Enhanced upregulation of smooth muscle related transcripts by TGFβ2 in asthmatic (myo) fibroblasts
- 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:
Dr R M Powell
The Brooke Laboratory, Level F, South Block, Southampton General Hospital, Southampton SO16 6YD, UK;
- Received 22 July 2005
- Accepted 14 January 2006
- Published Online First 31 January 2006
Background: Transforming growth factor beta (TGFβ) 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 TGFβ2 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 TGFβ2 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 α-smooth muscle actin (αSMA), heavy chain myosin (HCM), calponin 1 (CPN 1), desmin, and γ-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. TGFβ2 induced mRNA expression for all five smooth muscle related transcripts; αSMA, 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 TGFβ2 induced the transcription of several smooth muscle related genes, not all were translated into protein. Thus, while TGFβ2 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.
- 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
- TGFβ, transforming growth factor β
Published Online First 31 January 2006
This study was supported by the Medical Research Council (UK), the Wellcome Trust (London, UK), and the AAIR Charity (Southampton General Hospital, Southampton UK).
Competing interests: none.