KIF5A transports collagen vesicles of myofibroblasts during pleural fibrosis
Fibrosis involves the production of extracellular matrix proteins in tissues and is often preceded by injury or trauma. In pleural fibrosis excess collagen deposition results in pleural thickening, increased stiffness and impaired lung function. Myofibroblasts are responsible for increased collagen deposition, however the molecular mechanism of transportation of procollagen containing vesicles for secretion is unknown. Here, we studied the role of kinesin on collagen-1 (Col-1) containing vesicle transportation in human pleural mesothelial cells (HPMCs). Among a number of cargo transporting kinesins, KIF5A was notably upregulated during TGF-β induced mesothelial-mesenchymal transition (MesoMT). Using superresolution structured illumination microscopy and the DUO-Link technique, we found that KIF5A colocalized with Col-1 containing vesicles. KIF5A knock-down significantly reduced Col-1 secretion and attenuated TGF-β induced increment in Col-1 localization at cell peripheries. Live cell imaging revealed that GFP-KIF5A and mCherry-Col-1 containing vesicles moved together. Kymography showed that these molecules continuously move with a mean velocity of 0.56 μm/sec, suggesting that the movement is directional but not diffusion limited process. Moreover, KIF5A was notably upregulated along with Col-1 and α-smooth muscle actin in pleural thickening in the carbon-black bleomycin mouse model. These results support our hypothesis that KIF5A is responsible for collagen transportation and secretion from HPMCs.It was previously shown that HPMCs undergo MesoMT after TGF-β stimulation. Consequently, collagen secretion was enhanced in transitioning cells23, 24. We first studied what motor proteins can contribute collagen secretion. Serum starved HPMCs were stimulated with TGF-β for 24 hours. Total mRNA was isolated from the cells, transcribed into cDNA and then subjected to quantitative PCR analysis to determine changes in mRNA level. Among members of the kinesin superfamily, we examined isoforms that are suitable for cargo transport16, 19. KIF5A, specifically, was significantly upregulated after TGF-β stimulation (Fig. 1A). Western blot analyses also showed a marked increase in KIF5A protein expression after TGF-β stimulation (Fig. 1B). Markers of MesoMT α-SMA, PAI-1 and Col-1 (mRNA and protein) were likewise increased in the lysates of TGF-β treated cells (Fig. 1A,B)7, 8, 23, 25.HPMCs were collected in a deidentified manner and cultured from the pleural effusions of patients with congestive heart failure or who have undergone coronary artery bypass graft surgery, which would have otherwise been discarded55. All samples are collected in a deidentified manner through an exempt protocol approved by the Institutional Human Subjects Review Board of the University of Texas Health Science Center at Tyler (15-004). All experiments using these cells were performed in accordance with relevant guidelines and regulations. The cells were maintained on dishes with CellBIND surface (Corning) using LHC-8 culture medium (Gibco) containing 3% fetal bovine serum (Invitrogen), 2% antibiotic-antimycotic (Invitrogen), and 1% L-glutamine (Invitrogen) in a humidified incubator at 37 °C and 5% CO2/95% air.We thank O. Sato, S. Komatsu and T. Kitazawa for valuable suggestions and discussion. This work was supported by TEXAS STARS PLUS award.The authors declare that they have no competing interests.Hirotoshi Kamata and Yoshikazu Tsukasaki contributed equally to this work.Electronic supplementary materialSupplementary information accompanies this paper at doi:10.1038/s41598-017-04437-7Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.