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ASTHMA |
The Brooke Laboratories, Allergy and Inflammation Research, Division of Infection, Inflammation and Repair, School of Medicine, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK
Correspondence to:
Correspondence to:
Dr R M Powell
The Brooke Laboratory, Level F, South Block, Southampton General Hospital, Southampton SO16 6YD, UK; rmp2{at}soton.ac.uk
Background: Transforming growth factor beta (TGFß) upregulates a number of smooth muscle specific genes in (myo)fibroblasts. As asthma is characterised by an increase in airway smooth muscle, we postulated that TGFß2 favours differentiation of asthmatic (myo)fibroblasts towards a smooth muscle phenotype.
Methods: Primary fibroblasts were grown from bronchial biopsy specimens from normal (n = 6) and asthmatic (n = 7) donors and treated with TGFß2 to induce myofibroblast differentiation. The most stable genes for normalisation were identified using RT-qPCR and the geNorm software applied to a panel of 12 "housekeeping" genes. Expression of 
-smooth muscle actin (SMA), heavy chain myosin (HCM), calponin 1 (CPN 1), desmin, and 
-actin were measured by RT-qPCR. Protein expression was assessed by immunocytochemistry and western blotting.
Results: Phospholipase A2 and ubiquitin C were identified as the most stably expressed and practically useful genes for normalisation of gene expression during myofibroblast differentiation. TGFß2 induced mRNA expression for all five smooth muscle related transcripts; SMA, HCM and CPN 1 protein were also increased but desmin protein was not detectable. Although there was no difference in basal expression, HCM, CPN 1 and desmin were induced to a significantly greater extent in asthmatic fibroblasts than in those from normal controls (p = 0.041 and 0.011, respectively).
Conclusions: Although TGFß2 induced the transcription of several smooth muscle related genes, not all were translated into protein. Thus, while TGFß2 is unable to induce a bona fide smooth muscle cell phenotype, it may "prime" (myo)fibroblasts for further differentiation, especially if the cells are derived from asthmatic airways.
Abbreviations: CPN 1, calponin 1; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HCM, heavy chain myosin; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SFM, serum free medium; SMA, smooth muscle actin; TGFß, transforming growth factor ß
Keywords: asthma; smooth muscle markers; transforming growth factor ß
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