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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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Protein sequences of uPAR internal exon splice variants. Alternate splice variants which were identified and confirmed by real time PCR in the current analyses are shown. A signal peptide removed during processing (not included in the numbering of mature peptide) is highlighted green and the terminal domain (pink) is removed during processing to give a GPI anchor at the new C-terminus.
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Figure 5: Protein sequences of uPAR internal exon splice variants. Alternate splice variants which were identified and confirmed by real time PCR in the current analyses are shown. A signal peptide removed during processing (not included in the numbering of mature peptide) is highlighted green and the terminal domain (pink) is removed during processing to give a GPI anchor at the new C-terminus.

Mentions: To identify splice variants and determine the exon structure of uPAR, 5' and 3' RACE were completed in six different tissues/cell types (Figures 2 and 3). 5' RACE identified a localised TSS (see earlier) and demonstrated that exons 1 and 2 were conserved in all cells/tissues (data not shown). An exon 3 deletion variant was identified at low frequency in differentiated HBEC (4.5% of clones sequenced), PMN (4.5%) and PBMC (4.3%). 3' RACE confirmed the expression of the classical exon 7 as well as the alternative exon 7b, which was present with a truncated or extended 3'UTR (Figure 3). The pattern of expression of splice variants differed between tissues/cell types (Chi-square p < 0.001). Several exon deletion variants were identified including; exon 5, 6 and 5+6 deletions, although only the exon 6 deletion had a frequency >5% (Figure 3). In addition PCR products spanning exons 1–7 and 1–7b were cloned and sequenced to determine which exon deletions were found in combination with the two alternative terminal exons, demonstrating that an additional exon 4+5 deletion exists (data not shown). Interestingly, the structural cell types (HASM, HBEC) and lung appeared to express mostly exon 7b (55–87% of clones sequenced), encoding a soluble variant of uPAR, whilst more classical membrane bound uPAR (≈ 80%) was detected in the peripheral cells (PMN and PBMC) (when all exon deleted forms were pooled). In RACE, exon-deleted transcripts were cloned from all cell types except differentiated HBEC and HASM. These results were confirmed by full-length PCR, when no exon-deleted forms were obtained for differentiated HBEC and 2/48 clones for HASM, which would not have been detectable by RACE based on primer design (del3 and del4+5). Lung tissue showed 8% exon deleted clones in the RACE analysis; these were exon 5 or 6 deletions, in combination with exon 7b. Of the lung cells, undifferentiated HBEC showed the largest number of exon-deleted forms (7/44 clones analysed, 16%), which included three unique clones showing non-classical splice sites (data not shown). The two peripheral cell types analysed showed high frequencies of exon deleted forms in the RACE analysis (PMN: 14/22, 64%; PBMC: 10/21, 48%). These were predominantly exon 6 deletions, although del5 and del5+6 were also observed. PCR analysis of PBMC confirmed these data with 16/24 exon1–7 clones showing exon deletions (including del6 (5/24), del5 (4/34) and del5+6 (3/24)) and 4/24 exon 1–7b clones showing deletions (including del6 (1/24) and del5 (2/24)). All classical exon deletions are in frame deletions. See Table 1 for molecular details of splice variation and Figures 4 and 5 for predicted protein sequences.


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

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

Protein sequences of uPAR internal exon splice variants. Alternate splice variants which were identified and confirmed by real time PCR in the current analyses are shown. A signal peptide removed during processing (not included in the numbering of mature peptide) is highlighted green and the terminal domain (pink) is removed during processing to give a GPI anchor at the new C-terminus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Protein sequences of uPAR internal exon splice variants. Alternate splice variants which were identified and confirmed by real time PCR in the current analyses are shown. A signal peptide removed during processing (not included in the numbering of mature peptide) is highlighted green and the terminal domain (pink) is removed during processing to give a GPI anchor at the new C-terminus.
Mentions: To identify splice variants and determine the exon structure of uPAR, 5' and 3' RACE were completed in six different tissues/cell types (Figures 2 and 3). 5' RACE identified a localised TSS (see earlier) and demonstrated that exons 1 and 2 were conserved in all cells/tissues (data not shown). An exon 3 deletion variant was identified at low frequency in differentiated HBEC (4.5% of clones sequenced), PMN (4.5%) and PBMC (4.3%). 3' RACE confirmed the expression of the classical exon 7 as well as the alternative exon 7b, which was present with a truncated or extended 3'UTR (Figure 3). The pattern of expression of splice variants differed between tissues/cell types (Chi-square p < 0.001). Several exon deletion variants were identified including; exon 5, 6 and 5+6 deletions, although only the exon 6 deletion had a frequency >5% (Figure 3). In addition PCR products spanning exons 1–7 and 1–7b were cloned and sequenced to determine which exon deletions were found in combination with the two alternative terminal exons, demonstrating that an additional exon 4+5 deletion exists (data not shown). Interestingly, the structural cell types (HASM, HBEC) and lung appeared to express mostly exon 7b (55–87% of clones sequenced), encoding a soluble variant of uPAR, whilst more classical membrane bound uPAR (≈ 80%) was detected in the peripheral cells (PMN and PBMC) (when all exon deleted forms were pooled). In RACE, exon-deleted transcripts were cloned from all cell types except differentiated HBEC and HASM. These results were confirmed by full-length PCR, when no exon-deleted forms were obtained for differentiated HBEC and 2/48 clones for HASM, which would not have been detectable by RACE based on primer design (del3 and del4+5). Lung tissue showed 8% exon deleted clones in the RACE analysis; these were exon 5 or 6 deletions, in combination with exon 7b. Of the lung cells, undifferentiated HBEC showed the largest number of exon-deleted forms (7/44 clones analysed, 16%), which included three unique clones showing non-classical splice sites (data not shown). The two peripheral cell types analysed showed high frequencies of exon deleted forms in the RACE analysis (PMN: 14/22, 64%; PBMC: 10/21, 48%). These were predominantly exon 6 deletions, although del5 and del5+6 were also observed. PCR analysis of PBMC confirmed these data with 16/24 exon1–7 clones showing exon deletions (including del6 (5/24), del5 (4/34) and del5+6 (3/24)) and 4/24 exon 1–7b clones showing deletions (including del6 (1/24) and del5 (2/24)). All classical exon deletions are in frame deletions. See Table 1 for molecular details of splice variation and Figures 4 and 5 for predicted protein sequences.

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