STAT3-mediated coincidence detection regulates noncanonical immediate early gene induction.

Signaling pathways interact with one another to form dynamic networks in which the cellular response to one stimulus may depend on the presence, intensity, timing, or localization of other signals. In rare cases, two stimuli may be simultaneously required for cells to elicit a significant biological output. This phenomenon, generally termed "coincidence detection," requires a downstream signaling node that functions as a Boolean AND gate to restrict biological output from a network unless multiple stimuli are received within a specific window of time. Simultaneous activation of the EGF receptor (EGFR) and a thrombin receptor (protease-activated receptor-1, PAR-1) increases the expression of multiple immediate early genes (IEGs) associated with growth and angiogenesis. Using a bioinformatic comparison of IEG promoter regions, we identified STAT3 as a critical transcription factor for the detection of coincident EGFR/PAR-1 activation. EGFR activation induces classical STAT3 Tyr(705) phosphorylation but also initiates an inhibitory signal through the PI3K-AKT signaling axis that prevents STAT3 Ser(727) phosphorylation. Coincident PAR-1 signaling resolves these conflicting EGF-activated pathways by blocking AKT activation and permitting GSK-3α/ß-dependent STAT3 Ser(727) phosphorylation and STAT3-dependent gene expression. Functionally, combinatorial EGFR/PAR-1 signaling suppresses EGF-induced proliferation and thrombin-induced leukocyte adhesion and triggers a STAT3-dependent increase in endothelial cell migration. This study reveals a novel signaling role for STAT3 in which the simultaneous presence of extracellular EGF and thrombin is detected at the level of STAT3 post-translational modifications. Collectively, our results describe a novel regulatory mechanism in which combinatorial EGFR/PAR-1 signaling regulates STAT3-dependent IEG induction and endothelial cell migration.

Gßγ-independent recruitment of G-protein coupled receptor kinase 2 drives tumor necrosis factor α-induced cardiac ß-adrenergic receptor dysfunction.

BACKGROUND: Proinflammatory cytokine tumor necrosis factor-α (TNFα) induces ß-adrenergic receptor (ßAR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. METHODS AND RESULTS: Two different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNFα showed that TNFα alone is sufficient to mediate ßAR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling ßAR desensitization independent of sympathetic overdrive. TNFα-mediated ßAR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in ß2AR-overexpressing human embryonic kidney 293 cells showed significant ßAR desensitization, GRK2 upregulation, and recruitment to the ßAR complex following TNFα. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated ßAR phosphorylation and GRK2 recruitment on TNFα. Furthermore, TNFα-mediated ßAR phosphorylation was not blocked with ßAR antagonist propranolol. Additionally, TNFα administration in transgenic mice with cardiac overexpression of Gßγ-sequestering peptide ßARK-ct could not prevent ßAR desensitization or cardiac dysfunction showing that GRK2 recruitment to the ßAR is Gßγ independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNFα-mediated ßAR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNFα-mediated loss in contractility, showing that TNFα-induced ßAR desensitization is GRK2 dependent. CONCLUSIONS: TNFα-induced ßAR desensitization is mediated by GRK2 and is independent of Gßγ, uncovering a hitherto unknown cross-talk between TNFα and ßAR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.

Lack of mitogen-activated protein kinase phosphatase-1 protects ApoE-null mice against atherosclerosis.

RATIONALE: Multiple protein kinases have been implicated in cardiovascular disease; however, little is known about the role of their counterparts: the protein phosphatases. OBJECTIVE: To test the hypothesis that mitogen-activated protein kinase phosphatase (MKP)-1 is actively involved in atherogenesis. METHODS AND RESULTS: Mice with homozygous deficiency in MKP-1 (MKP-1(-/-)) were bred with apolipoprotein (Apo)E-deficient mice (ApoE(-/-)) and the 3 MKP-1 genotypes (MKP-1(+/+)/ApoE(-/-) ; MKP-1(+/-)/ApoE(-/-) and MKP-1(-/-)/ApoE(-/-)) were maintained on a normal chow diet for 16 weeks. The 3 groups of mice exhibited similar body weight and serum lipid profiles; however, both MKP-1(+/-) and MKP-1(-/-) mice had significantly less aortic root atherosclerotic lesion formation than MKP-1(+/+) mice. Less en face lesion was observed in 8-month-old MKP-1(-/-) mice. The reduction in atherosclerosis was accompanied by decreased plasma levels of interleukin-1alpha and tumor necrosis factor alpha, and preceded by increased antiinflammatory cytokine interleukin-10. In addition, MKP-1-null mice had higher levels of plasma stromal cell-derived factor-1a, which negatively correlated with atherosclerotic lesion size. Immunohistochemical analysis revealed that MKP-1 expression was enriched in macrophage-rich areas versus smooth muscle cell regions of the atheroma. Furthermore, macrophages isolated from MKP-1-null mice showed dramatic defects in their spreading/migration and impairment in extracellular signal-regulated kinase, but not c-Jun N-terminal kinase and p38, pathway activation. In line with this, MKP-1-null atheroma exhibited less macrophage content. Finally, transplantation of MKP-1-intact bone marrow into MKP-1-null mice fully rescued the wild-type atherosclerotic phenotype. CONCLUSION: These findings demonstrate that chronic deficiency of MKP-1 leads to decreased atherosclerosis via mechanisms involving impaired macrophage migration and defective extracellular signal-regulated kinase signaling.

Synergistic induction of mitogen-activated protein kinase phosphatase-1 by thrombin and epidermal growth factor requires vascular endothelial growth factor receptor-2.

OBJECTIVE: To determine the molecular mechanism underlying the synergistic response of mitogen-activated protein kinase phosphatase-1 (MKP-1), which is induced by thrombin and epidermal growth factor (EGF). METHODS AND RESULTS: MKP-1 induction by thrombin (approximately 6-fold) was synergistically increased (approximately 18-fold) by cotreatment with EGF in cultured endothelial cells. EGF alone did not induce MKP-1 substantially (<2-fold). The synergistic induction of MKP-1 was not mediated by matrix metalloproteinases. The EGF receptor kinase inhibitor AG1478 blocked approximately 70% of MKP-1 induction by thrombin plus EGF (from 18- to 6-fold) but not the response to thrombin alone. An extracellular signal-regulated kinase (ERK)-dependent protease-activated receptor-1 (PAR-1) signal was required for the thrombin alone effect; an ERK-independent PAR-1 signal was necessary for the approximately 12-fold MKP-1 induction by thrombin plus EGF. VEGF induction of MKP-1 was also approximately 12-fold and c-Jun N-terminal kinase (JNK) dependent. Inhibitors of extracellular signal-regulated kinase and JNK activation blocked thrombin plus EGF-induced MKP-1 completely. Furthermore, VEGF receptor 2 depletion blocked the synergistic response without affecting the induction of MKP-1 by thrombin alone. CONCLUSIONS: We have identified a novel signaling interaction between protease-activated receptor-1 and EGF receptor that is mediated by VEGF receptor 2 and results in synergistic MKP-1 induction.

Thrombin induces endothelial arginase through AP-1 activation.

Arterial thrombosis is a common disease leading to severe ischemia beyond the obstructing thrombus. Additionally, endothelial dysfunction at the site of thrombosis can be rescued by l-arginine supplementation or arginase blockade in several animal models. Exposure of rat aortic endothelial cells (RAECs) to thrombin upregulates arginase I mRNA and protein levels. In this study, we further investigated the molecular mechanism of thrombin-induced arginase changes in endothelial cells. Thrombin strikingly increased arginase I promoter and enzyme activity in primary cultured RAECs. Using different deletion and point mutations of the promoter, we demonstrated that the activating protein-1 (AP-1) consensus site located at -3,157 bp in the arginase I promoter was a thrombin-responsive element. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay further confirmed that upon thrombin stimulation, c-Jun and activating transcription factor-2 (ATF-2) bound to the AP-1 site, which initiated the transactivation. Moreover, loss-of-function studies using small interfering RNA confirmed that recruitment of these two transcription factors to the AP-1 site was required for thrombin-induced arginase upregulation. In the course of defining the signaling pathway leading to the activation of AP-1 by thrombin, we found thrombin-induced phosphorylation of stress-activated protein kinase/c-Jun-NH(2)-terminal kinase (SAPK/JNK or JNK1/2/3) and p38 mitogen-activated protein kinase, which were followed by the phosphorylation of both c-Jun and ATF-2. These findings reveal the basis for thrombin induction of endothelial arginase I and indicate that arginase inhibition may be an attractive therapeutic alternative in the setting of arterial thrombosis and its associated endothelial dysfunction.

Histone H3 as a novel substrate for MAP kinase phosphatase-1.

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a nuclear, dual-specificity phosphatase that has been shown to dephosphorylate MAP kinases. We used a "substrate-trap" technique involving a mutation in MKP-1 of the catalytically critical cysteine to a serine residue ("CS" mutant) to capture novel MKP-1 substrates. We transfected the MKP-1 (CS) mutant and control (wild-type, WT) constructs into phorbol 12-myristate 13-acetate (PMA)-activated COS-1 cells. MKP-1-substrate complexes were immunoprecipitated, which yielded four bands of 17, 15, 14, and 10 kDa with the CS MKP-1 mutant but not the WT MKP-1. The bands were identified by mass spectrometry as histones H3, H2B, H2A, and H4, respectively. Histone H3 was phosphorylated, and purified MKP-1 dephosphorylated histone H3 (phospho-Ser-10) in vitro; whereas, histone H3 (phospho-Thr-3) was unaffected. We have previously shown that thrombin and vascular endothelial growth factor (VEGF) upregulated MKP-1 in human endothelial cells (EC). We now show that both thrombin and VEGF caused dephosphorylation of histone H3 (phospho-Ser-10) and histone H3 (phospho-Thr-3) in EC with kinetics consistent with MKP-1 induction. Furthermore, MKP-1-specific small interfering RNA (siRNA) prevented VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation but had no effect on H3 (phospho-Thr-3 or Thr-11) dephosphorylation. In summary, histone H3 is a novel substrate of MKP-1, and VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation requires MKP-1. We propose that MKP-1-mediated H3 (phospho-Ser-10) dephosphorylation is a key regulatory step in EC activation by VEGF and thrombin.

Thrombospondin-1 up-regulates expression of cell adhesion molecules and promotes monocyte binding to endothelium.

Expression of cell adhesion molecules (CAM) responsible for leukocyte-endothelium interactions plays a crucial role in inflammation and atherogenesis. Up-regulation of vascular CAM-1 (VCAM-1), intracellular CAM-1 (ICAM-1), and E-selectin expression promotes monocyte recruitment to sites of injury and is considered to be a critical step in atherosclerotic plaque development. Factors that trigger this initial response are not well understood. As platelet activation not only promotes thrombosis but also early stages of atherogenesis, we considered the role of thrombospondin-1 (TSP-1), a matricellular protein released in abundance from activated platelets and accumulated in sites of vascular injury, as a regulator of CAM expression. TSP-1 induced expression of VCAM-1 and ICAM-1 on endothelium of various origins, which in turn, resulted in a significant increase of monocyte attachment. This effect could be mimicked by a peptide derived from the C-terminal domain of TSP-1 and known to interact with CD47 on the cell surface. The essential role of CD47 in the cellular responses to TSP-1 was demonstrated further using inhibitory antibodies and knockdown of CD47 with small interfering RNA. Furthermore, we demonstrated that secretion of endogenous TSP-1 and its interaction with CD47 on the cell surface mediates endothelial response to the major proinflammatory agent, tumor necrosis factor alpha (TNF-alpha). Taken together, this study identifies a novel mechanism regulating CAM expression and subsequent monocyte binding to endothelium, which might influence the development of anti-atherosclerosis therapeutic strategies.

Signal integration and gene induction by a functionally distinct STAT3 phosphoform.

Aberrant activation of the ubiquitous transcription factor STAT3 is a major driver of solid tumor progression and pathological angiogenesis. STAT3 activity is regulated by numerous posttranslational modifications (PTMs), including Tyr(705) phosphorylation, which is widely used as an indicator of canonical STAT3 function. Here, we report a noncanonical mechanism of STAT3 activation that occurs independently of Tyr(705) phosphorylation. Using quantitative liquid chromatography-tandem mass spectrometry, we have discovered and characterized a novel STAT3 phosphoform that is simultaneously phosphorylated at Thr(714) and Ser(727) by glycogen synthase kinase 3α and -ß (GSK-3α/ß). Both Thr(714) and Ser(727) are required for STAT3-dependent gene induction in response to simultaneous activation of epidermal growth factor receptor (EGFR) and protease-activated receptor 1 (PAR-1) in endothelial cells. In this combinatorial signaling context, preventing formation of doubly phosphorylated STAT3 by depleting GSK-3α/ß is sufficient to disrupt signal integration and inhibit STAT3-dependent gene expression. Levels of doubly phosphorylated STAT3 but not of Tyr(705)-phosphorylated STAT3 are remarkably elevated in clear-cell renal-cell carcinoma relative to adjacent normal tissue, suggesting that the GSK-3α/ß-STAT3 pathway is active in the disease. Collectively, our results describe a functionally distinct, noncanonical STAT3 phosphoform that positively regulates target gene expression in a combinatorial signaling context and identify GSK-3α/ß-STAT3 signaling as a potential therapeutic target in renal-cell carcinoma.

Release of nonmuscle myosin II from the cytosolic domain of tumor necrosis factor receptor 2 is required for target gene expression.

Tumor necrosis factor-α (TNF-α) elicits its biological activities through activation of TNF receptor 1 (TNFR1, also known as p55) and TNFR2 (also known as p75). The activities of both receptors are required for the TNF-α-induced proinflammatory response. The adaptor protein TNFR-associated factor 2 (TRAF2) is critical for either p55- or p75-mediated activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, as well as for target gene expression. We identified nonmuscle myosin II (myosin) as a binding partner of p75. TNF-α-dependent signaling by p75 and induction of target gene expression persisted substantially longer in cells deficient in myosin regulatory light chain (MRLC; a component of myosin) than in cells replete in myosin. In resting endothelial cells, myosin was bound constitutively to the intracellular region of p75, a region that overlaps with the TRAF2-binding domain, and TNF-α caused the rapid dissociation of myosin from p75. At early time points after exposure to TNF-α, p75 activated Rho-associated kinase 1 (ROCK1). Inhibition of ROCK1 activity blocked TNF-α-dependent phosphorylation of MRLC and the dissociation of myosin from p75. ROCK1-dependent release of myosin was necessary for the TNF-α-dependent recruitment of TRAF2 to p75 and for p75-specific activation of NF-κB and MAPK signaling. Thus, our findings have revealed a previously uncharacterized, noncanonical regulatory function of myosin in cytokine signaling.

VEGF and thrombin induce MKP-1 through distinct signaling pathways: role for MKP-1 in endothelial cell migration.

We have previously reported that MAPK phosphatase-1 (MKP-1/CL100) is a thrombin-responsive gene in endothelial cells (ECs). We now show that VEGF is another efficacious activator of MKP-1 expression in human umbilical vein ECs. VEGF-A and VEGF-E maximally induced MKP-1 expression in ECs; however, the other VEGF subtypes had no effect. Using specific neutralizing antibodies, we determined that VEGF induced MKP-1 specifically through VEGF receptor 2 (VEGFR-2), leading to the downstream activation of JNK. The VEGF-A(165) isoform stimulated MKP-1 expression, whereas the VEGF-A(162) isoform induced the gene to a lesser extent, and the VEGF-A(121) isoform had no effect. Furthermore, specific blocking antibodies against neuropilins, VEGFR-2 coreceptors, blocked MKP-1 induction. A Src kinase inhibitor (PP1) completely blocked both VEGF- and thrombin-induced MKP-1 expression. A dominant negative approach revealed that Src kinase was required for VEGF-induced MKP-1 expression, whereas Fyn kinase was critical for thrombin-induced MKP-1 expression. Moreover, VEGF-induced MKP-1 expression required JNK, whereas ERK was critical for thrombin-induced MKP-1 expression. In ECs treated with short interfering (si)RNA targeting MKP-1, JNK, ERK, and p38 phosphorylation were prolonged following VEGF stimulation. An ex vivo aortic angiogenesis assay revealed a reduction in VEGF- and thrombin-induced sprout outgrowth in segments from MKP-1-null mice versus wild-type controls. MKP-1 siRNA also significantly reduced VEGF-induced EC migration using a transwell assay system. Overall, these results demonstrate distinct MAPK signaling pathways for thrombin versus VEGF induction of MKP-1 in ECs and point to the importance of MKP-1 induction in VEGF-stimulated EC migration.

Tumor necrosis factor alpha (TNF-alpha) receptor-II is required for TNF-alpha-induced leukocyte-endothelial interaction in vivo.

Tumor necrosis factor-alpha (TNF-alpha) binds to 2 distinct cell-surface receptors: TNF-alpha receptor-I (TNFR-I: p55) and TNF-alpha receptor-II (TNFR-II: p75). TNF-alpha induces leukocyte adhesion molecules on endothelial cells (ECs), which mediate 3 defined steps of the inflammatory response; namely, leukocyte rolling, firm adhesion, and transmigration. In this study, we have investigated the role of p75 in TNF-alpha-induced leukocyte adhesion molecules using cultured ECs derived from wild-type (WT), p75-null (p75-/-), or p55-null (p55-/-) mice. We observed that p75 was essential for TNF-alpha-induced E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) expression. We also investigated the putative role of p75 in inflammation in vivo using an intravital microscopic approach with a mouse cremaster muscle model. TNF-alpha-stimulated leukocyte rolling, firm adhesion to ECs, and transmigration were dramatically reduced in p75-/- mice. Transplanted WT cremaster in p75-/- mice showed a robust leukocyte rolling and firm adhesion upon TNF-alpha activation, suggesting that the impairment in EC-leukocyte interaction in p75-/- mice is due to EC dysfunction. These results demonstrate, for the first time, that endothelial p75 is essential for TNF-alpha-induced leukocyte-endothelial-cell interaction. Our findings may contribute to the identification of novel p75-targeted therapeutic approaches for inflammatory diseases.

A VEGF165-induced phenotypic switch from increased vessel density to increased vessel diameter and increased endothelial NOS activity.

Although vascular endothelial growth factor-165 (VEGF(165)) regulates numerous angiogenic cellular activities, its complex effects on vascular morphology are not highly quantified. By fractal-based, multiparametric branching analysis of 2D vascular pattern in the quail chorioallantoic membrane (CAM), we report that vessel density increased maximally at lower VEGF concentrations, but that vessel diameter and activity of endothelial nitric oxide synthase (eNOS) increased maximally at higher VEGF concentrations. Following exogenous application of human VEGF(165) to the CAM at embryonic day 7, vessel density and diameter were measured after 24 h at arterial end points by the fractal dimension (D(f)) and generational branching parameters for vessel area density (A(v)), vessel length density (L(v)) and vessel diameter (D(v)) using the computer code VESGEN. The VEGF-dependent phenotypic switch from normal vessels displaying increased vessel density to abnormal, dilated vessels typical of tumor vasculature and other pathologies resulted from an approximate threefold increase in VEGF concentration (1.25 to 5 microg/CAM) and correlated positively with increased eNOS activity. Relative to control specimens, eNOS activity increased maximally to 60% following VEGF treatment at 5 microg/CAM, compared to 10% at 1.25 microg/CAM, and was accompanied by no significant change in activity of inducible NOS. In summary, VEGF(165) induced a phenotypic switch from increased vessel density associated with low VEGF concentration, to increased vessel diameter and increased eNOS activity at high VEGF concentration.

Role of CL-100, a dual specificity phosphatase, in thrombin-induced endothelial cell activation.

Using a cDNA microarray screening approach, we have identified seven novel thrombin-responsive genes in human umbilical vein endothelial cells that were verifiable by Northern blot analysis. Among them CL-100, a dual-specificity phosphatase also known as MAP kinase phosphatase-1 (MKP-1), showed greatest induction by thrombin. Steady-state levels of CL-100 mRNA induction by thrombin peaked at 1 h and declined rapidly (t1/2 approximately 45 min). Induction by thrombin was protease-activated receptor-1-mediated, protein synthesis-independent, and transcriptionally regulated. Metabolic labeling followed by immunoprecipitation verified that the thrombin-induced CL-100 mRNA was translated into protein. We found that both Src-kinase and p42/p44 ERK activity are critical for thrombin-induced CL-100 expression, whereas phosphatidylinositol 3-kinase and protein kinase C activity were not required. Antisense-mediated inhibition of CL-100 was shown to prolong thrombin-induced ERK activity in endothelial cells, concomitant with an inhibition in thrombin-induced PDGF-A (platelet-derived growth factor A) and PDGF-B gene expression and an up-regulation in thrombin-induced VCAM-1 and E-selectin gene expression. Inhibition of ERK activation by PD98059 in endothelial cells was shown to potentiate thrombin-induced expression of PDGF-B (approximately 3-fold) while inhibiting thrombin-induced VCAM-1 and E-selectin gene expression by 60 and 70%, respectively. These results suggested that induced expression of the CL-100 phosphatase and its subsequent regulation of ERK activity play a key regulatory role in the thrombin signaling pathway and in the transcriptional regulation of pathologically important "endothelial cell activation genes."