Connective tissue growth factor dependent collagen gene expression induced by MAS agonist AR234960 in human cardiac fibroblasts
Fig 3
MAS receptor present on adult human cardiac fibroblast primary cells induces CTGF and collagen expression in response to its agonist.
(A) Real-time PCR analysis shows significant upregulation of CTGF expression in response to MAS agonist (AR234960; 10μM); while MAS inverse-agonist (AR244555; 10μM) along with agonist (AR234960; 10μM) reduced CTGF expression significantly. CTGF expression decreases when MAS signaling is blocked by MEK1 inhibitor treatment. (B) Western-blot showing significant upregulation of CTGF in HCF cells treated with MAS agonist (AR234960; 10μM); the CTGF expression decreases when treated with inverse-agonist (AR244555; 10μM). MAS agonist (AR234960) activation also induces phosphorylation of ERK1/2 in HCF cells. In MAS agonist (AR234960) treated HCF cells, CTGF expression as well as ERK1/2 activation were significantly down-regulated in presence of MEK1 inhibitor (PD98059). CTGF expression and p-ERK1/2 levels were normalized by GAPDH and total ERK1/2 respectively. The western blot image shown is a representative of all the experiments done under similar experimental conditions and data from multiple experiments quantitated and cumulative data were presented as bar graphs. (C) Bar graphs showing real-time PCR analysis of different collagen sub-types (Col1A2 and Col3A1) [represented as fold increase (2‒ΔΔCt)] in HCF cells. Activated MAS induces collagen synthesis while inhibition of MAS receptor shows significant down-regulation of the same collagen sub-types. Inhibiting MEK1 also reduces expression of the same collagen sub-types. RT-qPCR was normalized by GAPDH. (*p<0.05; **p<0.01).