Lung fibrosis progression is closely associated with elevated levels of PAI-1 (Plasminogen Activator Inhibitor-1), a critical inhibitor of ECM (Extracellular Matrix) protein degradation that exacerbates fibrotic remodeling. Our previous study using confocal microscopy identified a close association between KIF5 and PAI-1-containing vesicles on microtubules. Knockdown of either CLSTN2 or KIF5c significantly reduced PAI-1 secretion from HPMCs and decreased the proximity signals between PAI-1 and KIF5c. Additionally, live imaging and kymograph analysis revealed that PAI-1 and KIF5c move together with directional manner along microtubules, indicating a collaborative role for CLSTN2 and KIF5c in PAI-1 containing vesicular transport to facilitate PAI-1 secretion to drive fibrosis progression. We further conducted immunofluorescent analyses on pleural tissue sections from CBB-treated and control mice. Using anti-CLSTN2 and anti-α-SMA antibodies, respectively, we found a marked increase in CLSTN2 expression along with α-SMA, a key fibrosis marker. Fluorescence in situ Hybridization (FISH) further revealed that CLSTN2 mRNA levels are substantially higher in the CBB-treated lung tissue in pleura compared to controls. These findings support the idea that myocardin-dependent CLSTN2 up-regulation facilitates the development of pleural fibrosis. To elucidate the mechanism of CLSTN2-dependent activation of PAI-1 transport, we studied the interaction among KIF5c, kinesin light chain (KLC), and CLSTN2 using the respective isolated proteins. We demonstrated that CLSTN2 associates with KIF5c through its binding to KLC. These results indicate that CLSTN2 functions as a linker to promote the association of KIF5 to PAI-1 containing vesicles. It is plausible that CLSTN2 may function as a regulator of KIF5 motor activity thus facilitating PAI-1 transport in addition to the function as a linker. Collectively, these results highlight the critical role of myocardin, CLSTN2 and KIF5c in modulating PAI-1 vesicular transport and secretion, thus playing a role as a key driver in the development of pleural fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an incurable lung disease of unknown cause. We previously reported that myocardin (MyoCD), a transcription co-activator of serum response factor (SRF) plays an important role in development of pleural fibrosis through mitigation of mesothelial-mesenchymal transition of pleural mesothelial cells. In the present study, we elucidated the role of MyoCD on myofibroblast differentiation of IPF cells. TGF-β induced up-regulation of a number of fibrosis marker genes including collagen-1, fibronectin-1, PAI-1 and α-smooth muscle actin (αSMA). Since MyoCD was markedly up-regulated after the stimulation, we examined the effect of MyoCD knock-down (KD) in the expression of fibrosis marker genes and found that MyoCD specific siRNA significantly diminished up-regulation of these genes. Moreover, MyoCD gene silencing markedly diminished TGF-b induced production of stress-fibers, suggesting that MyoCD is involved in cell contractility and motility. Supporting this view, TGF-b increased the expression of myosin IIB and smooth muscle myosin II. Moreover, phosphorylation of myosin regulatory light chain (MLC) that determines actomyosin contractile activity was significantly increased by TGF-b, which was diminished by MyoCD KD. It has been known that the relative activities of MLC phosphatases and MLC kinases regulate MLC phosphorylation. Our results indicated that TGF-b markedly increased the phosphorylation of MYPT1 (a regulatory subunit of MLC phosphatase) at the inhibitory sites, while MyoCD KD diminished MYPT1 phosphorylation. Moreover, the expression of CPI17, an MLC phosphatase inhibitor was markedly increased by TGF-b, which was diminished by MyoCD KD. These results suggest that MyoCD regulates the phosphorylation of MLC through controlling MLC phosphatase, thus regulating myosin II phosphorylation and actomyosin contractile activity in IPF cells. Using collagen gel contraction assay, we revealed that MyoCD KD attenuates the activation of cell contraction induced by TGF-β. Furthermore, using wound closure assay, we found that TGF-β suppressed the cell migration, and MyoCD silencing enhanced the wound closure, suggesting that MyoCD silencing facilitates the cell migration of IPF fibroblasts. These results suggest that MyoCD regulates myosin II phosphorylation, cell migration and cell contraction during myofibroblast differentiation of IPF cells. Further investigation on the involvement of MyoCD in cytoskeletal remodeling and cell phenotype switching during myofibroblast differentiation in IPF fibroblasts is underway.

Pleural conditions causing exudative effusions (empyema or complicated parapneumonia) can result in pathological pleural organization leading to pleural fibrosis (PF). Pleural mesothelial cells (PMCs) undergo mesenchymal transition (MesoMT) and acquire a profibrotic phenotype characterized by increased expression of ACTA2; collagen type I (Col-1); and phenotypic changes, including elongation, stress fiber formation, and contraction. Using RNA-sequencing analysis, we identified Tuftelin-1 (Tuft1) as a novel potential target. Although prior studies have shown that Tuft1 expression is associated with aggressive cellular phenotypes, its role in PF is unknown. Our prior studies show that inhibition of PI3K/Akt, mTORC2, or GSK-3β blocks MesoMT. In this study, we build on previous findings and suggest that Tuft1 plays a key role in promoting MesoMT. In human PMCs, various mediators that induce MesoMT result in upregulation of Tuft1 expression. Furthermore, we also found that Tuft1 was increased in human pleuritis tissues and in murine models of PF compared with normal lung. In our studies, TGF-β-mediated increase in Tuft1 was blocked by the GSK-3β inhibitor 9-ING-41. Knockdown of Tuft1 in vitro blocked TGF-β-mediated MesoMT. Conversely, Tuft1 overexpression induced mTORC2 signaling and promoted MesoMT in the absence of TGF-β. In vivo analyses showed that mesothelial cell-specific Tuft1 knockout mice (Tuft1PMC-/-) were protected from Streptococcus pneumoniae-mediated pleural injury. Histological analysis showed that pleural thickening and profibrotic markers were significantly reduced in Tuft1PMC-/- mice compared with wild-type control animals. These studies strongly support therapeutic targeting of Tuft1 as a novel means to mitigate PF.

Progressive lung scarring due to persistent pleural organization often results in pleural fibrosis (PF). This process affects patients with complicated parapneumonic pleural effusions, empyema, and other pleural diseases prone to loculation. In PF, pleural mesothelial cells undergo mesomesenchymal transition (MesoMT) to become profibrotic, characterized by increased expression of α-smooth muscle actin (α-SMA) and matrix proteins, including collagen (Col)-1. In our previous study, we showed that blocking PI3K/Akt signaling inhibits MesoMT induction in human pleural mesothelial cells (HPMCs). However, the downstream signaling pathways leading to MesoMT induction remain obscure. Here, we investigated the role of mammalian target of rapamycin (mTOR) complexes (mTORC1/2) in MesoMT induction. Our studies show that activation of the downstream mediator mTORC1/2 complex is likewise a critical component of MesoMT. Specific targeting of mTORC1/2 complex using pharmacological inhibitors, such as INK128 and AZD8055, significantly inhibited TGF-β-induced MesoMT markers in HPMCs. We further identified mTORC2/Rictor complex as the principal contributor to MesoMT progression induced by TGF-β. Knockdown of Rictor, but not Raptor, attenuated TGF-β induced MesoMT in these cells. In these studies, we further show that concomitant activation of the SGK1/NDRG1 signaling cascade is essential for inducing MesoMT. Targeting SGK1 and NDRG1 with siRNA and small molecular inhibitors attenuated TGF-β-induced MesoMT in HPMCs. Additionally, preclinical studies in our Streptococcus pneumoniae mediated mouse model of PF showed that inhibition of mTORC1/2 with INK128 significantly attenuated the progression of PF in sub-acute and chronic injury. In conclusion, our studies demonstrate that mTORC2/Rictor-mediated activation of SGK1/NDRG1 are critical for MesoMT induction, and targeting this pathway could inhibit or even reverse the progression of MesoMT and PF.

The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis ( Mtb ) infections. In this study, we show that bacteriophage strains D29 and DS6A can efficiently lyse Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently kills H37Rv in liquid culture and in Mtb -infected human primary macrophages. We further show in subsequent experiments that, after the humanized mice were infected with aerosolized H37Rv, then treated with DS6A intravenously, the DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduces Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrate the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.

Electrospun drug-eluting fibers have demonstrated potentials in topical drug delivery applications, where drug releases can be modulated by polymer fiber compositions. In this study, blend fibers of polycaprolactone (PCL) and polyethylene oxide (PEO) at various compositions were electrospun from 10 wt% of polymer solutions to encapsulate a model drug of ibuprofen (IBP). The results showed that the average polymer solution viscosities determined the electrospinning parameters and the resulting average fiber diameters. Increasing PEO contents in the blend PCL/PEO fibers decreased the average elastic moduli, the average tensile strength, and the average fracture strains, where IBP exhibited a plasticizing effect in the blend PCL/PEO fibers. Increasing PEO contents in the blend PCL/PEO fibers promoted the surface wettability of the fibers. The in vitro release of IBP suggested a transition from a gradual release to a fast release when increasing PEO contents in the blend PCL/PEO fibers up to 120 min. The in vitro viability of blend PCL/PEO fibers using MTT assays showed that the fibers were compatible with MEF-3T3 fibroblasts. In conclusion, our results explained the scientific correlations between the solution properties and the physicomechanical properties of electrospun fibers. These blend PCL/PEO fibers, having the ability to modulate IBP release, are suitable for topical drug delivery applications.

During phenotype switching from mesothelial to mesenchymal, pleural mesothelial cells (PMCs) produce extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1), critical components in the development of fibrosis. Here, we found that myocardin, a transcriptional coactivator of serum response factor (SRF), strongly activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression. This study provides insights about the regulation of FN1 that could lead to the development of novel interventional approaches to prevent pleural fibrosis.

During the development of pleural fibrosis, pleural mesothelial cells (PMCs) undergo phenotypic switching from differentiated mesothelial cells to mesenchymal cells (MesoMT). Here, we investigated how external stimuli such as TGF-β induce HPMC-derived myofibroblast differentiation to facilitate the development of pleural fibrosis. TGF-β significantly increased di-phosphorylation but not mono-phosphorylation of myosin II regulatory light chain (RLC) in HPMCs. An increase in RLC di-phosphorylation was also found at the pleural layer of our carbon black bleomycin (CBB) pleural fibrosis mouse model, where it showed filamentous localization that coincided with alpha smooth muscle actin (αSMA) in the cells in the pleura. Among the protein kinases that can phosphorylate myosin II RLC, ZIPK (zipper-interacting kinase) protein expression was significantly augmented after TGF-β stimulation. Furthermore, ZIPK gene silencing attenuated RLC di-phosphorylation, suggesting that ZIPK is responsible for di-phosphorylation of myosin II in HPMCs. Although TGF-β significantly increased the expression of ZIP kinase protein, the change in ZIP kinase mRNA was marginal, suggesting a posttranscriptional mechanism for the regulation of ZIP kinase expression by TGF-β. ZIPK gene knockdown (KD) also significantly reduced TGF-β-induced upregulation of αSMA expression. This finding suggests that siZIPK attenuates myofibroblast differentiation of HPMCs. siZIPK diminished TGF-β-induced contractility of HPMCs consistent with siZIPK-induced decrease in the di-phosphorylation of myosin II RLC. The present results implicate ZIPK in the regulation of the contractility of HPMC-derived myofibroblasts, phenotype switching, and myofibroblast differentiation of HPMCs.NEW & NOTEWORTHY Here, we highlight that ZIP kinase is responsible for di-phosphorylation of myosin light chain, which facilitates stress fiber formation and actomyosin-based cell contraction during mesothelial to mesenchymal transition in human pleural mesothelial cells. This transition has a significant impact on tissue remodeling and subsequent stiffness of the pleura. This study provides insight into a new therapeutic strategy for the treatment of pleural fibrosis.

Abstract Purpose: The goal of this study was to understand the role of altered mitochondrial function in breast cancer progression and determine the potential of the molecular alteration signature in developing exosome-based biomarkers. Experimental Design: This study was designed to characterize the critical components regulating mitochondrial function in breast tumorigenesis. Experiments were conducted to assess the potential of these molecules for exosome-based biomarker development. Results: We observed a remarkable reduction in spontaneous metastases through the interplay in mitochondria by SH3GL2, vesicular endocytosis–associated protein and MFN2, an important regulator of mitochondrial fusion. Following its overexpression in breast cancer cells, SH3GL2 translocated to mitochondria and induced the production of superoxide and release of cytochrome C from mitochondria to the cytoplasm. These molecular changes were accompanied by decreased lung and liver metastases and primary tumor growth. SH3GL2 depletion reversed the above phenotypic and associated molecular changes in nontumorigenic and tumorigenic breast epithelial cells. Loss of SH3GL2 and MFN2 expression was evident in primary human breast cancer tissues and their positive lymph nodes, which was associated with disease progression. SH3GL2 and MFN2 expression was detected in sera exosomes of normal healthy women, but barely detectable in the majority of the women with breast cancer exhibiting SH3GL2 and MFN2 loss in their primary tumors. Conclusions: This study identified a new mitochondria reprogramming pathway influencing breast cancer progression through SH3GL2 and MFN2. These proteins were frequently lost in breast cancer, which was traceable in the circulating exosomes. Clin Cancer Res; 22(13); 3348–60. ©2016 AACR.

SH3GL2 depletion in BCa cells.

Association between SH3GL2/MFN2 loss and progression.

SH3GL2 introduction alters various growth regulatory molecules.

Influence of SH3GL2 overexpression in BCa cells.

Percent change in E-cadherin expression after treatment.

Legends for Supplementary Figures 1-6.

Phosphorylation sites in SH3GL2.

Impact of SH3GL2 overexpression in vivo.

<p>supplemental legend</p>

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by an excess deposition of extracellular matrix in the pulmonary interstitium. Caveolin-1 scaffolding domain peptide (CSP) has been found to mitigate pulmonary fibrosis in several animal models. However, its pathophysiological role in IPF is obscure, and it remains critical to understand the mechanism by which CSP protects against pulmonary fibrosis. We first studied the delivery of CSP into cells and found that it is internalized and accumulated in the Endoplasmic Reticulum (ER). Furthermore, CSP reduced ER stress via suppression of inositol requiring enzyme1α (IRE1α) in transforming growth factor β (TGFβ)-treated human IPF lung fibroblasts (hIPF-Lfs). Moreover, we found that CSP enhanced the gelatinolytic activity of TGFβ-treated hIPF-Lfs. The IRE1α inhibitor; 4µ8C also augmented the gelatinolytic activity of TGFβ-treated hIPF-Lfs, supporting the concept that CSP induced inhibition of the IRE1α pathway. Furthermore, CSP significantly elevated expression of MMPs in TGFβ-treated hIPF-Lfs, but conversely decreased the secretion of collagen 1. Similar results were observed in two preclinical murine models of PF, bleomycin (BLM)- and adenovirus expressing constitutively active TGFβ (Ad-TGFβ)-induced PF. Our findings provide new insights into the mechanism by which lung fibroblasts contribute to CSP dependent protection against lung fibrosis.