Transcriptional Regulation Of CTGF And Runx2 In Airway Smooth Muscle Cells In Asthma

BackgroundAsthma is a chronic inflammatory disease of the airways affecting more than 300 million people worldwide and characterized by airway inflammation,airway hyperresponsiveness and airway remodeling.Airway remodeling is one of the key features contributing to the progressiveness of Asthma.Airway remodeling is the structural changes of the airway wall and can include a variety of effects such as alteration of the extracellular matrix(ECM),increasing in thickness of the basal membrane and epithelial cell metaplasia.Airway remodeling can increase the volume of the airway smooth muscle bundles due to hypertrophy of hyperplasia and an increase in the number of blood vessels.Airway smooth muscle cell bundles are responsible for the contractibility and relaxation of the airways,the production and secretion of cytokines,chemokines and ECM proteins and are the main target for β-agonist treatment.The ASM mass of asthmatic airways is significantly increased when compared to non-asthmatic airways.The increased mass of airway smooth muscle cell bundle,the migration of ASMC towards the epithelial layer is a major contributor to pulmonary obstruction.Angiogenesis is the formation of new blood vessels from pre-existing ones.Angiogenesis is balanced by pro-angiogenic and anti-angiogenic factors.In asthma several pro-angiogenic factors(VEGF,angiopoeitin)are increased when compared to healthy controls.The disturbance in the pro-/anti-angiogenic balance can lead to an overgrowth or lack of new blood vessels.Angiogenesis is recognized as a major contributor in progressing severity of Asthma.The increasing number of blood vessels is correlated with the increased severity of Asthma.Connective Tissue Growth Factor(CTGF)is an important modulator of VEGF activity.In the lung,airway smooth muscle cells are predominantly responsible for the production and secretion to the ECM of VEGF and CTGF.CTGF and VEGF165 are both released form airway smooth muscle cells and co-localize in the ECM.CTGF could potentially interact through with a variety of pro-angiogenic factor such as platelet derived growth factor(PDGF),VEGF-A,slit3,von Willebrand factor and TGF-β.CTGF is responsible for many physiological functions,like wound repair,cell migration,cell proliferation,cell adhesion and skeletal development.CTGF induces the expression of VEGF and ECM proteins,such as fibronectin and collagen Ⅰ.TGF-β is able to bind to TGF-β receptor and phosphorylate receptor regulated Smads.The phosphorylation of the Smads including Smad2/3,can bind to the target gene recognizing the Smad Binding Element(SBE)then regulate the target gene expression.TGF-β could induce CTGF expression.The CTGF promoter has a SBE specific region and in other cell types,TGF-β/Smad signalling is often mentioned to be responsible for the induction of CTGF expression by binding to the SBE.There are several binding partners which have been identified for Smad2 and Smad3.One of the binding partners is Several the Runt-related transcription factor 2.Runx2 is an essential protein in the regulation of genes responsible for the formation of skeletal structures and bone.Runx2 can activate transcription through binding to the transcription factor binding site in target genes.In vascular smooth muscle cells(VSMC),Runx2 could inhibit TGF-induced CTGF promoter activity and expression.However,the mechanisms underlying the regulation of CTGF expression in A-ASM are not currently known,as well as the expression and biological activity of Runx2.In this study,we investigated the mechanisms that enhance TGF-P induction of CTGF release from A-ASM cells and the potential links to airway remodeling in asthma.And examined the Runx2 isoforms expression in ASM cells,and biopsies,from non-asthmatic and asthmatic subjects and to investigate the transcriptional function role of RUNX2 in the regulation of airway remodelingMethods1.CTGF and Runx2 expression in animal model,OVA-induced mice allergic airway disease lung tissue was provided by prof.Philip Hansbro in Newcastle University;HDM-induced sheep allergic airway disease lung tissue was provided by prof.Kenneth J Snibson in Melbourne university;Immunohistochemistry was conduct to detect CTGF and Runx2 expression in lung tissue of different animal models.2.CTGF and Runx2 expression in non-asthmatic and asthmatic human lung tissue.Human lung tissue were from resection or transplantation tissue provided by St Vincent’s Hospital Sydney,Strathfield Private Hospital,Royal Prince Alfred Hospital.CTGF and Runx2 expression in lung tissue were determined by immunohistochemistry.3.Primary Airway Smooth Muscle Cell culture and TGF-β treatment.Airway smooth muscle cells were dissected from airways of asthmatic and non,asthmatic airways and treated with TGF-β.CTGF、Runx2 and Smad3 mRNA and protein were detected by q-PCR and western blot.4.Translational and post-translational regulation of CTGF in airway smooth muscle cells.A Gluc-on reporter plasmid containing the full length CTGF promoter(-1500 to+200 bp)driving expression of a secreted Gaussia luciferase and a series of 5’deleted constructs were transfected into non-asthmatic and asthmatic ASM cells,stimulated with TGF-β1 and the supernatant was collected for luciferase detection.To investigate other potential transcription factor binding sites in close proximity to the CTGF promotor,H3K27Ac binding(a marker of transcription factor binding)was investigated.To measure CTGF mRNA stability,ASM cells were treated with TGF-β1(1ng/ml)and then stimulated with actinomycin D.Total RNA was isolated and CTGF mRNA was quantified by PCR.5.Runx2 variants transfection and effects on inflammation and airway remodeling markers.Runx2 variants were transfected into iA-ASM cells and CTGF、VEGFa、FN-1、IL-6 mRNA were measured by PCR,ELISA and western blot.6.CTGF gene expression relationship with clinical factors.We obtained high quality RNAseq data from 184 biopsies.Biopsies were derived from 77 healthy subjects and 107 current or former asthma patients.A linear model was fitted to CTGF gene expression as FEV1%predicted,basement membrane(BM)thickness,log2(PC20 to methacholine)and log2(%of eosinophils)in sputum of asthmatics.Result1.CTGF and Runx2 expression in animal model.Immunohistochemistry showed that CTGF was detected in both sham and house dust mite(HDM)-sensitised sheep lung tissues and the staining was concentrated in the airway smooth muscle layer.Runx2 expression was concentrated in epithelial layer in sham and OVA induced mice model.It is hardly see Runx2 expression in airway smooth muscle in OVA-induced mice airway tissue.Unfortunately,we could not really compare that to sham mice because the muscle layer in sham mice is hardly to recognize.2.CTGF and Runx2 expression in non-asthmatic and asthmatic human lung tissueImmunohistochemical staining showed CTGF protein expression was concentrated in the ASM area in human lung tissue,with enhanced detection visible in asthmatic tissues,particularly from severe asthmatics.Runx2 expression was concentrated in epithelial layer in non-asthmatic and asthmatic airways.However,if we amplify the airway smooth muscle section,we could observe there was obvious Runx2 expression in smooth muscle cells’ cytoplasma and nucleus in the non-asthmatic airways while only little could be seen in asthmatic airways3.CTGF and Runx2 expression as well as Smad3 activity in non-asthmatic and asthmatic airway smooth muscle cells.The increase of CTGF mRNA in A-ASM cells was 3-and 2.5-fold greater than in NA-ASM cells at 12 and 24 hours after TGF-β1 treatment,respectively.Runx2 mRNA was upregulated only in the NA-ASM cells at 8 hours and 12 hours after TGF-β treatment and there was greater Runx2 mRNA expression in NA-ASM cells than A-ASM cells at 12 hours.More smad3 was activated and transfer into nuclear in A-ASM cells after TGF-β treated4.Translational and post-translational regulation of CTGF in airway smooth muscle cells Transfection of CTGF promoter-luciferase reporter constructs into NA-and A-ASM cells indicated that no region of the CTGF promoter(-1500 to +200 bp)displayed enhanced activity in the presence of TGF-β.However,in silico analysis of the CTGF promoter suggested that distant transcription factor binding sites may influence CTGF promoter activation by TGF-β in ASM cells.The discord between promoter activity and mRNA expression was also explained,in part,by differential post-transcriptional regulation in A-ASM cells due to enhanced mRNA stability for CTGF.5.Runx2 variants transfection and effects on inflammation and airway remodeling markers.We found CTGF mRNA expression decreased in Runx2 variantl group at 12 hours after TGF-β treatment but increased again at 24 hours.we didn’t get any difference on FN1,VEGFa and IL-6 mRNA expression after transfection and TGF-β treatment compared to pcDNA3.1 group.6.CTGF gene expression relationship with clinical factors.Within asthmatic subjects,we found a significant relationship between CTGF expression and basement membrane(BM)thickness.In contrast,higher CTGF expression in asthmatic patients was not associated with lower FEV1%predicted,more severe bronchial hyperresponsiveness or higher%eosinophil levels in sputum.Conclusion.1.TGF-β signal is stronger in asthmatic airway smooth muscle cells.2.Basal promoter activity is the same between asthmatic and non-asthmatic airway smooth muscle cells.Unique transcription regulation mechanism exists in airway smooth muscle cells.3.In addition to transcription regulation,post-transcription regulation also works on CTGF expression in asthmatic airway smooth muscle cells.4.Significant relationship between CTGF expression and basement membrane(BM)thickness suggesting ASM-derived CTGF expression may influence airway narrowing and remodeling in asthma which represents an underappreciated target for future therapeutic intervention addressing an aspect of disease pathogenesis currently not effectively treated by existing approaches.5.TGF-β selectively increases Runx2 expression in non-asthmatic airway smooth muscle cells.And Runx2 could repress TGF-β induced CTGF expression witch provide new therapeutic target for asthma.