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PDGF isoform-induced proliferation and receptor expression in human cultured airway smooth muscle cells
S J Hirst1, P J Barnes, C H Twort
1Department of Allergy and Respiratory Medicine, United Medical and Dental Schools, St. Thomas' Hospital, London, United Kingdom.
Abstract:
The effect of recombinant platelet-derived growth factor (PDGF) isoforms (PDGF-AA, -BB and -AB) on mitogenesis of human cultured airway smooth muscle (ASM) was examined using the MTT-reduction assay and [3H]thymidine incorporation. Results were correlated with expression of PDGF receptor (PDGFR)-alpha and -beta subunits in the absence and presence of fetal calf serum (FCS). When FCS was absent PDGF-AB and -BB were potent mitogens, whereas PDGF-AA was weakly mitogenic, evoking <20% of the maximum response induced by the B-chain isoforms. When FCS (2.5%) was present, all PDGF isoforms stimulated marked ASM proliferation with similar efficacy and potency. Cross-competition binding analysis in FCS-deprived cells revealed that ASM cells in culture express mainly PDGFR-beta. Preincubation with PDGF-AA or PDGFR-alpha neutralizing antiserum abolished PDGF-AA binding and decreased total receptor number by approximately 15%. The ratio of PDGFR-alpha to beta subunits was approximately 1:8, supported by intense immunofluorescence staining for PDGFR-beta and weak staining for PDGFR-alpha. In parallel studies, uptake of [3H]thymidine stimulated by PDGF-AA, but not PDGF-AB or -BB, was inhibited by PDGFR-alpha immobilization. Western immunoblot analysis confirmed expression of mature PDGFR-alpha and -beta subunits in ASM cells. FCS did not cause any detectable increase in PDGFR-alpha expression or in PDGF-AA binding. These data support a role for PDGFR-beta mediating ASM mitogenesis during FCS-free conditions, but in the presence of FCS, both PDGFR-alpha and -beta subunits are linked to mitogenesis. The enhanced mitogenicity of PDGF-AA in the presence of FCS was independent of any detectable upregulation of PDGFR-alpha, suggesting that the inability of PDGF-AA to promote mitogenesis in the absence of FCS is not simply due to relative numbers of PDGFR-alpha and PDGFR-beta.
Insights
Platelet-derived growth factor (PDGF) isoforms differentially affect airway smooth muscle (ASM) cell proliferation. PDGFR-beta mediates ASM mitogenesis without fetal calf serum (FCS), while both PDGFR-alpha and -beta are involved with FCS.
Area of Science:
- Cell biology
- Molecular signaling
- Respiratory medicine
Background:
- Airway smooth muscle (ASM) cell proliferation is crucial in respiratory diseases.
- Platelet-derived growth factor (PDGF) signaling through its receptors (PDGFRs) influences cell growth.
- The specific roles of PDGF isoforms and PDGFR subunits in ASM mitogenesis require clarification.
Purpose of the Study:
- To investigate the effects of PDGF isoforms (PDGF-AA, -BB, -AB) on human ASM cell proliferation.
- To correlate mitogenic responses with the expression and function of PDGFR-alpha and -beta subunits.
- To determine the influence of fetal calf serum (FCS) on PDGF-mediated ASM mitogenesis.
Main Methods:
- MTT-reduction assay and [3H]thymidine incorporation were used to measure ASM proliferation.
- Cross-competition binding, immunofluorescence, and Western immunoblotting assessed PDGFR expression and subunit ratios.
- Neutralizing antisera and receptor immobilization were employed to investigate PDGFR function.
Main Results:
- Without FCS, PDGF-AB and -BB were potent mitogens, while PDGF-AA was weakly mitogenic.
- In the presence of FCS, all PDGF isoforms stimulated ASM proliferation similarly.
- ASM cells predominantly express PDGFR-beta, with a lower ratio of PDGFR-alpha to -beta (approx. 1:8).
Conclusions:
- PDGFR-beta mediates ASM mitogenesis in the absence of FCS.
- Both PDGFR-alpha and -beta subunits contribute to ASM proliferation when FCS is present.
- The enhanced mitogenicity of PDGF-AA with FCS is not solely due to PDGFR-alpha expression levels.