Aberrant activation of the ubiquitous transcription factor STAT3 is a major driver of solid tumor... more 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 -β (GSK-3α/β). 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 depl...
We have previously reported that MAPK phosphatase-1 (MKP-1/CL100) is a thrombin-responsive gene i... more 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-A165 isoform stimulated MKP-1 expression, whereas the VEGF-A162 isoform induced the gene to a lesser extent, and the VEGF-A121 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 wa...
Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a nuclear, dual-specificity phosp... more 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...
Rationale: Multiple protein kinases have been implicated in cardiovascular disease; however, litt... more 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-1α and tumor necrosis factor α, and precede...
ABSTRACT Signaling interactions between G-protein coupled receptors (GPCRs) and receptor tyrosine... more ABSTRACT Signaling interactions between G-protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) function in a number of pathophysiological processes, including cellular proliferation, invasion, and acquired resistance to chemotherapeutic agents. Here, we examine the consequences of simultaneous EGF receptor (EGFR) and thrombin receptor (protease-activated receptor-1, PAR-1) activation in human endothelial cells (EC). We observe that concurrent EGFR and PAR-1 activation causes the synergistic induction of at least six pro-angiogenic immediate early genes (IEGs), including the transcription factor early growth response-1 (EGR-1). In contrast to other cell types, matrix metalloprotease-mediated transactivation of EGFR by PAR-1 does not appear to be a major signaling mechanism in EC. Instead, an intracellular mechanism involving glycogen synthase kinase-3 (GSK-3α/β) mediates receptor crosstalk by integrating signals from EGFR and PAR-1. These findings provide a mechanistic basis for how cells integrate information of concurrent receptor activation to enhance EGR-1 expression in EC.
The current model of serine protease diversity theorizes that the earliest protease molecules wer... more The current model of serine protease diversity theorizes that the earliest protease molecules were simple digestive enzymes that gained complex regulatory functions and restricted substrate specificities through evolution. Among the chymase group of serine proteases are enzymes that convert angiotensin I to angiotensin II, as well as others that simply degrade angiotensins. An ancestral chymase reconstructed with the use of phylogenetic inference, total gene synthesis, and protein expression had efficient and specific angiotensin II-forming activity (turnover number, about 700 per second). Thus, angiotensin II-forming activity is the more primitive state for chymases, and the loss of such activity occurred later in the evolution of some of these serine proteases.
Tumor necrosis factor-α (TNF-α) elicits its biological activities through activation of TNF recep... more 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.
Interleukin 17 (IL-17) is a signature cytokine of Th17 cells. We previously reported that deletio... more Interleukin 17 (IL-17) is a signature cytokine of Th17 cells. We previously reported that deletion of NF-κB activator 1 (Act1), the key transducer of IL-17 receptor signaling, from the neuroectodermal lineage in mice (neurons, oligodendrocytes and astrocytes) results in attenuated severity of experimental autoimmune encephalomyelitis (EAE). Here we examined the cellular basis of this observation. EAE disease course was unaffected by deletion of Act1 in neurons or mature oligodendrocytes, and Act1 deletion in astrocytes only modestly affected disease course. Deletion of Act1 in NG2(+) glia resulted in markedly reduced EAE severity. Furthermore, IL-17 induced characteristic inflammatory mediator expression in NG2(+) glial cells. IL-17 also exhibited strong inhibitory effects on the maturation of oligodendrocyte lineage cells in vitro and reduced their survival. These data identify NG2(+) glia as the major CNS cellular target of IL-17 in EAE. The sensitivity of oligodendrocyte lineage cells to IL-17-mediated toxicity further suggests a direct link between inflammation and neurodegeneration in multiple sclerosis.
Tumor necrosis factor-α (TNF-α) binds to 2 distinct cell-surface receptors: TNF-α receptor-I (TNF... more Tumor necrosis factor-α (TNF-α) binds to 2 distinct cell-surface receptors: TNF-α receptor-I (TNFR-I: p55) and TNF-α receptor-II (TNFR-II: p75). TNF-α 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-α–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-α–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-α–stimulated leukocyte rolling, firm adhesion to ECs, and transmigration were dramatically reduced in p75−/− mice. Transplanted WT cremaster in p75...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2007
... Unni M. Chandrasekharan Lori Mavrakis Jonathan D. Smith Paul E. DiCorleto Department of Cell ... more ... Unni M. Chandrasekharan Lori Mavrakis Jonathan D. Smith Paul E. DiCorleto Department of Cell Biology Lerner Research Institute and Cleveland Clinic ... 2. Branen L, Hovgaard L, Nitulescu M, Bengtsson E, Nilsson J, Jovinge S. Inhibition of tumor necrosis factor-alpha reduces ...
Aberrant activation of the ubiquitous transcription factor STAT3 is a major driver of solid tumor... more 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 -β (GSK-3α/β). 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 depl...
We have previously reported that MAPK phosphatase-1 (MKP-1/CL100) is a thrombin-responsive gene i... more 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-A165 isoform stimulated MKP-1 expression, whereas the VEGF-A162 isoform induced the gene to a lesser extent, and the VEGF-A121 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 wa...
Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a nuclear, dual-specificity phosp... more 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...
Rationale: Multiple protein kinases have been implicated in cardiovascular disease; however, litt... more 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-1α and tumor necrosis factor α, and precede...
ABSTRACT Signaling interactions between G-protein coupled receptors (GPCRs) and receptor tyrosine... more ABSTRACT Signaling interactions between G-protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) function in a number of pathophysiological processes, including cellular proliferation, invasion, and acquired resistance to chemotherapeutic agents. Here, we examine the consequences of simultaneous EGF receptor (EGFR) and thrombin receptor (protease-activated receptor-1, PAR-1) activation in human endothelial cells (EC). We observe that concurrent EGFR and PAR-1 activation causes the synergistic induction of at least six pro-angiogenic immediate early genes (IEGs), including the transcription factor early growth response-1 (EGR-1). In contrast to other cell types, matrix metalloprotease-mediated transactivation of EGFR by PAR-1 does not appear to be a major signaling mechanism in EC. Instead, an intracellular mechanism involving glycogen synthase kinase-3 (GSK-3α/β) mediates receptor crosstalk by integrating signals from EGFR and PAR-1. These findings provide a mechanistic basis for how cells integrate information of concurrent receptor activation to enhance EGR-1 expression in EC.
The current model of serine protease diversity theorizes that the earliest protease molecules wer... more The current model of serine protease diversity theorizes that the earliest protease molecules were simple digestive enzymes that gained complex regulatory functions and restricted substrate specificities through evolution. Among the chymase group of serine proteases are enzymes that convert angiotensin I to angiotensin II, as well as others that simply degrade angiotensins. An ancestral chymase reconstructed with the use of phylogenetic inference, total gene synthesis, and protein expression had efficient and specific angiotensin II-forming activity (turnover number, about 700 per second). Thus, angiotensin II-forming activity is the more primitive state for chymases, and the loss of such activity occurred later in the evolution of some of these serine proteases.
Tumor necrosis factor-α (TNF-α) elicits its biological activities through activation of TNF recep... more 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.
Interleukin 17 (IL-17) is a signature cytokine of Th17 cells. We previously reported that deletio... more Interleukin 17 (IL-17) is a signature cytokine of Th17 cells. We previously reported that deletion of NF-κB activator 1 (Act1), the key transducer of IL-17 receptor signaling, from the neuroectodermal lineage in mice (neurons, oligodendrocytes and astrocytes) results in attenuated severity of experimental autoimmune encephalomyelitis (EAE). Here we examined the cellular basis of this observation. EAE disease course was unaffected by deletion of Act1 in neurons or mature oligodendrocytes, and Act1 deletion in astrocytes only modestly affected disease course. Deletion of Act1 in NG2(+) glia resulted in markedly reduced EAE severity. Furthermore, IL-17 induced characteristic inflammatory mediator expression in NG2(+) glial cells. IL-17 also exhibited strong inhibitory effects on the maturation of oligodendrocyte lineage cells in vitro and reduced their survival. These data identify NG2(+) glia as the major CNS cellular target of IL-17 in EAE. The sensitivity of oligodendrocyte lineage cells to IL-17-mediated toxicity further suggests a direct link between inflammation and neurodegeneration in multiple sclerosis.
Tumor necrosis factor-α (TNF-α) binds to 2 distinct cell-surface receptors: TNF-α receptor-I (TNF... more Tumor necrosis factor-α (TNF-α) binds to 2 distinct cell-surface receptors: TNF-α receptor-I (TNFR-I: p55) and TNF-α receptor-II (TNFR-II: p75). TNF-α 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-α–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-α–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-α–stimulated leukocyte rolling, firm adhesion to ECs, and transmigration were dramatically reduced in p75−/− mice. Transplanted WT cremaster in p75...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2007
... Unni M. Chandrasekharan Lori Mavrakis Jonathan D. Smith Paul E. DiCorleto Department of Cell ... more ... Unni M. Chandrasekharan Lori Mavrakis Jonathan D. Smith Paul E. DiCorleto Department of Cell Biology Lerner Research Institute and Cleveland Clinic ... 2. Branen L, Hovgaard L, Nitulescu M, Bengtsson E, Nilsson J, Jovinge S. Inhibition of tumor necrosis factor-alpha reduces ...
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