Calponin 1 contributes to myofibroblast differentiation of human pleural mesothelial cells

Pleural mesothelial cells (PMCs) can become myofibroblasts via mesothelial-mesenchymal transition (MesoMT) and contribute to pleural organization, fibrosis, and rind formation. However, how these transformed mesothelial cells contribute to lung fibrosis remains unclear. Here, we investigated the mechanism of contractile myofibroblast differentiation of PMCs. TGF-b induced marked upregulation of calponin 1 expression, which was correlated with notable cytoskeletal rearrangement in human PMCs (HPMCs) to produce stress fibers. Downregulation of calponin 1 expression reduced stress fiber formation. Interestingly, induced stress fibers predominantly contain αSMA associated with calponin 1 but not b-actin. Calponin 1 associated stress fibers also contained myosin II and α-actinin. Further, focal adhesions were aligned with the produced stress fibers. These results suggest that calponin 1 facilitates formation of stress fibers that resemble contractile myofibrils. Supporting this notion, TGF-b significantly increased the contractile activity of HPMCs, an effect that was abolished by downregulation of calponin 1 expression. We infer that differentiation of HPMCs to contractile myofibroblasts facilitates stiffness of scar tissue in pleura to promote pleural fibrosis and that upregulation of calponin 1 plays a central role in this process.

TGF-β regulation of the uPA/uPAR axis modulates mesothelial-mesenchymal transition (MesoMT)

Pleural fibrosis (PF) is a chronic and progressive lung disease which affects approximately 30,000 people per year in the United States. Injury and sustained inflammation of the pleural space can result in PF, restricting lung expansion and impairing oxygen exchange. During the progression of pleural injury, normal pleural mesothelial cells (PMCs) undergo a transition, termed mesothelial mesenchymal transition (MesoMT). While multiple components of the fibrinolytic pathway have been investigated in pleural remodeling and PF, the role of the urokinase type plasminogen activator receptor (uPAR) is unknown. We found that uPAR is robustly expressed by pleural mesothelial cells in PF. Downregulation of uPAR by siRNA blocked TGF-β mediated MesoMT. TGF-β was also found to significantly induce uPA expression in PMCs undergoing MesoMT. Like uPAR, uPA downregulation blocked TGF-β mediated MesoMT. Further, uPAR is critical for uPA mediated MesoMT. LRP1 downregulation likewise blunted TGF-β mediated MesoMT. These findings are consistent with in vivo analyses, which showed that uPAR knockout mice were protected from S. pneumoniae-mediated decrements in lung function and restriction. Histological assessments of pleural fibrosis including pleural thickening and α-SMA expression were likewise reduced in uPAR knockout mice compared to WT mice. These studies strongly support the concept that uPAR targeting strategies could be beneficial for the treatment of PF.

Anaphylatoxins orchestrate Th17 response via interactions between CD16+ monocytes and pleural mesothelial cells in tuberculous pleural effusion

The complement system is activated in tuberculous pleural effusion (TPE), with increased levels of the anaphylatoxins stimulating pleural mesothelial cells (PMCs) to secrete chemokines, which recruit nonclassical monocytes to the pleural cavity. The differentiation and recruitment of naive CD4+ T cells are induced by pleural cytokines and PMC-produced chemokines in TPE. However, it is unclear whether anaphylatoxins orchestrate CD4+ T cell response via interactions between PMCs and monocytes in TPE. In this study, CD16+ and CD16- monocytes isolated from TPE patients were cocultured with PMCs pretreated with anaphylatoxins. After removing the PMCs, the conditioned monocytes were cocultured with CD4+ T cells. The levels of the cytokines were measured in PMCs and monocyte subsets treated separately with anaphylatoxins. The costimulatory molecules were assessed in conditioned monocyte subsets. Furthermore, CD4+ T cell response was evaluated in different coculture systems. The results indicated that anaphylatoxins induced PMCs and CD16+ monocytes to secrete abundant cytokines capable of only inducing Th17 expansion, but Th1 was feeble. In addition, costimulatory molecules were more highly expressed in CD16+ than in CD16− monocytes isolated from TPE. The interactions between monocytes and PMCs enhanced the ability of PMCs and monocytes to produce cytokines and that of monocytes to express HLA-DR, CD40, CD80 and CD86, which synergistically induced Th17 expansion. In the above process, anaphylatoxins enhanced the interactions between monocytes and PMCs by increasing the level of the cytokines IL-1β, IL-6, IL-23 and upregulating the phenotype of CD40 and CD80 in CD16+ monocytes. Collectively, these data indicate that anaphylatoxins play a central role in orchestrating Th17 response mainly via interactions between CD16+ monocytes and PMCs in TPE.

Folliculin haploinsufficiency causes cellular dysfunction of pleural mesothelial cells

Birt–Hogg–Dubé syndrome (BHDS), an autosomal dominant inheritance disease caused by folliculin (FLCN) mutations, is associated with lung cysts and spontaneous pneumothorax. The possibility of FLCN haploinsufficiency in pleural mesothelial cells (PMCs) contributing to development of pneumothorax has not yet been clarified. Electron microscopy revealed exposed intercellular boundaries between PMCs on visceral pleura and decreased electron density around the adherens junctions in BHDS. To characterize cellular function of PMCs in BHDS patients (BHDS-PMCs), during surgery for pneumothorax, we established the flow cytometry-based methods of isolating high-purity PMCs from pleural lavage fluid. BHDS-PMCs showed impaired cell attachment and a significant decrease in proliferation and migration, but a significant increase in apoptosis compared with PMCs from primary spontaneous pneumothorax (PSP) patients (PSP-PMCs). Microarray analysis using isolated PMCs revealed a significant alteration in the expression of genes belonging to Gene Ontology terms “cell–cell adhesion junction” and “cell adhesion molecule binding”. Gene set enrichment analysis demonstrated that CDH1, encoding E-cadherin, was identified in the down-regulated leading edge of a plot in BHDS-PMCs. AMPK and LKB1 activation were significantly impaired in BHDS-PMCs compared with PSP-PMCs. Our findings indicate that FLCN haploinsufficiency may affect the E-cadherin-LKB1-AMPK axis and lead to abnormal cellular function in BHDS-PMCs.

Metformin Induces Apoptosis and Inhibits Notch1 in Malignant Pleural Mesothelioma Cells

Malignant pleural mesothelioma (MPM) is an aggressive asbestos-related cancer arising from the mesothelial cells lining the pleural cavity. MPM is characterized by a silent clinical progression and a highly resistance to conventional chemo/radio-therapies. MPM patients die in a few months/years from diagnosis. Notch signaling is a well-conserved cell communication system, which regulates many biological processes. In humans, the dysregulation of Notch pathway potentially contributes to cancer onset/progression, including MPM. Metformin is the first-line drug used to treat type 2 diabetes mellitus. Metformin is proven to be an effective antitumor drug in preclinical models of different types of cancer. To date, clinical efficacy is being studied in many clinical trials. In this study, the anti-proliferative effect of metformin on MPM cells and the putative involvement of Notch1 as a mediator of metformin activities, were investigated. MPM cells showed high levels of Notch1 activation compared to normal pleural mesothelial cells. Furthermore, metformin treatment hampered MPM cell proliferation and enhanced the apoptotic process, accompanied by decreased Notch1 activation.

Folliculin Haploinsufficiency Causes Cellular Dysfunction of Pleural Mesothelial Cells

Birt–Hogg–Dubé syndrome (BHDS), an autosomal dominant inheritance disease caused by folliculin (FLCN) mutations, is associated with lung cysts and spontaneous pneumothorax. The possibility of FLCN haploinsufficiency in pleural mesothelial cells (PMCs) contributing to development of pneumothorax has not yet been clarified. Electron microscopy revealed exposed intercellular boundaries between PMCs on visceral pleura and decreased electron density around the adherens junctions in BHDS. To characterize cellular function of PMCs in BHDS patients (BHDS-PMCs), during surgery for pneumothorax, we established the flow cytometry-based methods of isolating high-purity PMCs from pleural lavage fluid. BHDS-PMCs showed impaired cell attachment and a significant decrease in proliferation and migration, but a significant increase in apoptosis compared with PMCs from primary spontaneous pneumothorax (PSP) patients (PSP-PMCs). Microarray analysis using isolated PMCs revealed a significant alteration in the expression of genes belonging to Gene Ontology terms “cell-cell adhesion junction” and “cell adhesion molecule binding”. Gene set enrichment analysis demonstrated that CDH1, encoding E-cadherin, was identified in the down-regulated leading edge of a plot in BHDS-PMCs. AMPK and LKB1 activation were significantly impaired in BHDS-PMCs compared with PSP-PMCs. Our findings indicate that FLCN haploinsufficiency may affect the E-cadherin-LKB1-AMPK axis and lead to abnormal cellular function in BHDS-PMCs.