Myocardin regulates fibronectin expression and secretion from human pleural mesothelial cells.

During the progression of pleural fibrosis, pleural mesothelial cells (PMCs) undergo a phenotype switching process known as mesothelial-mesenchymal transition (MesoMT). During MesoMT, transformed PMCs become myofibroblasts that produce increased extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1) that is critical to develop fibrosis. Here, we studied the mechanism that regulates FN1 expression in myofibroblasts derived from human pleural mesothelial cells (HPMCs). We found that myocardin (Myocd), a transcriptional coactivator of serum response factor (SRF) and a master regulator of smooth muscle and cardiac muscle differentiation, strongly controls FN1 gene expression. Myocd gene silencing markedly inhibited FN1 expression. FN1 promoter analysis revealed that deletion of the Smad3-binding element diminished FN1 promoter activity, whereas deletion of the putative SRF-binding element increased FN1 promoter activity. Smad3 gene silencing decreased FN1 expression, whereas SRF gene silencing increased FN1 expression. Moreover, SRF competes with Smad3 for binding to Myocd. These results indicate that Myocd activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression in HPMCs. In HPMCs, TGF-ß induced Smad3 nuclear localization, and the proximity ligation signal between Myocd and Smad3 was markedly increased after TGF-ß stimulation at nucleus, suggesting that TGF-ß facilitates nuclear translocation of Smad3 and interaction between Smad3 and Myocd. Moreover, Myocd and Smad3 were coimmunoprecipitated and isolated Myocd and Smad3 proteins directly bound each other. Chromatin immunoprecipitation assays revealed that Myocd interacts with the FN1 promoter at the Smad3-binding consensus sequence. The results indicate that Myocd regulates FN1 gene activation through interaction and activation of the Smad3 transcription factor.NEW & NOTEWORTHY 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.

Role of ZIP kinase in development of myofibroblast differentiation from HPMCs.

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-ß induce HPMC-derived myofibroblast differentiation to facilitate the development of pleural fibrosis. TGF-ß 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-ß stimulation. Furthermore, ZIPK gene silencing attenuated RLC di-phosphorylation, suggesting that ZIPK is responsible for di-phosphorylation of myosin II in HPMCs. Although TGF-ß 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-ß. ZIPK gene knockdown (KD) also significantly reduced TGF-ß-induced upregulation of αSMA expression. This finding suggests that siZIPK attenuates myofibroblast differentiation of HPMCs. siZIPK diminished TGF-ß-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.

Myo5b Transports Fibronectin-Containing Vesicles and Facilitates FN1 Secretion from Human Pleural Mesothelial Cells.

Pleural mesothelial cells (PMCs) play a central role in the progression of pleural fibrosis. As pleural injury progresses to fibrosis, PMCs transition to mesenchymal myofibroblast via mesothelial mesenchymal transition (MesoMT), and produce extracellular matrix (ECM) proteins including collagen and fibronectin (FN1). FN1 plays an important role in ECM maturation and facilitates ECM-myofibroblast interaction, thus facilitating fibrosis. However, the mechanism of FN1 secretion is poorly understood. We report here that myosin 5b (Myo5b) plays a critical role in the transportation and secretion of FN1 from human pleural mesothelial cells (HPMCs). TGF-ß significantly increased the expression and secretion of FN1 from HPMCs and facilitates the close association of Myo5B with FN1 and Rab11b. Moreover, Myo5b directly binds to GTP bound Rab11b (Rab11b-GTP) but not GDP bound Rab11b. Myo5b or Rab11b knockdown via siRNA significantly attenuated the secretion of FN1 without changing FN1 expression. TGF-ß also induced Rab11b-GTP formation, and Rab11b-GTP but not Rab11b-GDP significantly activated the actin-activated ATPase activity of Myo5B. Live cell imaging revealed that Myo5b- and FN1-containing vesicles continuously moved together in a single direction. These results support that Myo5b and Rab11b play an important role in FN1 transportation and secretion from HPMCs, and consequently may contribute to the development of pleural fibrosis.

Mitochondria-associated myosin 19 processively transports mitochondria on actin tracks in living cells.

Mitochondria are fundamentally important in cell function, and their malfunction can cause the development of cancer, cardiovascular disease, and neuronal disorders. Myosin 19 (Myo19) shows discrete localization with mitochondria and is thought to play an important role in mitochondrial dynamics and function; however, the function of Myo19 in mitochondrial dynamics at the cellular and molecular levels is poorly understood. Critical missing information is whether Myo19 is a processive motor that is suitable for transportation of mitochondria. Here, we show for the first time that single Myo19 molecules processively move on actin filaments and can transport mitochondria in cells. We demonstrate that Myo19 dimers having a leucine zipper processively moved on cellular actin tracks in demembraned cells with a velocity of 50 to 60 nm/s and a run length of ∼0.4 µm, similar to the movement of isolated mitochondria from Myo19 dimer-transfected cells on actin tracks, suggesting that the Myo19 dimer can transport mitochondria. Furthermore, we show single molecules of Myo19 dimers processively moved on single actin filaments with a large step size of ∼34 nm. Importantly, WT Myo19 single molecules without the leucine zipper processively move in filopodia in living cells similar to Myo19 dimers, whereas deletion of the tail domain abolished such active movement. These results suggest that Myo19 can processively move on actin filaments when two Myo19 monomers form a dimer, presumably as a result of tail-tail association. In conclusion, Myo19 molecules can directly transport mitochondria on actin tracks within living cells.

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. Transforming growth factor-ß (TGF-ß) 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 α-smooth muscle actin (αSMA) associated with calponin 1 but not ß-actin. Calponin 1-associated stress fibers also contained myosin II and α-actinin. Furthermore, 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-ß 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 (PF) and that upregulation of calponin 1 plays a central role in this process.

Sappanone A inhibits RANKL-induced osteoclastogenesis in BMMs and prevents inflammation-mediated bone loss.

Receptor activator of nuclear factor-kB ligand (RANKL) is a key factor in the differentiation and activation of osteoclasts. Suppressing osteoclastogenesis is considered an effective therapeutic approach for bone-destructive diseases, such as osteoporosis and rheumatoid arthritis. Sappanone A (SPNA), a homoisoflavanone compound isolated from the heartwood of Caesalpinia sappan, has been reported to exert anti-inflammatory effects; however, the effects of SPNA on osteoclastogenesis have not been investigated. In the present study, we describe for the first time that SPNA inhibits RANKL-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs) and suppresses inflammation-induced bone loss in a mouse model. SPNA inhibited the formation of osteoclasts from BMMs, osteoclast actin-ring formation, and bone resorption in a concentration-dependent manner. At the molecular level, SPNA significantly inhibited RANKL-induced activation of the AKT/glycogen synthase kinase-3ß (GSK-3ß) signaling pathway without affecting its activation of the mitogen-activated protein kinases (MAPKs) JNK, p38, and ERK. In addition, SPNA suppressed the induction of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), which is a crucial transcription factor in osteoclast differentiation. As a result, SPNA decreased osteoclastogenesis-related marker gene expression, including CtsK, TRAP, dendritic cell-specific transmembrane protein (DC-STAMP), MMP-9 and osteoclast-associated receptor (OSCAR). In a mouse inflammatory bone loss model, SPNA significantly inhibited lipopolysaccharide (LPS)-induced bone loss by suppressing the number of osteoclasts. Taken together, these findings suggest that SPNA inhibits osteoclastogenesis and bone resorption by inhibiting the AKT/GSK-3ß signaling pathway and may be a potential candidate compound for the prevention and/or treatment of inflammatory bone loss.

7-Methoxy-(9H-ß-Carbolin-1-il)-(E)-1-Propenoic Acid, a ß-Carboline Alkaloid From Eurycoma longifolia, Exhibits Anti-Inflammatory Effects by Activating the Nrf2/Heme Oxygenase-1 Pathway.

Eurycoma longifolia is an herbal medicinal plant popularly used in Southeast Asian countries. In the present study, we show that 7-methoxy-(9H-ß-carbolin-1-il)-(E)-1-propenoic acid (7-MCPA), a ß-carboline alkaloid isolated from E. longifolia, exerted anti-inflammatory effects by activating the nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. 7-MCPA inhibited lipopolysaccharide (LPS)-induced production of nitric oxide (NO), prostaglandin E2 (PGE2 ), and interleukin-6 (IL-6) in RAW264.7 cells and rescued C57BL/6 mice from LPS-induced lethality in vivo. LPS-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and IL-6 was also significantly suppressed by treatment of 7-MCPA in RAW264.7 cells. 7-MCPA induced nuclear translocation of Nrf2 and increased transcription of its target genes, such as HO-1. Treating RAW264.7 cells with 7-MCPA increased the intracellular level of reactive oxygen species (ROS) and the phosphorylation level of p38 mitogen-activated protein kinase (MAPK); however, co-treatment with the antioxidant N-acetyl-cysteine (NAC) blocked 7-MCPA-induced p38 MAPK phosphorylation. Moreover, NAC or SB203580 (p38 MAPK inhibitor) blocked 7-MCPA-induced nuclear translocation of Nrf2, suggesting that 7-MCPA activated Nrf2 via a ROS-dependent p38 pathway. 7-MCPA induced HO-1 protein and mRNA expression and knockdown of Nrf2 with siRNA or SB203580 blocked 7-MCPA-mediated induction of HO-1 expression. Inhibiting Nrf2 or HO-1 abrogated the anti-inflammatory effects of 7-MCPA in LPS-stimulated RAW264.7 cells. We also demonstrated that 7-MCPA suppressed LPS-induced nuclear factor κB (NF-κB) activation. These results provide the first evidence that 7-MCPA exerts its anti-inflammatory effect by modulating the Nrf2 and NF-κB pathways and may be a potential Nrf2 activator to prevent or treat inflammatory diseases.

Sappanone A exhibits anti-inflammatory effects via modulation of Nrf2 and NF-κB.

Homoisoflavonoids constitute a small class of natural products. In the present study, we investigated the anti-inflammatory effect of sappanone A (SPNA), a homoisoflavanone that is isolated from the heartwood of Caesalpinia sappan (Leguminosae), in murine macrophages. SPNA inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2) and interleukin-6 (IL-6) as well as the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and IL-6 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Moreover, SPNA protected C57BL/6 mice from LPS-induced mortality. Treatment of RAW264.7 cells with SPNA induced heme oxygenase (HO)-1 protein and mRNA expression and increased nuclear translocation of the nuclear factor-E2-related factor 2 (Nrf2) as well as the expression of Nrf2 target genes such as NAD(P)H: quinone oxidoreductase 1 (NQO1). Knockdown of Nrf2 by siRNA blocked SPNA-mediated HO-1 induction. SB203580, p38 mitogen-activated protein kinase (MAPK) inhibitor, blocked SPNA-induced HO-1 expression and nuclear translocation of Nrf2, suggesting that SPNA induces HO-1 expression by activating Nrf2 through the p38 MAPK pathway. Consistent with the notion that the Nrf2/HO-1 pathway has anti-inflammatory properties, inhibiting HO-1 significantly abrogated the anti-inflammatory effects of SPNA in LPS-stimulated RAW264.7 cells. Moreover, SPNA suppressed LPS-induced nuclear factor κB (NF-κB) activation via inhibiting Ser 536 phosphorylation and transcriptional activity of RelA/p65 subunit of NF-κB. Taken together, these findings suggest that SPNA exerts its anti-inflammatory effect by modulating the Nrf2 and NF-κB pathways, and may be a valuable compound to prevent or treat inflammatory diseases.

The anti-inflammatory effect of 3-deoxysappanchalcone is mediated by inducing heme oxygenase-1 via activating the AKT/mTOR pathway in murine macrophages.

3-Deoxysappanchalcone (3-DSC), isolated from Caesalpinia sappan (Leguminosae), is a chalcone that exerts a variety of pharmacological activities. In the present study, we demonstrated that 3-DSC exerts anti-inflammatory activity in murine macrophages by inducing heme oxygenase-1 (HO-1) expression at the translational level. Treatment of RAW264.7 cells with 3-DSC induced HO-1 protein expression in a dose- and time-dependent manner without affecting HO-1 mRNA expression. Mitogen-activated protein kinase inhibitors or actinomycin D, a transcriptional inhibitor, did not block 3-DSC-mediated HO-1 induction. However, 3-DSC-mediated HO-1 induction was completely blocked by treatment with cycloheximide, a translational inhibitor, or rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR). Strikingly, 3-DSC increased the phosphorylation level of mTOR downstream target molecules such as eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase 1 (S6K1), as well as AKT in a dose- and time-dependent manner, suggesting that the 3-DSC induces HO-1 expression by activating the AKT/mTOR pathway. Consistent with the notion that HO-1 has anti-inflammatory properties, 3-DSC inhibited the production of nitric oxide (NO) and interleukin (IL)-6 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Inhibition of HO-1 activity by treatment with tin protoporphyrin IX, a specific HO-1 inhibitor, abrogated the inhibitory effects of 3-DSC on the production of NO and IL-6 in LPS-stimulated RAW264.7 cells. Taken together, 3-DSC may be an effective HO-1 inducer at the translational level that has anti-inflammatory effects, and a valuable compound for modulating inflammatory conditions.