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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 normal adult human lung tissue. Low cytoplasmic and membranous expression of 5-HT4R was found in the pneumocytes of the alveoli in all three normal lung samples (a-c). The epithelium showed variable staining; donor 1 had very weak cytoplasmic staining (g), donor 2 had strong cytoplasmic and nuclear staining of >50% of nuclei (h), whilst donor 3 had weak staining in <20% of epithelial cells (i). All isotype controls were negative (d-f and j-l). X40 magnification.
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Figure 1: Immunohistochemistry of 5-HT4R in normal adult human lung tissue. Low cytoplasmic and membranous expression of 5-HT4R was found in the pneumocytes of the alveoli in all three normal lung samples (a-c). The epithelium showed variable staining; donor 1 had very weak cytoplasmic staining (g), donor 2 had strong cytoplasmic and nuclear staining of >50% of nuclei (h), whilst donor 3 had weak staining in <20% of epithelial cells (i). All isotype controls were negative (d-f and j-l). X40 magnification.

Mentions: Immunohistochemistry performed with an anti-5-HT4R antibody in normal lung tissue from three donors identified specific staining for 5-HT4R in alveolar pneumocytes (Figure 1a-c). The staining in these cells was both cytoplasmic and membranous, which is consistent with the anticipated sub-cellular expression profile for a GPCR. Some specific 5-HT4R staining was also detected in bronchial epithelial cells, but substantial variation between donors was observed in this location. While strong staining was observed in the epithelial cells of donor 2 (Figure 1h), only weak staining was observed in the other 2 donors (Figure 1g and 1i). In addition, the strong staining in donor 2 was cytoplasmic and nuclear, with little apparent staining at the plasma membrane (Figure 1h). No staining was observed in any of the isotype controls (Figure 1d-f and 1j-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 normal adult human lung tissue. Low cytoplasmic and membranous expression of 5-HT4R was found in the pneumocytes of the alveoli in all three normal lung samples (a-c). The epithelium showed variable staining; donor 1 had very weak cytoplasmic staining (g), donor 2 had strong cytoplasmic and nuclear staining of >50% of nuclei (h), whilst donor 3 had weak staining in <20% of epithelial cells (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 1: Immunohistochemistry of 5-HT4R in normal adult human lung tissue. Low cytoplasmic and membranous expression of 5-HT4R was found in the pneumocytes of the alveoli in all three normal lung samples (a-c). The epithelium showed variable staining; donor 1 had very weak cytoplasmic staining (g), donor 2 had strong cytoplasmic and nuclear staining of >50% of nuclei (h), whilst donor 3 had weak staining in <20% of epithelial cells (i). All isotype controls were negative (d-f and j-l). X40 magnification.
Mentions: Immunohistochemistry performed with an anti-5-HT4R antibody in normal lung tissue from three donors identified specific staining for 5-HT4R in alveolar pneumocytes (Figure 1a-c). The staining in these cells was both cytoplasmic and membranous, which is consistent with the anticipated sub-cellular expression profile for a GPCR. Some specific 5-HT4R staining was also detected in bronchial epithelial cells, but substantial variation between donors was observed in this location. While strong staining was observed in the epithelial cells of donor 2 (Figure 1h), only weak staining was observed in the other 2 donors (Figure 1g and 1i). In addition, the strong staining in donor 2 was cytoplasmic and nuclear, with little apparent staining at the plasma membrane (Figure 1h). No staining was observed in any of the isotype controls (Figure 1d-f and 1j-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