Limits...
HTR4 gene structure and altered expression in the developing lung.

Hodge E, Nelson CP, Miller S, Billington CK, Stewart CE, Swan C, Malarstig A, Henry AP, Gowland C, Melén E, Hall IP, Sayers I - Respir. Res. (2013)

Bottom Line: Meta-analyses of genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) spanning the 5-hydroxytryptamine receptor 4 (5-HT₄R) gene (HTR4) associated with lung function.Radioligand binding experiments also indicated that HBEC and HASM cells did not express a significant 5-HT₄R population. 5' RACE in brain identified a novel N-terminal variant, containing an extended N-terminal sequence.Taken together, these data suggest a role for HTR4 in lung development, which may at least in part explain the genetic association with lung function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Respiratory Medicine, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.

ABSTRACT

Background: Meta-analyses of genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) spanning the 5-hydroxytryptamine receptor 4 (5-HT₄R) gene (HTR4) associated with lung function. The aims of this study were to i) investigate the expression profile of HTR4 in adult and fetal lung tissue and cultured airway cells, ii) further define HTR4 gene structure and iii) explore the potential functional implications of key SNPs using a bioinformatic approach.

Methods: Following reverse transcription (RT)-PCR in human brain, 5' rapid amplification of cDNA ends (5' RACE) was used to examine the exonic structure of HTR4 at the 5' end. Quantitative (Q)-PCR was used to quantify HTR4 mRNA expression in total RNA from cultured airway cells and whole lung tissue. Publically available gene microarray data on fetal samples of estimated gestational age 7-22 weeks were mined for HTR4 expression. Immunohistochemistry (IHC; in adult and fetal lung tissue) and a radioligand binding assay (in cultured airway cells) were used to analyze 5-HT₄R protein expression.

Results: IHC in adult lung, irrespective of the presence of chronic obstructive pulmonary disease (COPD), suggested low level expression of 5-HT₄R protein, which was most prominent in alveolar pneumocytes. There was evidence of differential 5-HT₄R protein levels during gestation in fetal lung, which was also evident in gene expression microarray data. HTR4 mRNA expression, assessed by Q-PCR, was <0.5% relative to brain in total adult lung tissue and in human airway smooth muscle (HASM) and bronchial epithelial cells (HBEC) derived from adult donors. Radioligand binding experiments also indicated that HBEC and HASM cells did not express a significant 5-HT₄R population. 5' RACE in brain identified a novel N-terminal variant, containing an extended N-terminal sequence. The functional significance of key HTR4 SNPs was investigated using the encyclopedia of DNA elements consortium (ENCODE) dataset. These analyses identified multiple alterations in regulatory motifs for transcription factors implicated in lung development, including Foxp1.

Conclusions: Taken together, these data suggest a role for HTR4 in lung development, which may at least in part explain the genetic association with lung function.

Show MeSH

Related in: MedlinePlus

Expression profiling of HTR4 in airway cells. RT-PCR and sequencing confirmed expression of HTR4 transcripts a (NM_001040169.2) and b (NM_000870.5) in total brain tissue (a). Sequence encoding exon 1 was found in both transcripts, which contradicts information provided by NCBI for transcript a, however transcripts a and b were differentiated by the presence of exon 13 or 9 respectively. Q-PCR analysis (b) indicated highest expression in total brain tissue, but ≤0.26% relative to this in total lung tissue, HBEC, HASM and the BEAS2B-R1 cell line. Error bars indicate SEM (n ≥3). Radioligand binding experiments (c), using a saturating concentration of [3H]-GR113808 (approx. 1 nM), defined 5-HT4R expression in CHO-K1 cells transiently transfected with pcDNA3-HTR4a, un-transfected CHO-K1 cells, HBEC, HASM and BEAS2B-R1 cells. Error bars indicate SEM (n ≥3). While the pcDNA3-HTR4a-transfected CHO-K1 cells expressed a substantial 5-HT4R population (approx. 460 fmol/mg protein), no other cell type expressed a significant receptor population (all ≤50 fmol/mg protein).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750317&req=5

Figure 5: Expression profiling of HTR4 in airway cells. RT-PCR and sequencing confirmed expression of HTR4 transcripts a (NM_001040169.2) and b (NM_000870.5) in total brain tissue (a). Sequence encoding exon 1 was found in both transcripts, which contradicts information provided by NCBI for transcript a, however transcripts a and b were differentiated by the presence of exon 13 or 9 respectively. Q-PCR analysis (b) indicated highest expression in total brain tissue, but ≤0.26% relative to this in total lung tissue, HBEC, HASM and the BEAS2B-R1 cell line. Error bars indicate SEM (n ≥3). Radioligand binding experiments (c), using a saturating concentration of [3H]-GR113808 (approx. 1 nM), defined 5-HT4R expression in CHO-K1 cells transiently transfected with pcDNA3-HTR4a, un-transfected CHO-K1 cells, HBEC, HASM and BEAS2B-R1 cells. Error bars indicate SEM (n ≥3). While the pcDNA3-HTR4a-transfected CHO-K1 cells expressed a substantial 5-HT4R population (approx. 460 fmol/mg protein), no other cell type expressed a significant receptor population (all ≤50 fmol/mg protein).

Mentions: Previously described gene arrangements for HTR4 are summarized in Figure 4. Transcripts a, b, i, d and g are currently reported by NCBI (December 2012); transcripts h, n, e, f and c have been described in the literature [10]. These data illustrate the complex splicing that exists at this gene locus, including multiple functionally relevant C-terminal variants, leading to alterations in cyclic AMP signalling responses [10]. Analysis by RT-PCR indicated expression of mRNA encoding HTR4 in total brain tissue, but failed to detect expression in HASM, undifferentiated HBEC, PBMC or total lung tissue (data not shown). Targeted sequencing of PCR products derived from brain cDNA confirmed the presence of both variants a (accession NM_001040169.2) and b (accession NM_000870.5), distinguished by the presence of exons 9 and 13 respectively. Additionally, exon 1 was present in variant a, although this contradicts data currently reported by NCBI (Figure 5a). Q-PCR data indicated very low levels of mean HTR4 mRNA expression in airway cells and tissues when compared to total brain tissue: total lung (0.26%), undifferentiated HBEC (0.05%), HASM (0.02%) and BEAS2B-R1 (0.16%) (Figure 5b).


HTR4 gene structure and altered expression in the developing lung.

Hodge E, Nelson CP, Miller S, Billington CK, Stewart CE, Swan C, Malarstig A, Henry AP, Gowland C, Melén E, Hall IP, Sayers I - Respir. Res. (2013)

Expression profiling of HTR4 in airway cells. RT-PCR and sequencing confirmed expression of HTR4 transcripts a (NM_001040169.2) and b (NM_000870.5) in total brain tissue (a). Sequence encoding exon 1 was found in both transcripts, which contradicts information provided by NCBI for transcript a, however transcripts a and b were differentiated by the presence of exon 13 or 9 respectively. Q-PCR analysis (b) indicated highest expression in total brain tissue, but ≤0.26% relative to this in total lung tissue, HBEC, HASM and the BEAS2B-R1 cell line. Error bars indicate SEM (n ≥3). Radioligand binding experiments (c), using a saturating concentration of [3H]-GR113808 (approx. 1 nM), defined 5-HT4R expression in CHO-K1 cells transiently transfected with pcDNA3-HTR4a, un-transfected CHO-K1 cells, HBEC, HASM and BEAS2B-R1 cells. Error bars indicate SEM (n ≥3). While the pcDNA3-HTR4a-transfected CHO-K1 cells expressed a substantial 5-HT4R population (approx. 460 fmol/mg protein), no other cell type expressed a significant receptor population (all ≤50 fmol/mg protein).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750317&req=5

Figure 5: Expression profiling of HTR4 in airway cells. RT-PCR and sequencing confirmed expression of HTR4 transcripts a (NM_001040169.2) and b (NM_000870.5) in total brain tissue (a). Sequence encoding exon 1 was found in both transcripts, which contradicts information provided by NCBI for transcript a, however transcripts a and b were differentiated by the presence of exon 13 or 9 respectively. Q-PCR analysis (b) indicated highest expression in total brain tissue, but ≤0.26% relative to this in total lung tissue, HBEC, HASM and the BEAS2B-R1 cell line. Error bars indicate SEM (n ≥3). Radioligand binding experiments (c), using a saturating concentration of [3H]-GR113808 (approx. 1 nM), defined 5-HT4R expression in CHO-K1 cells transiently transfected with pcDNA3-HTR4a, un-transfected CHO-K1 cells, HBEC, HASM and BEAS2B-R1 cells. Error bars indicate SEM (n ≥3). While the pcDNA3-HTR4a-transfected CHO-K1 cells expressed a substantial 5-HT4R population (approx. 460 fmol/mg protein), no other cell type expressed a significant receptor population (all ≤50 fmol/mg protein).
Mentions: Previously described gene arrangements for HTR4 are summarized in Figure 4. Transcripts a, b, i, d and g are currently reported by NCBI (December 2012); transcripts h, n, e, f and c have been described in the literature [10]. These data illustrate the complex splicing that exists at this gene locus, including multiple functionally relevant C-terminal variants, leading to alterations in cyclic AMP signalling responses [10]. Analysis by RT-PCR indicated expression of mRNA encoding HTR4 in total brain tissue, but failed to detect expression in HASM, undifferentiated HBEC, PBMC or total lung tissue (data not shown). Targeted sequencing of PCR products derived from brain cDNA confirmed the presence of both variants a (accession NM_001040169.2) and b (accession NM_000870.5), distinguished by the presence of exons 9 and 13 respectively. Additionally, exon 1 was present in variant a, although this contradicts data currently reported by NCBI (Figure 5a). Q-PCR data indicated very low levels of mean HTR4 mRNA expression in airway cells and tissues when compared to total brain tissue: total lung (0.26%), undifferentiated HBEC (0.05%), HASM (0.02%) and BEAS2B-R1 (0.16%) (Figure 5b).

Bottom Line: Meta-analyses of genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) spanning the 5-hydroxytryptamine receptor 4 (5-HT₄R) gene (HTR4) associated with lung function.Radioligand binding experiments also indicated that HBEC and HASM cells did not express a significant 5-HT₄R population. 5' RACE in brain identified a novel N-terminal variant, containing an extended N-terminal sequence.Taken together, these data suggest a role for HTR4 in lung development, which may at least in part explain the genetic association with lung function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Respiratory Medicine, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.

ABSTRACT

Background: Meta-analyses of genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) spanning the 5-hydroxytryptamine receptor 4 (5-HT₄R) gene (HTR4) associated with lung function. The aims of this study were to i) investigate the expression profile of HTR4 in adult and fetal lung tissue and cultured airway cells, ii) further define HTR4 gene structure and iii) explore the potential functional implications of key SNPs using a bioinformatic approach.

Methods: Following reverse transcription (RT)-PCR in human brain, 5' rapid amplification of cDNA ends (5' RACE) was used to examine the exonic structure of HTR4 at the 5' end. Quantitative (Q)-PCR was used to quantify HTR4 mRNA expression in total RNA from cultured airway cells and whole lung tissue. Publically available gene microarray data on fetal samples of estimated gestational age 7-22 weeks were mined for HTR4 expression. Immunohistochemistry (IHC; in adult and fetal lung tissue) and a radioligand binding assay (in cultured airway cells) were used to analyze 5-HT₄R protein expression.

Results: IHC in adult lung, irrespective of the presence of chronic obstructive pulmonary disease (COPD), suggested low level expression of 5-HT₄R protein, which was most prominent in alveolar pneumocytes. There was evidence of differential 5-HT₄R protein levels during gestation in fetal lung, which was also evident in gene expression microarray data. HTR4 mRNA expression, assessed by Q-PCR, was <0.5% relative to brain in total adult lung tissue and in human airway smooth muscle (HASM) and bronchial epithelial cells (HBEC) derived from adult donors. Radioligand binding experiments also indicated that HBEC and HASM cells did not express a significant 5-HT₄R population. 5' RACE in brain identified a novel N-terminal variant, containing an extended N-terminal sequence. The functional significance of key HTR4 SNPs was investigated using the encyclopedia of DNA elements consortium (ENCODE) dataset. These analyses identified multiple alterations in regulatory motifs for transcription factors implicated in lung development, including Foxp1.

Conclusions: Taken together, these data suggest a role for HTR4 in lung development, which may at least in part explain the genetic association with lung function.

Show MeSH
Related in: MedlinePlus