RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis.
BACKGROUND
Idiopathic Pulmonary Fibrosis (IPF) is a debilitating disease characterized by exaggerated extracellular matrix deposition and aggressive lung structural remodeling. Disease pathogenesis is driven by fibroblastic foci formation, consequent on growth factor overexpression and myofibroblast proliferation. We have previously shown that both CTGF overexpression and myofibroblast formation in IPF cell lines are dependent on RhoA signaling. As RhoA-mediated regulation is also involved in cell cycle progression, we hypothesise that this pathway is key to lung fibroblast turnover through modulation of cyclin D1 kinetic expression.


METHODS
Cyclin D1 expression was compared in primary IPF patient-derived fibroblasts and equivalent normal control cells. Quantitative real time PCR was employed to examine relative expression levels of cyclin D1 mRNA; protein expression was confirmed by western blotting. Effects of Rho signaling were investigated using transient transfection of constitutively active and dominant negative RhoA constructs as well as pharmacological inhibitors. Cellular proliferation of lung fibroblasts was determined by BrdU incorporation ELISA. To further explore RhoA regulation of cyclin D1 in lung fibroblasts and associated cell cycle progression, an established Rho inhibitor, Simvastatin, was incorporated in our studies.


RESULTS
Cyclin D1 expression was upregulated in IPF compared to normal lung fibroblasts under exponential growth conditions (p < 0.05). Serum deprivation inhibited cyclin D1 expression, which was restored following treatment with fibrogenic growth factors (TGF-beta1 and CTGF). RhoA inhibition, using a dominant negative mutant and a pharmacological inhibitor (C3 exotoxin), suppressed levels of cyclin D1 mRNA and protein in IPF fibroblasts, with significant abrogation of cell turnover (p < 0.05). Furthermore, Simvastatin dose-dependently inhibited fibroblast cyclin D1 gene and protein expression, inducing G1 cell cycle arrest. Similar trends were observed in control experiments using normal lung fibroblasts, though exhibited responses were lower in magnitude.


CONCLUSIONS
These findings report for the first time that cyclin D1 expression is deregulated in IPF through a RhoA dependent mechanism that influences lung fibroblast proliferation. This potentially unravels new molecular targets for future anti-IPF strategies; accordingly, Simvastatin inhibition of Rho-mediated cyclin D1 expression in IPF fibroblasts merits further exploitation.
Watts
K L
KL
Lung Research, Institute of Science and Technology in Medicine, University Hospital of North Staffordshire/Keele University, Staffordshire, UK. keira_watts@yahoo.co.uk
Cottrell
E
E
Hoban
P R
PR
Spiteri
M A
MA
D036002:ADP Ribose Transferases / Q000494:pharmacology; D001905:Botulinum Toxins / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D055513:Connective Tissue Growth Factor; D019938:Cyclin D1 / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D017874:Immediate-Early Proteins / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D008168:Lung / Q000166:cytology; D011658:Pulmonary Fibrosis / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D019821:Simvastatin / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
C526317:CCN2 protein, human; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D019938:Cyclin D1; D055513:Connective Tissue Growth Factor; D019821:Simvastatin; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein