RNase-free H2O. All reagents used in the RT-PCR were purchased from Perkin-Elmer. The RT reaction was performed for 30 minutes at 48°C using MultiScribe reverse transcriptase (final concentration, 1.25 U). AmpliTaq Gold Polymerase (final concentration, 1.25 U) was activated by incubating the reactions at 95°C for 10 minutes followed by 40 cycles of amplification (95°C for 15 seconds and 60°C for 1 minute). The TaqMan ribosomal RNA control reagents (Perkin-Elmer) were used as a standard to quantify the initial concentration and ascertain the quality of the total RNA and to assure reproducibility of the RT-PCR. Human total RNA dilutions from 10 ng/μl down to 0.0001 ng/μl were used as templates to create a standard curve. Each experiment was run twice in duplicate and the data reported are the average results for duplicate samples from each of the two experiments. The initial amount of rRNA in the sample was assessed using a threshold cycle (Ct) value. The Ct value is defined as the first cycle at which a statistically significant increase is detected in the difference between the emission intensity of the reporter dye in the sample and the emission intensity of the controls without RNA. This value is then used to compare one sample value to another. To quantitate the copy numbers of HOX genes present in the lung samples, we used plasmids (pGEM-T vector systems, JM 109 cells; Promega Corp.) containing cloned HOXA5, HOXB2, HOXB5, HOXB6, and HOXC5 sequences in concentrations ranging from 0.1 ng to 0.0001 ng/μl. We compared the Ct of the unknown samples against the standard curve of the plasmid with known copy numbers. Plasmids and lung samples were run in duplicate and a control reaction without reverse transcriptase was also run for each sample. In addition, reactions without RNA were used to establish baseline levels for fluorescence. Five μl (300 ng of total RNA from lung tissue samples) was used as a template for the one-step RT-PCR. We used fresh adult lung fragments frozen in Tissue Tek embedding medium (Sakura, Torrance, CA) and maintained at −70°C until sectioning, and paraffin-embedded fetal lung tissue (pseudoglandular and canalicular stage). Seven-mm cryostat sections were mounted onto Superfrost slides (Fisher Scientific, Pittsburgh, PA). At least three different sections of each lung were processed for in situ hybridization. HOXA5, HOXB2, and HOXB6 were PCR amplified as described before and cloned into the pGEM T vector (Promega Corp.). Digoxigenin-labeled antisense and sense cRNA probes were synthesized from the T7 and SP6 polymerase promoters, respectively (Genius IV kit; Boehringer Mannheim Biochemicals, Indianapolis, IN). Paraffin sections (fetal lung) and frozen sections of normal human adult lung (n = 3), emphysematous lungs (n = 3), and lungs with severe pulmonary hypertension (n = 3) were submitted to in situ detection of HOXA5, HOXB2, and HOXB6 mRNA according to the protocol by Crabb and colleagues28Crabb ID Hughes SS Hicks DG Puzas JE Tsao GJ Rosier RN Nonradioactive in situ hybridization using digoxigenin-labeled oligonucleotides. Applications to musculoskeletal tissues.Am J Pathol. 1992; 141: 579-589PubMed Google Scholar and Boehringer Mannheim's guide for filter hybridization. The slides were prehybridized with 50% formamide hybridization cocktail (Amresco, Solon, OH) for 2 hours at room temperature. Hybridization (probe concentration, 400 ng/ml) was performed at 42°C overnight. Immunodetection of the hybridized product was performed with a sheep anti-digoxigenin antibody at 1:2,000 dilution (Boehringer Mannheim). The color solution consisted of 4-tetrazolium chloride, 4.5 μg/ml, and X-phosphate, 3.5 μl in buffer 3, according to the manufacturer's protocol (Boehringer Mannheim). The reaction was allowed to develop for ∼7 hours. The slides were then mounted in Immuno-mount (Shandon, Pittsburgh, PA). All data underwent variance analysis and significant differences between two groups were consecutively compared using Student's t-tests. Differences were considered significant at P < 0.05 and the values represent the mean ± SD. To survey the expression of HOX genes in human lung tissue, we used a microarray gene chip technique (Hu GeneFL array, Affymetrix gene chip). We screened three normal human adult lungs, three emphysematous lungs, three lungs with severe pulmonary hypertension, and a sample of pooled fetal lung tissue (12 weeks of gestation age). To compare the expression of HOX genes in human and mouse lung, we additionally screened lung tissue from adult mice (pooled samples of five FVB/N mice) using a murine-specific microarray (Mu 6500, Affymetrix gene chip). The human microarray contained probes for 17 of the 39 known HOX genes (murine microarray contains 32 out of 39 known Hox genes). The 17 HOX genes represented on the human microarray chip were also represented on the murine microarray, allowing thus a direct comparison of the corresponding HOX gene lung expression between species. Figure 1 demonstrates the expression pattern of HOX genes in normal adult human and mouse lung tissue. Of the 17 HOX genes only three HOX genes were detected in both mice and human lungs (HOXA5, HOXB6, HOXC6). However, five HOX genes were detected in human lungs but not in mice lungs (HOXA1, HOXA4, HOXA10, HOXB2, and HOXB13). Three Hox genes (Hox-A2, Hox-C8, and Hox-D10) were only detected in the mouse but not in the normal human-adult lung tissue. Table 1 shows the expression pattern by microarray technique of HOX genes in human normal adult lung, fetal, emphysematous, and PPH lung tissue. Of the 17 HOX genes represented on the chip, six HOX genes (HOXA4, HOXA5, HOXB2, HOXB6, HOXC6, and HOXC8) were expressed in fetal lung tissue. In normal human-adult lung tissue, eight HOX genes were detected. Compared to the fetal lung, HOXC8 was not expressed in adult lung tissue and three additional HOX genes (HOXA1, HOXA10, and HOXB13) were present in adult but not fetal lung tissue. Interestingly, the expression pattern of HOX genes in normal adult lung tissue was not consistent in all three examined lungs. For example HOXA1 was detectable in one lung sample but it was below the detection level in the other two samples. HOXA4, HOXA5, and HOXC6 were detected in two out of three samples of normal adult lung tissue. A variation of HOX gene expression was also noted in the samples of emphysematous and PPH lung tissue. In lung emphysema only HOXC8 was present in each of three examined tissue samples. HOXA5, HOXB2, and HOXB13 were detected in two tissue samples whereas HOXA4 was present in only one of the samples. In PPH lung tissue HOXB2 was detected in two of three samples (HOXA5, HOXA10, HOXB13 = one of three samples). Compared to the normal adult lung, a reduction in the number of detectable HOX genes was noted for fetal, emphysematous, and PPH lung tissue. Only four of the 17 represented HOX genes were detectable in PPH lung tissue (emphysema, 5 of 17; fetal lung, 6 of 17) whereas eight HOX genes were detected in normal adult lung tissue. Among those eight HOX genes, only HOXA5 and HOXB2 were detectable in all examined adult lungs.Table 1HOX Genes in PPH and EmphysemaFetal lung (12 weeks) pooled samplesNormal adult lung n = 3Emphysema n = 3PPH n = 3HOXA1ap (1/3)aaHOXA2aaaaHOXA3aaaaHOXA4pp (2/3)p (1/3)aHOXA5pp (2/3)p (2/3)p (1/3)HOXA9aaaaHOXA10ap (3/3)ap (1/3)HOXB1aaaaHOXB2pp (3/3)p (2/3)p (2/3)HOXB6pp (3/3)aaHOXB13ap (3/3)p (2/3)p (1/3)HOXC4aaaaHOXC5aaaaHOXC6pp (2/3)aaHOXC8pap (3/3)aHOXD9aaaaHOXD10aaaaa, absent; p, present; n, number of samples. Open table in a new tab a, absent; p, present; n, number of samples. To provide an independent confirmation of the expression pattern found by the microarray gene chip technique, we used a degenerate RT-PCR technique followed by cloning and DNA sequencing. Figure 2 shows the expression pattern of HOX genes found in fetal lung tissue (pooled sample, 12 weeks of gestation age), human normal adult lung (n = 2),
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