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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.

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Related in: MedlinePlus

Immunohistochemistry of 5-HT4R in COPD lung tissue. Weak 5-HT4R staining was found in the nuclei and cytoplasm of pneumoctyes in the alveolar regions of all COPD samples (a-c). In the bronchial epithelium COPD1 had weak nuclear staining (g), COPD2 had moderate nuclear and cytoplasmic staining (h) whilst COPD3 was negative for the 5-HT4R protein (i). All isotype controls were negative (d-f and j-l). X40 magnification.
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Figure 2: Immunohistochemistry of 5-HT4R in COPD lung tissue. Weak 5-HT4R staining was found in the nuclei and cytoplasm of pneumoctyes in the alveolar regions of all COPD samples (a-c). In the bronchial epithelium COPD1 had weak nuclear staining (g), COPD2 had moderate nuclear and cytoplasmic staining (h) whilst COPD3 was negative for the 5-HT4R protein (i). All isotype controls were negative (d-f and j-l). X40 magnification.

Mentions: 5-HT4R protein expression was analyzed by IHC in three COPD donors. Donor 1 (COPD1) was a 61 year old female, heavy smoker with moderate COPD; donor 2 (COPD2) was a 55 year old male, heavy smoker with severe COPD; donor 3 (COPD3) was a 48 year old female, heavy smoker with severe COPD. Weak 5-HT4R immunopositivity was found in the nuclei and cytoplasm of pneumoctyes in the alveolar regions of all three COPD samples (Figure 2a-c). The epithelium showed variable staining between donors: COPD1 had weak nuclear staining (Figure 2g), COPD2 had moderate nuclear and cytoplasmic staining (Figure 2h) whilst COPD3 was negative for the 5-HT4R protein (Figure 2i). All isotype controls were negative (Figure 2d-f and 2j-l).


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)

Immunohistochemistry of 5-HT4R in COPD lung tissue. Weak 5-HT4R staining was found in the nuclei and cytoplasm of pneumoctyes in the alveolar regions of all COPD samples (a-c). In the bronchial epithelium COPD1 had weak nuclear staining (g), COPD2 had moderate nuclear and cytoplasmic staining (h) whilst COPD3 was negative for the 5-HT4R protein (i). All isotype controls were negative (d-f and j-l). X40 magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Immunohistochemistry of 5-HT4R in COPD lung tissue. Weak 5-HT4R staining was found in the nuclei and cytoplasm of pneumoctyes in the alveolar regions of all COPD samples (a-c). In the bronchial epithelium COPD1 had weak nuclear staining (g), COPD2 had moderate nuclear and cytoplasmic staining (h) whilst COPD3 was negative for the 5-HT4R protein (i). All isotype controls were negative (d-f and j-l). X40 magnification.
Mentions: 5-HT4R protein expression was analyzed by IHC in three COPD donors. Donor 1 (COPD1) was a 61 year old female, heavy smoker with moderate COPD; donor 2 (COPD2) was a 55 year old male, heavy smoker with severe COPD; donor 3 (COPD3) was a 48 year old female, heavy smoker with severe COPD. Weak 5-HT4R immunopositivity was found in the nuclei and cytoplasm of pneumoctyes in the alveolar regions of all three COPD samples (Figure 2a-c). The epithelium showed variable staining between donors: COPD1 had weak nuclear staining (Figure 2g), COPD2 had moderate nuclear and cytoplasmic staining (Figure 2h) whilst COPD3 was negative for the 5-HT4R protein (Figure 2i). All isotype controls were negative (Figure 2d-f and 2j-l).

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