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Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells.

Stewart CE, Sayers I - BMC Mol. Biol. (2009)

Bottom Line: Real-time PCR confirmed expression of uPAR mRNA in lung, as well as airway and peripheral cell types with ~50-100 fold greater expression in peripheral cells versus airway cells and confirmed RACE data.The pattern of expression did not directly reflect that seen at the mRNA level, indicating that post-translational mechanisms of regulation may also play an important role.These data provide a novel mechanism for uPAR regulation, as different exon splicing may determine uPAR function e.g. alternative E7b results in a soluble isoform due to the loss of the GPI anchor and exon deletions may affect uPA (ligand) and/or integrin binding and therefore influence downstream pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Therapeutics and Molecular Medicine, Nottingham Respiratory Biomedical Research Unit, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK. ceri.stewart@nottingham.ac.uk

ABSTRACT

Background: Expression of the urokinase plasminogen activator receptor (UPAR) has been shown to have clinical relevance in various cancers. We have recently identified UPAR as an asthma susceptibility gene and there is evidence to suggest that uPAR may be upregulated in lung diseases such as COPD and asthma. uPAR is a key receptor involved in the formation of the serine protease plasmin by interacting with uPA and has been implicated in many physiological processes including proliferation and migration. The current aim was to determine key regulatory regions and splice variants of UPAR and quantify its expression in primary human tissues and cells (including lung, bronchial epithelium (HBEC), airway smooth muscle (HASM) and peripheral cells).

Results: Using Rapid Amplification of cDNA Ends (RACE) a conserved transcription start site (-42 to -77 relative to ATG) was identified and multiple transcription factor binding sites predicted. Seven major splice variants were identified (>5% total expression), including multiple exon deletions and an alternative exon 7b (encoding a truncated, soluble, 229aa protein). Variants were differentially expressed, with a high proportion of E7b usage in lung tissue and structural cells (55-87% of transcripts), whereas classical exon 7 (encoding the GPI-linked protein) was preferentially expressed in peripheral cells (approximately 80% of transcripts), often with exon 6 or 5+6 deletions. Real-time PCR confirmed expression of uPAR mRNA in lung, as well as airway and peripheral cell types with ~50-100 fold greater expression in peripheral cells versus airway cells and confirmed RACE data. Protein analysis confirmed expression of multiple different forms of uPAR in the same cells as well as expression of soluble uPAR in cell supernatants. The pattern of expression did not directly reflect that seen at the mRNA level, indicating that post-translational mechanisms of regulation may also play an important role.

Conclusion: We have identified multiple uPAR isoforms in the lung and immune cells and shown that expression is cell specific. These data provide a novel mechanism for uPAR regulation, as different exon splicing may determine uPAR function e.g. alternative E7b results in a soluble isoform due to the loss of the GPI anchor and exon deletions may affect uPA (ligand) and/or integrin binding and therefore influence downstream pathways. Expression of different isoforms within the lung should be taken into consideration in studies of uPAR in respiratory disease.

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Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR. A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, PMN, PBMC, THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2-ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.
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Figure 8: Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR. A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, PMN, PBMC, THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2-ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.

Mentions: Normalisation of these data for total uPAR expression allowed direct comparison with our previous RACE data which highlighted proportional expression of different splice variants in tissues/cells (Figure 8). The correlation between RACE data (Figure 3) and classical uPAR proportional expression (Figure 8A) was not clear as it may be expected that the proportional expression of this isoform should be elevated in e.g. PBMC/PMN vs. lung/HASM/HBEC as shown in the absolute expression data (Figure 7A). However, uPAR(E7b) was proportionally highly expressed in cultured cells (THP1 and BEAS2B) > lung, HASM and HBEC (about 2/3×) >> (1/50×) PBMC (not detected in PMN) (ANOVA not significant) (Figure 8E). These data correspond with RACE data (Figure 3), suggesting lower proportional expression of this variant in primary peripheral cells versus lung cells and highlighted differences between cultured cell lines (BEAS2B, THP1) and the equivalent primary cells i.e. HBEC and PBMC. Comparison of the proportional expression of the exon 4+5 deleted uPAR(E7b) form to total uPAR(E7b) expression, suggests lower expression in lung, PBMC and THP1 than HASM, HBEC and BEAS2B (Figure 8F), potentially suggesting that this splicing event occurs preferentially in structural cells. The predicted protein sequences of all confirmed splice variants are shown in Figures 4 and 5.


Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells.

Stewart CE, Sayers I - BMC Mol. Biol. (2009)

Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR. A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, PMN, PBMC, THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2-ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724484&req=5

Figure 8: Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR. A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, PMN, PBMC, THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2-ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.
Mentions: Normalisation of these data for total uPAR expression allowed direct comparison with our previous RACE data which highlighted proportional expression of different splice variants in tissues/cells (Figure 8). The correlation between RACE data (Figure 3) and classical uPAR proportional expression (Figure 8A) was not clear as it may be expected that the proportional expression of this isoform should be elevated in e.g. PBMC/PMN vs. lung/HASM/HBEC as shown in the absolute expression data (Figure 7A). However, uPAR(E7b) was proportionally highly expressed in cultured cells (THP1 and BEAS2B) > lung, HASM and HBEC (about 2/3×) >> (1/50×) PBMC (not detected in PMN) (ANOVA not significant) (Figure 8E). These data correspond with RACE data (Figure 3), suggesting lower proportional expression of this variant in primary peripheral cells versus lung cells and highlighted differences between cultured cell lines (BEAS2B, THP1) and the equivalent primary cells i.e. HBEC and PBMC. Comparison of the proportional expression of the exon 4+5 deleted uPAR(E7b) form to total uPAR(E7b) expression, suggests lower expression in lung, PBMC and THP1 than HASM, HBEC and BEAS2B (Figure 8F), potentially suggesting that this splicing event occurs preferentially in structural cells. The predicted protein sequences of all confirmed splice variants are shown in Figures 4 and 5.

Bottom Line: Real-time PCR confirmed expression of uPAR mRNA in lung, as well as airway and peripheral cell types with ~50-100 fold greater expression in peripheral cells versus airway cells and confirmed RACE data.The pattern of expression did not directly reflect that seen at the mRNA level, indicating that post-translational mechanisms of regulation may also play an important role.These data provide a novel mechanism for uPAR regulation, as different exon splicing may determine uPAR function e.g. alternative E7b results in a soluble isoform due to the loss of the GPI anchor and exon deletions may affect uPA (ligand) and/or integrin binding and therefore influence downstream pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Therapeutics and Molecular Medicine, Nottingham Respiratory Biomedical Research Unit, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK. ceri.stewart@nottingham.ac.uk

ABSTRACT

Background: Expression of the urokinase plasminogen activator receptor (UPAR) has been shown to have clinical relevance in various cancers. We have recently identified UPAR as an asthma susceptibility gene and there is evidence to suggest that uPAR may be upregulated in lung diseases such as COPD and asthma. uPAR is a key receptor involved in the formation of the serine protease plasmin by interacting with uPA and has been implicated in many physiological processes including proliferation and migration. The current aim was to determine key regulatory regions and splice variants of UPAR and quantify its expression in primary human tissues and cells (including lung, bronchial epithelium (HBEC), airway smooth muscle (HASM) and peripheral cells).

Results: Using Rapid Amplification of cDNA Ends (RACE) a conserved transcription start site (-42 to -77 relative to ATG) was identified and multiple transcription factor binding sites predicted. Seven major splice variants were identified (>5% total expression), including multiple exon deletions and an alternative exon 7b (encoding a truncated, soluble, 229aa protein). Variants were differentially expressed, with a high proportion of E7b usage in lung tissue and structural cells (55-87% of transcripts), whereas classical exon 7 (encoding the GPI-linked protein) was preferentially expressed in peripheral cells (approximately 80% of transcripts), often with exon 6 or 5+6 deletions. Real-time PCR confirmed expression of uPAR mRNA in lung, as well as airway and peripheral cell types with ~50-100 fold greater expression in peripheral cells versus airway cells and confirmed RACE data. Protein analysis confirmed expression of multiple different forms of uPAR in the same cells as well as expression of soluble uPAR in cell supernatants. The pattern of expression did not directly reflect that seen at the mRNA level, indicating that post-translational mechanisms of regulation may also play an important role.

Conclusion: We have identified multiple uPAR isoforms in the lung and immune cells and shown that expression is cell specific. These data provide a novel mechanism for uPAR regulation, as different exon splicing may determine uPAR function e.g. alternative E7b results in a soluble isoform due to the loss of the GPI anchor and exon deletions may affect uPA (ligand) and/or integrin binding and therefore influence downstream pathways. Expression of different isoforms within the lung should be taken into consideration in studies of uPAR in respiratory disease.

Show MeSH
Related in: MedlinePlus