Karl Deacon

Characterization of the Mitogen-activated Protein Kinase Kinase 4 (MKK4)/c-Jun NH2-terminal kinase 1 and MKK3/p38 Pathways Regulated by MEK Kinases 2 and 3 MEK KINASE 3 ACTIVATES MKK3 BUT DOES NOT CAUSE ACTIVATION OF p38 KINASE IN VIVO

We previously reported the isolation of cDNAs encoding two mammalian mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK)kinase kinases, designated MEKK2 and MEKK3 (Blank, J.L., Gerwins, P., Elliott, E.M., Sather, S. and Johnson, G.L. (1996) J. Biol. Chem. 271, 5361–5368). In the present study, cotransfection experiments were used to examine the regulation by MEKK2 and MEKK3 of the dual specificityMAP kinase kinases, MKK3 and MKK4. MKK3 specifically phosphorylates and activates p38, whereas MKK4 phosphorylates and activates both p38 and JNK. Coexpression of MEKK2 or MEKK3 with MKK4 in COS-7 cells resulted in activation of MKK4, as assessed by enhanced autophosphorylation and by its ability to phosphorylate and activate recombinant JNK1 or p38 in vitro. MKK3 autophosphorylation and activation of p38 was also observed following coexpression of MKK3 with MEKK3, but not with MEKK2. Consistent with these observations, immunoprecipitated MEKK2 directly activated recombinant MKK4 in vitro but failed to activate MKK3. The sites of activating phosphorylation in MKK3 and MKK4 were identified within kinase subdomains VII and VIII. Replacement of Ser189 or Thr193 in MKK3 with Ala abolished autophosphorylation and activation of MKK3 by MEKK3. Analogous mutations in MKK4 indicated that Ser221 and, to a lesser extent, Thr225 were necessary for MKK4 activation by MEKK2 and MEKK3. These data indicate that MKK3 is preferentially activated by MEKK3, whereas MKK4 is activated both by MEKK2 and MEKK3. Consistent with these observations, MEKK2 and MEKK3 also activated JNK1 in vivo. However, MEKK3 failed to activate p38 when coexpressed in either the absence or presence of MKK3, indicating that MEKK3 is not coupled to p38 activation in vivo. These observations suggest that regulation of p38 and JNK1 pathways by MEKK3 may involve distinct mechanisms to prevent p38 activation but to allow JNK1 activation.

MEK Kinase 3 Directly Activates MKK6 and MKK7, Specific Activators of the p38 and c-Jun NH2-terminal Kinases

Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinasekinase kinase 3 (MEKK3) activates the c-Jun NH2-terminal kinase (JNK) pathway, although no substrates for MEKK3 have been identified. We have examined the regulation by MEKK3 of MAPK kinase 7 (MKK7) and MKK6, two novel MAPK kinases specific for JNK and p38, respectively. Coexpression of MKK7 with MEKK3 in COS-7 cells enhanced MKK7 autophosphorylation and its ability to activate recombinant JNK1 in vitro. MKK6 autophosphorylation and in vitro activation of p38α were also observed following coexpression of MKK6 with MEKK3. MEKK2, a closely related homologue of MEKK3, also activated MKK7 and MKK6 in COS-7 cells. Importantly, immunoprecipitates of either MEKK3 or MEKK2 directly activated recombinant MKK7 and MKK6 in vitro. These data identify MEKK3 as a MAPK kinase kinase specific for MKK7 and MKK6 in the JNK and p38 pathways. We have also examined whether MEKK3 or MEKK2 activates p38 in intact cells using MAPK-activated protein kinase-2 (MAPKAPK2) as an affinity ligand and substrate. Anisomycin, sorbitol, or the expression of MEKK3 in HEK293 cells enhanced MAPKAPK2 phosphorylation, whereas MEKK2 was less effective. Furthermore, MAPKAPK2 phosphorylation induced by MEKK3 or cellular stress was abolished by the p38 inhibitor SB-203580, suggesting that MEKK3 is coupled to p38 activation in intact cells.

Novel Regulation of Vascular Endothelial Growth Factor-A (VEGF-A) by Transforming Growth Factor β1 REQUIREMENT FOR Smads, β-CATENIN, AND GSK3β

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.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-β (TGFβ) are critical. The Wnt/β-catenin signaling pathway is emerging as necessary for vascular development. Although VEGF, TGFβ, 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 TGFβ1, in human pulmonary artery smooth muscle cells (PASMCs), depends on a direct interaction between TGFβ signaling proteins, Smads, and members of the Wnt/β-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 TGFβ1-induced VEGF transcription. Mutation of TCF sites and expression of dominant negative TCF4 abolished TGFβ1-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 TGFβ1, 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 β-catenin at the VEGF promoter, whereas co-immunoprecipitation showed a direct physical interaction between Smad2 and β-catenin in the nucleus of PASMCs. Finally, we demonstrate that TGFβ1 regulates TCF by modifying β-catenin phosphorylation via regulation of glycogen synthase kinase 3β. These results provide new insight into the molecular regulation of VEGF by two interacting pathways necessary for vascular development, maintenance, and disease.

Endothelin-1 (ET-1) Increases the Expression of Remodeling Genes in Vascular Smooth Muscle through Linked Calcium and cAMP Pathways ROLE OF A PHOSPHOLIPASE A2(cPLA2)/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-β-A, and CYR61. ET-1 also activated two other genes epiregulin and HB-EGF. Amphiregulin, follistatin, and inhibin-β-A and epiregulin were activated by an autocrine loop involving cPLA2, arachidonic acid release, COX-2-dependent PGI2 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 ETa 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.

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

Background: VEGF is central to cancer angiogenesis; however, we have a poor understanding of how VEGF is regulated in lung tumors. Results: High levels of SP-1 transcription factor expression amplify basal and hypoxia-induced VEGF expression. Conclusion: SP-1 plays a key role in both genetic and hypoxic microenvironment regulation of VEGF in cancer. Significance: Targeting of both VEGF and SP-1 may provide a more effective cancer therapy. 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-1β-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 β2-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 β2-agonist) on MCP-1 production from human ASM cells. TNFα and IL-1β induced MCP-1 secretion from human ASM cells. Formoterol had no effect on MCP-1 expression, while ciclesonide significantly inhibited IL-1β- and TNFα-induced MCP-1. Furthermore, ciclesonide inhibited IL-1β- and TNFα-induced MCP-1 mRNA and IL-1β- 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-1β- and TNFα-induced MCP-1 production from human ASM cells via a transcriptional mechanism involving inhibition of NF-κB binding.

Gene Expression at Sites of Wounding, Pathogen Invasion & Phenylpropanoid Synthesis in Monocots and Dicots

We have evaluated a novel system which allows the facile isolation of proteins and mRNA found specifically in wound site cells (Harikrishna et al. [1991] J.Ex. Bot. 42: 791–799) and has enabled the construction of cDNA libraries enriched for genes expressed at the wound surface. Standard differential and “cold plaque” screening indicated that over 40% of the clones in our cDNA libraries are differentially expressed in seedlings and wounded tissue. Sequence analysis of a number of wound-inducible clones has revealed that a diversity of novel genes are upregulated at wound sites, including a cytosolic Zn/Cu superoxide dismutase, a glycine-rich protein, S-Adenosyl-homocysteine hydrolase and a family of genes (AoPRl-3) related to the ‘intracellular’ class of PR proteins. These latter three gene families have been found by other laboratories to be upregulated in fungal elicitor-treated suspension cultured cells (Somssich et al. [1986], PNAS 83:2427–2430; Walter et al. [1990] MGG 222:353–360) and the data suggests that genes activated transiently at the wound surface (local induction) may be regulated by similar signal transduction pathways responsible for upregulating genes specifically induced around sites of attempted pathogen invasion in plants. The AoPRl gene family (Warner et al. [1992] PMB 19: 555–561) is massively and rapidly induced by wounding, its expression persisting for at least one week. A 1.1 kb upstream region of the AoPRl gene was fused to the GUS coding region and transferred to tobacco and potato. Histochemical analysis of transgenic plants (Warner et al [1992] The Plant J. In Press) has indicated that GUS activity is rapidly induced in the locality of the wound surface and around hypersensitive and necrotic lesions caused by fungal (Botrytis cinerea) and viral attack (PVY and TNV).