Abstract Rationale Pleural fibrosis (PF) is a rare disease which shows thickening of pleura and can lead to respiratory failure and death. We previously demonstrated that myocardin (MyoCD) is a regulator of genes upregulated by TGFβ. Factor Xa (FXa) is a coagulation factor that contributes to initial pathogenesis of pleural fibrosis. We attempted to clarify the mechanism of by which MyoCD regulates FXa dependent phenotypic modulation of human pleural mesothelial cells (HPMCs). Methods HPMCs were isolated from donors and RNA sequencing was performed in FXa-stimulated cells with or without MyoCD knockdown (KD). Bioinformatic analysis, quantitative RT-PCR, western-blotting, and cell migration assays were conducted to study the role of MyoCD on FXa stimulated HPMCs. Furthermore, in vivo study was conducted using Carbon Black/Bleomycin (CBB) treated mice for 3, 7, 14 days, respectively. Lung samples were collected and tissue immunofluorescence (IF) analysis was performed to investigate protein up-regulation. Results RNA-seq analysis revealed the genes activated by FXa that are dependent on MyoCD. The results showed that a group of genes related to cell migration are activated by FXa and attenuated by MyoCD knock-down (KD). Cell migration assays revealed that migration of HPMCs was promoted by FXa which was diminished by MyoCD KD. Among these genes, we focused on KIF20A, which is significantly up-regulated in the lung of bleomycin mouse disease model and human pleuritis patients. We found that KIF20A is notably up-regulated at the pleura of CBB mouse disease model and pleuritis patients. Next, we investigated the role of KIF20A in the progression of PF. First, we examined the effect of KIF20A KD on the expression of fibrosis marker genes. However, KIF20A KD did not significantly affect the expression of these genes. Next, we investigated the effect of KIF20A KD on the secretion of these markers. Interestingly, KIF20A KD markedly diminished the secretion of FN1 and PAI-1 proteins. Since MyoCD KD also diminished the secretion of these proteins, KIF20A is likely involved in the regulation of MyoCD dependent secretion of these proteins. Conclusion FXa facilitates the migratory activity of HPMCs. Moreover, we found that MyoCD regulates genes upregulated by HPMCs stimulated by FXa. Among these genes, KIF20A, a novel MyoCD effector protein, plays a role in the secretion of extracellular matrix proteins and might be related to migration of cells. These findings clearly highlight that MyoCD is a master regulator of FXa induced cell migration and PF progression. This abstract is funded by: NIH
Choi et al. (Fri,) studied this question.