PINX1 and TERT Are Required for TNF-α-Induced Airway Smooth Muscle Chemokine Gene Expression.

Airway smooth muscle (ASM) cells contribute to asthmatic lung pathology with chemokine hypersecretion and increased ASM cell mass. With little recent progress in the development of asthma therapies, a greater understanding of lung inflammation mechanisms has become a priority. Chemokine gene expression in ASM cells is dependent upon NF-κB transcription factor activity. The telomerase/shelterin complex maintains chromosomal telomere ends during cell division. Telomerase is a possible cofactor for NF-κB activity, but its role in NF-κB activity in airway tissue inflammation is not known. In this study, we sought to address two key questions: whether telomerase is involved in inflammation in ASM cells, and whether components of the shelterin complex are also required for an inflammatory response in ASM cells. Telomerase inhibitors and telomerase small interfering RNA (siRNA) reduced TNF-α-induced chemokine expression in ASM cells. Telomerase siRNA and inhibitors reduced NF-κB activity. An siRNA screen of shelterin components identified a requirement for PIN2/TERF1 interacting-telomerase inhibitor 1 (PINX1) in chemokine gene expression. High-level PINX1 overexpression reduced NF-κB reporter activity, but low-level expression amplified NF-κB activity. Coimmunoprecipitation studies showed association of PINX1 and p65. Overexpression of the N terminus (2-252 aa) of PINX1, but not the C-terminal telomerase-inhibitor domain (253-328 aa), amplified TNF-α-induced NF-κB activity. GST pull-downs demonstrated that the N terminus of PINX1 bound more p65 than the C-terminal telomerase-inhibitor domain; these observations were confirmed in whole cells with N-terminal and C-terminal PINX1 immunoprecipitation. We conclude that telomerase and PINX1 are required for chemokine expression in ASM cells and represent significant new targets for future anti-inflammatory therapies for lung diseases, such as asthma.

Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE2, inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells.

Human airway smooth muscle cells (HASMC) contribute to asthma pathophysiology through an increased smooth muscle mass and elevated cytokine/chemokine output. Little is known about how HASMC and the airway epithelium interact to regulate chronic airway inflammation and remodeling. Amphiregulin is a member of the family of epidermal growth factor receptor (EGFR) agonists with cell growth and proinflammatory roles and increased expression in the lungs of asthma patients. Here we show that bradykinin (BK) stimulation of HASMC increases amphiregulin secretion in a mechanism dependent on BK-induced COX-2 expression, increased PGE2 output, and the stimulation of HASMC EP2 and EP4 receptors. Conditioned medium from BK treated HASMC induced CXCL8, VEGF, and COX-2 mRNA and protein accumulation in airway epithelial cells, which were blocked by anti-amphiregulin antibodies and amphiregulin siRNA, suggesting a paracrine effect of HASMC-derived amphiregulin on airway epithelial cells. Consistent with this, recombinant amphiregulin induced CXCL8, VEGF, and COX-2 in airway epithelial cells. Finally, we found that conditioned media from amphiregulin-stimulated airway epithelial cells induced amphiregulin expression in HASMC and that this was dependent on airway epithelial cell COX-2 activity. Our study provides evidence of a dynamic axis of interaction between HASMC and epithelial cells that amplifies CXCL8, VEGF, COX-2, and amphiregulin production.

Elevated SP-1 transcription factor expression and activity drives basal and hypoxia-induced vascular endothelial growth factor (VEGF) expression in non-small cell lung cancer.

VEGF plays a central role in angiogenesis in cancer. Non-small cell lung cancer (NSCLC) tumors have increased microvascular density, localized hypoxia, and high VEGF expression levels; however, there is a lack of understanding of how oncogenic and tumor microenvironment changes such as hypoxia lead to greater VEGF expression in lung and other cancers. We show that NSCLC cells secreted higher levels of VEGF than normal airway epithelial cells. Actinomycin D inhibited all NSCLC VEGF secretion, and VEGF minimal promoter-luciferase reporter constructs were constitutively active until the last 85 base pairs before the transcription start site containing three SP-1 transcription factor-binding sites; mutation of these VEGF promoter SP-1-binding sites eliminated VEGF promoter activity. Furthermore, dominant negative SP-1, mithramycin A, and SP-1 shRNA decreased VEGF promoter activity, whereas overexpression of SP-1 increased VEGF promoter activity. Chromatin immunoprecipitation assays demonstrated SP-1, p300, and PCA/F histone acetyltransferase binding and histone H4 hyperacetylation at the VEGF promoter in NSCLC cells. Cultured NSCLC cells expressed higher levels of SP-1 protein than normal airway epithelial cells, and double-fluorescence immunohistochemistry showed a strong correlation between SP-1 and VEGF in human NSCLC tumors. In addition, hypoxia-driven VEGF expression in NSCLC cells was SP-1-dependent, with hypoxia increasing SP-1 activity and binding to the VEGF promoter. These studies are the first to demonstrate that overexpression of SP-1 plays a central role in hypoxia-induced VEGF secretion.

Ciclesonide inhibits TNFα- and IL-1ß-induced monocyte chemotactic protein-1 (MCP-1/CCL2) secretion from human airway smooth muscle cells.

Monocyte chemotactic protein-1 (MCP-1) is a member of the CC family of cytokines. It has monocyte and lymphocyte chemotactic activity and stimulates histamine release from basophils. MCP-1 is implicated in the pathogenesis of inflammatory diseases, including asthma. The airway smooth muscle (ASM) layer is thickened in asthma, and the growth factors and cytokines secreted by ASM cells play a role in the inflammatory response of the bronchial wall. Glucocorticoids and ß(2)-agonists are first-line drug treatments for asthma. Little is known about the effect of asthma treatments on MCP-1 production from human ASM cells. Here, we determined the effect of ciclesonide (a glucocorticoid) and formoterol (a ß(2)-agonist) on MCP-1 production from human ASM cells. TNFα and IL-1ß induced MCP-1 secretion from human ASM cells. Formoterol had no effect on MCP-1 expression, while ciclesonide significantly inhibited IL-1ß- and TNFα-induced MCP-1. Furthermore, ciclesonide inhibited IL-1ß- and TNFα-induced MCP-1 mRNA and IL-1ß- and TNFα-induced MCP-1 promoter and enhancer luciferase reporters. Western blots showed that ciclesonide had no effect on IκB degradation. Finally, ciclesonide inhibited an NF-κB luciferase reporter. Our data show that ciclesonide inhibits IL-1ß- and TNFα-induced MCP-1 production from human ASM cells via a transcriptional mechanism involving inhibition of NF-κB binding.

Endothelin-1 (ET-1) increases the expression of remodeling genes in vascular smooth muscle through linked calcium and cAMP pathways: role of a phospholipase A(2)(cPLA(2))/cyclooxygenase-2 (COX-2)/prostacyclin receptor-dependent autocrine loop.

Several important genes that are involved in inflammation and tissue remodeling are switched on by virtue of CRE response elements in their promoters. The upstream signaling mechanisms that inflammatory mediators use to activate cAMP response elements (CREs) are poorly understood. Endothelin (ET) is an important vasoactive mediator that plays roles in inflammation, vascular remodeling, angiogenesis, and carcinogenesis by activating 7 transmembrane G protein-coupled receptors (GPCR). Here we characterized the mechanisms ET-1 uses to regulate CRE-dependent remodeling genes in pulmonary vascular smooth muscle cells. These studies revealed activation pathways involving a cyclooxygenase-2 (COX-2)/prostacyclin receptor (IP receptor) autocrine loop and an interlinked calcium-dependent pathway. We found that ET-1 activated several CRE response genes in vascular smooth muscle cells, particularly COX-2, amphiregulin, follistatin, inhibin-beta-A, and CYR61. ET-1 also activated two other genes epiregulin and HB-EGF. Amphiregulin, follistatin, and inhibin-beta-A and epiregulin were activated by an autocrine loop involving cPLA2, arachidonic acid release, COX-2-dependent PGI(2) synthesis, and IP receptor-linked elevation of cAMP leading to CRE transcription activation. In contrast COX-2, CYR61, and HB-EGF transcription were regulated in a calcium-dependent, COX-2 independent, manner. Observations with IP receptor antagonists and COX-2 inhibitors were confirmed with IP receptor or COX-2-specific small interfering RNAs. ET-1 increases in intracellular calcium and gene transcription were dependent upon ET(a) activation and calcium influx through T type voltage-dependent calcium channels. These studies give important insights into the upstream signaling mechanisms used by G protein-coupled receptor-linked mediators such as ET-1, to activate CRE response genes involved in angiogenesis, vascular remodeling, inflammation, and carcinogenesis.

Novel regulation of vascular endothelial growth factor-A (VEGF-A) by transforming growth factor (beta)1: requirement for Smads, (beta)-CATENIN, AND GSK3(beta).

Vascular endothelial growth factor (VEGF) is a vital angiogenic effector, regulating key angiogenic processes. Vascular development relies on numerous signaling pathways, of which those induced by transforming growth factor-beta (TGFbeta) are critical. The Wnt/beta-catenin signaling pathway is emerging as necessary for vascular development. Although VEGF, TGFbeta, and Wnt signal transductions are well studied individually, it has not been demonstrated previously that all three can interact or be dependent on each other. We show that regulation of VEGF by TGFbeta(1), in human pulmonary artery smooth muscle cells (PASMCs), depends on a direct interaction between TGFbeta signaling proteins, Smads, and members of the Wnt/beta-catenin signaling family. VEGF promoter reporter constructs identified a region of the VEGF promoter containing two T cell factor (TCF)-binding sites as necessary for TGFbeta(1)-induced VEGF transcription. Mutation of TCF sites and expression of dominant negative TCF4 abolished TGFbeta(1)-induced VEGF promoter activity. Studies in Smad2 and Smad3 knock-out mouse embryonic fibroblasts demonstrated that one or both are required for VEGF regulation by TGFbeta(1), with transfection of dominant negative Smad2 or Smad3 into PASMCs confirming this. Chromatin immunoprecipitation assays showed in cell interactions of Smad2 and Smad3 with TCF4 and beta-catenin at the VEGF promoter, whereas co-immunoprecipitation showed a direct physical interaction between Smad2 and beta-catenin in the nucleus of PASMCs. Finally, we demonstrate that TGFbeta(1) regulates TCF by modifying beta-catenin phosphorylation via regulation of glycogen synthase kinase 3beta. These results provide new insight into the molecular regulation of VEGF by two interacting pathways necessary for vascular development, maintenance, and disease.

PKCbetaII augments NF-kappaB-dependent transcription at the CCL11 promoter via p300/CBP-associated factor recruitment and histone H4 acetylation.

The transcription factor NF-kappaB plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators, including histone acetyltransferases (HATs) such as CREB-binding protein/p300 and p300/CBP-associated factor (p/CAF). The molecular mechanisms regulating cofactor recruitment are poorly understood. In this study, we describe a novel role for protein kinase C (PKC) betaIotaIota in augmenting NF-kappaB-mediated TNF-alpha-induced transcription of the target gene CCL11 in human airway smooth muscle cells by phosphorylating the HAT p/CAF. Studies using reporters, overexpression strategies, kinase-dead and HAT-defective mutants, and chromatin immunoprecipitation showed that PKCbetaII activation was not involved in NF-kappaB translocation, but facilitated NF-kappaB-mediated CCL11 transcription by colocalizing with and phosphorylating p/CAF, and thereby acetylating histone H4 and promoting p65 association with the CCL11 promoter. The effect was dependent on p/CAF's HAT activity. Furthermore, mouse embryonic fibroblasts from PKCbeta knockout mice showed markedly reduced TNF-alpha-induced CCL11 expression and NF-kappaB reporter activity that was restored on PKCbetaII overexpression, suggesting a critical role for this pathway. These data suggest a novel important biological role for PKCbetaIotaIota in NF-kappaB-mediated CCL11 transcription by p/CAF activation and histone H4 acetylation.

NF-kappaB promotes survival during mitotic cell cycle arrest.

By activating the mitotic checkpoint, anti-microtubule drugs such as nocodazole cause mammalian cells to arrest in mitosis and then undergo apoptosis. Microtubule depolymerization is rapid and results in the activation of the transcription factor NF-kappaB and induction of NF-kappaB-dependent gene expression. However, the functional consequence of NF-kappaB activation has remained unclear. Evidence has accumulated to suggest that NF-kappaB transcriptional activity is required to suppress apoptosis. In the present study, we confirm and extend previous findings that microtubule depolymerization leads to the rapid activation of NF-kappaB and test the hypothesis that the induction of NF-kappaB regulates cell survival during mitotic cell cycle arrest in order to define its role. Using a range of functional assays, we have shown that microtubule depolymerization correlates with the activation of IKKalpha and IKKbeta; the phosphorylation, ubiquitination, and degradation of IkappaBalpha; the translocation of native p65 (RelA) into the nucleus; and increased NF-kappaB transcriptional activity. By inhibiting either the activation of the IKKs or the degradation of IkappaBalpha, we find that the level of apoptosis is significantly increased in the mitotically arrested cells. Inhibition of NF-kappaB signaling in the nonmitotic cells did not affect their survival. We establish that although NF-kappaB is activated rapidly in response to microtubule depolymerization, its cell survival function is not required until mitotic cell cycle arrest, when the mitotic checkpoint is activated and apoptosis is triggered. We conclude that NF-kappaB may regulate the transcription of one or more antiapoptotic proteins that may regulate cell survival during mitotic cell cycle arrest.

p38 Mitogen-activated protein kinase mediates cell death and p21-activated kinase mediates cell survival during chemotherapeutic drug-induced mitotic arrest.

Activation of the mitotic checkpoint by chemotherapeutic drugs such as taxol causes mammalian cells to arrest in mitosis and then undergo apoptosis. However, the biochemical basis of chemotherapeutic drug-induced cell death is unclear. Herein, we provide new evidence that both cell survival and cell death-signaling pathways are concomitantly activated during mitotic arrest by microtubule-interfering drugs. Treatment of HeLa cells with chemotherapeutic drugs activated both p38 mitogen-activated protein kinase (MAPK) and p21-activated kinase (PAK). p38 MAPK was necessary for chemotherapeutic drug-induced cell death because the p38 MAPK inhibitors SB203580 or SB202190 suppressed cell death. Dominant-active MKK6, a direct activator of p38 MAPK, also induced cell death by stimulating translocation of Bax from the cytosol to the mitochondria in a p38 MAPK-dependent manner. Dominant active PAK suppressed this MKK6-induced cell death. PAK seems to mediate cell survival by phosphorylating Bad, and inhibition of PAK in mitotically arrested cells reduced Bad phosphorylation and increased apoptosis. Our results suggest that therapeutic strategies that suppress PAK-mediated survival signals may improve the efficacy of current cancer chemotherapies by enhancing p38 MAPK-mediated cell death.