Enhanced upregulation of smooth muscle related transcripts by TGF beta2 in asthmatic (myo) fibroblasts.
BACKGROUND
Transforming growth factor beta (TGF beta) 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 TGFbeta(2) 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 TGF beta2 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 alpha-smooth muscle actin (alpha SMA), heavy chain myosin (HCM), calponin 1 (CPN 1), desmin, and gamma-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. TGF beta2 induced mRNA expression for all five smooth muscle related transcripts; alpha 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 TGF beta2 induced the transcription of several smooth muscle related genes, not all were translated into protein. Thus, while TGF beta2 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.
Wicks
J
J
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.
Haitchi
H M
HM
Holgate
S T
ST
Davies
D E
DE
Powell
R M
RM
D000328:Adult; D001249:Asthma / Q000378:metabolism* / Q000473:pathology; D001980:Bronchi / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D032342:Muscle Cells / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism* / Q000473:pathology; D010741:Phospholipases A; D054467:Phospholipases A2; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053781:Transforming Growth Factor beta2; D025822:Ubiquitin C / Q000378:metabolism; D015854:Up-Regulation
C509041:TGFB2 protein, human; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D025822:Ubiquitin C; D012313:RNA; D010741:Phospholipases A; D054467:Phospholipases A2