Programmed translational readthrough generates antiangiogenic VEGF-Ax.

Translational readthrough, observed primarily in less complex organisms from viruses to Drosophila, expands the proteome by translating select transcripts beyond the canonical stop codon. Here, we show that vascular endothelial growth factor A (VEGFA) mRNA in mammalian endothelial cells undergoes programmed translational readthrough (PTR) generating VEGF-Ax, an isoform containing a unique 22-amino-acid C terminus extension. A cis-acting element in the VEGFA 3' UTR serves a dual function, not only encoding the appended peptide but also directing the PTR by decoding the UGA stop codon as serine. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 binds this element and promotes readthrough. Remarkably, VEGF-Ax exhibits antiangiogenic activity in contrast to the proangiogenic activity of VEGF-A. Pathophysiological significance of VEGF-Ax is indicated by robust expression in multiple human tissues but depletion in colon adenocarcinoma. Furthermore, genome-wide analysis revealed AGO1 and MTCH2 as authentic readthrough targets. Overall, our studies reveal a novel protein-regulated PTR event in a vertebrate system.

Inactivation of the enzyme GSK3α by the kinase IKKi promotes AKT-mTOR signaling pathway that mediates interleukin-1-induced Th17 cell maintenance.

Interleukin-1 (IL-1)-induced activation of the mTOR kinase pathway has major influences on Th17 cell survival, proliferation, and effector function. Via biochemical and genetic approaches, the kinases IKKi and GSK3α were identified as the critical intermediate signaling components for IL-1-induced AKT activation, which in turn activated mTOR. Although insulin-induced AKT activation is known to phosphorylate and inactivate GSK3α and GSK3ß, we found that GSK3α but not GSK3ß formed a constitutive complex to phosphorylate and suppress AKT activation, showing that a reverse action from GSK to AKT can take place. Upon IL-1 stimulation, IKKi was activated to mediate GSK3α phosphorylation at S21, thereby inactivating GSK3α to promote IL-1-induced AKT-mTOR activation. Thus, IKKi has a critical role in Th17 cell maintenance and/or proliferation through the GSK-AKT-mTOR pathway, implicating the potential of IKKi as a therapeutic target.

IRAK-M mediates Toll-like receptor/IL-1R-induced NFκB activation and cytokine production.

Toll-like receptors transduce their signals through the adaptor molecule MyD88 and members of the IL-1R-associated kinase family (IRAK-1, 2, M and 4). IRAK-1 and IRAK-2, known to form Myddosomes with MyD88-IRAK-4, mediate TLR7-induced TAK1-dependent NFκB activation. IRAK-M was previously known to function as a negative regulator that prevents the dissociation of IRAKs from MyD88, thereby inhibiting downstream signalling. However, we now found that IRAK-M was also able to interact with MyD88-IRAK-4 to form IRAK-M Myddosome to mediate TLR7-induced MEKK3-dependent second wave NFκB activation, which is uncoupled from post-transcriptional regulation. As a result, the IRAK-M-dependent pathway only induced expression of genes that are not regulated at the post-transcriptional levels (including inhibitory molecules SOCS1, SHIP1, A20 and IκBα), exerting an overall inhibitory effect on inflammatory response. On the other hand, through interaction with IRAK-2, IRAK-M inhibited TLR7-mediated production of cytokines and chemokines at translational levels. Taken together, IRAK-M mediates TLR7-induced MEKK3-dependent second wave NFκB activation to produce inhibitory molecules as a negative feedback for the pathway, while exerting inhibitory effect on translational control of cytokines and chemokines.

Tumor necrosis factor (TNF)-α induction of CXCL10 in endothelial cells requires protein arginine methyltransferase 5 (PRMT5)-mediated nuclear factor (NF)-κB p65 methylation.

The chemokine CXCL10/IP-10 facilitates recruitment of Th1-type leukocytes to inflammatory sites. In this study, we show that the arginine methyltransferase PRMT5 is critical for CXCL10 transcription in TNF-α-activated human endothelial cells (EC). We found that depletion of PRMT5 results in significantly reduced levels of CXCL10 mRNA, demonstrating a positive role for PRMT5 in CXCL10 induction. Chromatin immunoprecipitation experiments revealed the presence of the symmetrical dimethylarginine modification catalyzed by PRMT5 associated with the CXCL10 promoter in response to TNF-α. However, symmetrical dimethylarginine-modified proteins were not detected at the promoter in the absence of PRMT5, indicating that PRMT5 is essential for methylation to occur. Furthermore, NF-κB p65, a critical driver of TNF-α-mediated CXCL10 induction, was determined to be methylated at arginine residues. Crucially, RNAi-mediated PRMT5 depletion abrogated p65 methylation and CXCL10 promoter binding. Mass spectrometric analysis in EC identified five dimethylated arginine residues in p65, four of which are uncharacterized in the literature. Expression of Arg-to-Lys point mutants of p65 demonstrated that both Arg-30 and Arg-35 must be dimethylated to achieve full CXCL10 expression. In conclusion, we have identified previously uncharacterized p65 post-translational modifications critical for CXCL10 induction.

Mitogen-activated protein kinase phosphatase-1 promotes neovascularization and angiogenic gene expression.

OBJECTIVE: Angiogenesis is the formation of new blood vessels through endothelial cell sprouting. This process requires the mitogen-activated protein kinases, signaling molecules that are negatively regulated by the mitogen-activated protein kinase phosphatase-1 (MKP-1). The purpose of this study was to evaluate the role of MKP-1 in neovascularization in vivo and identify associated mechanisms in endothelial cells. APPROACH AND RESULTS: We used murine hindlimb ischemia as a model system to evaluate the role of MKP-1 in angiogenic growth, remodeling, and arteriogenesis in vivo. Genomic deletion of MKP-1 blunted angiogenesis in the distal hindlimb and microvascular arteriogenesis in the proximal hindlimb. In vitro, endothelial MKP-1 depletion/deletion abrogated vascular endothelial growth factor-induced migration and tube formation, and reduced proliferation. These observations establish MKP-1 as a positive mediator of angiogenesis and contrast with the canonical function of MKP-1 as a mitogen-activated protein kinase phosphatase, implying an alternative mechanism for MKP-1-mediated angiogenesis. Cloning and sequencing of MKP-1-bound chromatin identified localization of MKP-1 to exonic DNA of the angiogenic chemokine fractalkine, and MKP-1 depletion reduced histone H3 serine 10 dephosphorylation on this DNA locus and blocked fractalkine expression. In vivo, MKP-1 deletion abrogated ischemia-induced fractalkine expression and macrophage and T-lymphocyte infiltration in distal hindlimbs, whereas fractalkine delivery to ischemic hindlimbs rescued the effect of MKP-1 deletion on neovascular hindlimb recovery. CONCLUSIONS: MKP-1 promoted angiogenic and arteriogenic neovascular growth, potentially through dephosphorylation of histone H3 serine 10 on coding-region DNA to control transcription of angiogenic genes, such as fractalkine. These observations reveal a novel function for MKP-1 and identify MKP-1 as a potential therapeutic target.

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.

Phosphorylation of glutamyl-prolyl tRNA synthetase by cyclin-dependent kinase 5 dictates transcript-selective translational control.

Cyclin-dependent kinase 5 (Cdk5) is an atypical but essential member of the Cdk kinase family, and its dysregulation or deletion has been implicated in inflammation-related disorders by an undefined mechanism. Here we show that Cdk5 is an indispensable activator of the GAIT (IFN-γ-activated inhibitor of translation) pathway, which suppresses expression of a posttranscriptional regulon of proinflammatory genes in myeloid cells. Through induction of its regulatory protein, Cdk5R1 (p35), IFN-γ activates Cdk5 to phosphorylate Ser(886) in the linker domain of glutamyl-prolyl tRNA synthetase (EPRS), the initial event in assembly of the GAIT complex. Cdk5/p35 also induces, albeit indirectly via a distinct kinase, phosphorylation of Ser(999), the second essential event in GAIT pathway activation. Diphosphorylated EPRS is released from its residence in the tRNA multisynthetase complex for immediate binding to NS1-associated protein and subsequent binding to ribosomal protein L13a and GAPDH. The mature heterotetrameric GAIT complex binds the 3' UTR GAIT element of VEGF-A and other target mRNAs and suppresses their translation in myeloid cells. Inhibition of Cdk5/p35 inhibits both EPRS phosphorylation events, prevents EPRS release from the tRNA multisynthetase complex, and blocks translational suppression of GAIT element-bearing mRNAs, resulting in increased expression of inflammatory proteins. Our study reveals a unique role of Cdk5/p35 in activation of the major noncanonical function of EPRS, namely translational control of macrophage inflammatory gene expression.

The critical role of IL-1 receptor-associated kinase 4-mediated NF-κB activation in modified low-density lipoprotein-induced inflammatory gene expression and atherosclerosis.

Exciting discoveries related to IL-1R/TLR signaling in the development of atherosclerosis plaque have triggered intense interest in the molecular mechanisms by which innate immune signaling modulates the onset and development of atherosclerosis. Previous studies have clearly shown the definitive role of proinflammatory cytokine IL-1 in the development of atherosclerosis. Recent studies have provided direct evidence supporting a link between innate immunity and atherogenesis. Although it is still controversial about whether infectious pathogens contribute to cardiovascular diseases, direct genetic evidence indicates the importance of IL-1R/TLR signaling in atherogenesis. In this study, we examined the role of IL-1R-associated kinase 4 (IRAK4) kinase activity in modified low-density lipoprotein (LDL)-mediated signaling using bone marrow-derived macrophage as well as an in vivo model of atherosclerosis. First, we found that the IRAK4 kinase activity was required for modified LDL-induced NF-κB activation and expression of a subset of proinflammatory genes but not for the activation of MAPKs in bone marrow-derived macrophage. IRAK4 kinase-inactive knockin (IRAK4KI) mice were bred onto ApoE(-/-) mice to generate IRAK4KI/ApoE(-/-) mice. Importantly, the aortic sinus lesion formation was impaired in IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Furthermore, proinflammatory cytokine production was reduced in the aortic sinus region of IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Taken together, our results indicate that the IRAK4 kinase plays an important role in modified LDL-mediated signaling and the development of atherosclerosis, suggesting that pharmacological inhibition of IRAK4 kinase activity might be a feasible approach in the development of antiatherosclerosis drugs.

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.

TNFR2 Depletion Reduces Psoriatic Inflammation in Mice by Downregulating Specific Dendritic Cell Populations in Lymph Nodes and Inhibiting IL-23/IL-17 Pathways.

TNF-α, a proinflammatory cytokine, is a crucial mediator of psoriasis pathogenesis. TNF-α functions by activating TNFR1 and TNFR2. Anti-TNF drugs that neutralize TNF-α, thus blocking the activation of TNFR1 and TNFR2, have been proven highly therapeutic in psoriatic diseases. TNF-α also plays an important role in host defense; thus, anti-TNF therapy can cause potentially serious adverse effects, including opportunistic infections and latent tuberculosis reactivation. These adverse effects are attributed to TNFR1 inactivation. Therefore, understanding the relative contributions of TNFR1 and TNFR2 has clinical implications in mitigating psoriasis versus global TNF-α blockade. We found a significant reduction in psoriasis lesions as measured by epidermal hyperplasia, characteristic gross skin lesion, and IL-23 or IL-17A levels in Tnfr2-knockout but not in Tnfr1-knockout mice in the imiquimod psoriasis model. Furthermore, imiquimod-mediated increase in the myeloid dendritic cells, TNF/inducible nitric oxide synthase‒producing dendritic cells, and IL-23 expression in the draining lymph nodes were dependent on TNFR2 but not on TNFR1. Together, our results support that psoriatic inflammation is not dependent on TNFR1 activity but is driven by a TNFR2-dependent IL-23/IL-17 pathway activation. Thus, targeting the TNFR2 pathway may emerge as a potential next-generation therapeutic approach for psoriatic diseases.

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.

ADP stimulates human endothelial cell migration via P2Y1 nucleotide receptor-mediated mitogen-activated protein kinase pathways.

Extensive research on the role of ADP in platelet activation led to the design of new anti-thrombotic drugs, such as clopidogrel (Plavix; sanofi-aventis); however, very little is known about the ADP-preferring nucleotide receptors (P2Y1, P2Y12, and P2Y13) in endothelium. Here, we show that ADP stimulates migration of cultured human umbilical vein endothelial cells (HUVECs) in both Boyden chamber and in vitro wound repair assays. This promigratory effect was mimicked by 2-MeSADP, but not by AMP, and was inhibited by MRS2179 (P2Y1 receptor antagonist) but not by AR-C69931MX (P2Y12/13 receptor antagonist). RT-PCR revealed abundant P2Y1, barely detectable P2Y12, and absent P2Y13 receptor message in these cells. In addition, both ADP and 2-MeSADP, but not AMP, activated the mitogen-activated protein kinase pathways as evidenced by increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK), and p38 kinase. ADP also stimulated phosphorylation of p90RSK, a downstream substrate of phosphorylated ERK1/2, and induced phosphorylation of such transcription factors downstream of the JNK and p38 pathways as c-Jun and activating transcription factor-2. These signaling events were inhibited by MRS2179 but not by AR-C69931MX. Furthermore, blockade of the ERK or JNK pathways by U0126 and SP600125, respectively, abolished ADP- and 2-MeSADP-stimulated HUVEC migration. However, inhibition of the p38 pathway by SB203580 partially suppressed ADP- and 2-MeSADP-induced HUVEC migration. We conclude that ADP promotes human endothelial cell migration by activating P2Y1 receptor-mediated MAPK pathways, possibly contributing to reendothelialization and angiogenesis after vascular injury.

HOXA9 participates in the transcriptional activation of E-selectin in endothelial cells.

The homeobox gene HOXA9 has recently been shown to be an important regulator of endothelial cell (EC) differentiation and activation in addition to its role in embryonic development and hematopoiesis. In this report, we have determined that the EC-leukocyte adhesion molecule E-selectin is a key target for HOXA9. The depletion of HOXA9 protein in ECs resulted in a significant and specific decrease in tumor necrosis factor alpha (TNF-alpha)-induced E-selectin gene expression. In addition, HOXA9 specifically activated the E-selectin gene promoter in ECs. Progressive deletional analyses together with site-specific mutagenesis of the E-selectin promoter indicated that the Abd-B-like HOX DNA-binding motif, CAATTTTATTAA, located in the proximal region spanning bp -210 to -221 upstream of the transcription start site was crucial for the promoter induction by HOXA9. Both HOXA9 in EC nuclear extract and recombinant HOXA9 protein bound to this sequence in vitro. Moreover, we showed that HOXA9 binds temporally, in a TNF-alpha-dependent manner, to the region containing this Abd-B-like element in vivo. We have thus identified a novel and functionally critical cis-regulatory element for TNF-alpha-mediated transient expression of the E-selectin gene. Further, we provide evidence that HOXA9 acts as an obligate proinflammatory factor by mediating cytokine induction of E-selectin.

Identification of an angiogenic factor that when mutated causes susceptibility to Klippel-Trenaunay syndrome.

Angiogenic factors are critical to the initiation of angiogenesis and maintenance of the vascular network. Here we use human genetics as an approach to identify an angiogenic factor, VG5Q, and further define two genetic defects of VG5Q in patients with the vascular disease Klippel-Trenaunay syndrome (KTS). One mutation is chromosomal translocation t(5;11), which increases VG5Q transcription. The second is mutation E133K identified in five KTS patients, but not in 200 matched controls. VG5Q protein acts as a potent angiogenic factor in promoting angiogenesis, and suppression of VG5Q expression inhibits vessel formation. E133K is a functional mutation that substantially enhances the angiogenic effect of VG5Q. VG5Q shows strong expression in blood vessels and is secreted as vessel formation is initiated. VG5Q can bind to endothelial cells and promote cell proliferation, suggesting that it may act in an autocrine fashion. We also demonstrate a direct interaction of VG5Q with another secreted angiogenic factor, TWEAK (also known as TNFSF12). These results define VG5Q as an angiogenic factor, establish VG5Q as a susceptibility gene for KTS, and show that increased angiogenesis is a molecular pathogenic mechanism of KTS.

Fibulin-4 regulates expression of the tropoelastin gene and consequent elastic-fibre formation by human fibroblasts.

Elastic fibres are essential for normal physiology in numerous tissues, including arteries, lungs and skin. Fibulin-4 is an elastic-fibre-associated glycoprotein that is indispensable for elastic-fibre formation in mice. However, the mechanism by which fibulin-4 executes this function remains to be determined. Here, we established an in vitro functional assay system in which fibulin-4 was knocked down in human foreskin fibroblasts using siRNA (small interfering RNA) technology. With two different siRNAs, substantial knockdown of fibulin-4 was achieved, and this suppression was associated with impaired elastic-fibre formation by the fibroblasts. Real-time reverse transcription-PCR analysis showed that knockdown of fibulin-4 expression was accompanied by reduced expression of tropoelastin mRNA. Further analysis showed that this decrease was caused by transcriptional down-regulation of tropoelastin. This effect was selective, since the mRNA level of other elastic-fibre-associated proteins, including fibrillin-1, lysyl oxidase and lysyl oxidase-like-1, was not affected. Moreover, addition of conditioned medium from cultures of CHO (Chinese-hamster ovary) cells overexpressing fibulin-4 stimulated tropoelastin expression and elastic-fibre formation in cultures of Williams-Beuren-syndrome fibroblasts. Knocking down or knocking out fibulin-4 in mice led to a decrease in tropoelastin expression in the aorta. These results indicate that fibulin-4, considered as a structural protein, may also participate in regulating elastic-fibre formation in human cells through an unanticipated mechanism, namely the regulation of tropoelastin expression.

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.

Arginase blockade lessens endothelial dysfunction after thrombosis.

INTRODUCTION: Acute arterial thrombosis causes endothelial dysfunction due to decreased nitric oxide bioactivity. Increased arginase activity may modulate intracellular L-arginine levels, the substrate for nitric oxide. The purpose of this study was to identify the role of arginase in endothelial dysfunction in cell culture and in the vasomotor response of arteries exposed to thrombus. METHODS: Rat aortic endothelial cells were exposed to thrombin at different time points. The cell extract was analyzed by immunoblotting and real-time polymerase chain reaction. Adult male rats underwent infrarenal aortic thrombosis by clip ligature for 1 hour. Infrarenal aortic ring segments were harvested and placed in physiologic buffer baths, and a force transducer was used to measure endothelial-dependent relaxation (EDR) and endothelial-independent relaxation (EIR). Arginase blockade was performed by incubating infrarenal aortic ring segments with arginase inhibitors for 1 hour before measuring EDR. Whole tissue extracts also underwent immunoblot analysis. The EDR and EIR curves were compared with analyses of variance. RESULTS: A 6.76 +/- 1.4-fold induction in arginase I message levels (P = .001) was found in rat aortic endothelial cells exposed to thrombin (30 U/mL), and arginase I protein levels increased 2.1 times. The eight infrarenal aortic ring segments exposed to thrombosis for 1 hour had diminished EDR curves compared with 14 nonthrombosed normal segments (controls). The maximum (+/- SEM) EDR (acetylcholine 10(-5)M dose) in control infrarenal aortic ring segments was 108% +/- 4.3% compared with 63% +/- 6.2% for thrombosed infrarenal aortic ring segments (P < .001). Exposure to arterial thrombosis resulted in a 3.8-times increase in arginase I protein levels in infrarenal aortic ring segments. Preincubation of nine infrarenal aortic ring segments with the nonspecific (difluoromethylornithine) and six with specific ([S]-[2-boronoethyl]-L-Cysteine-HCl [BEC]) arginase inhibitor for 1 hour significantly increased the maximum EDR compared with untreated thrombosed segments (104 +/- 5.2, 108 +/- 7.6 vs 63% +/- 6.2, P < .001). EDR curves for difluoromethylornithine- and BEC-treated infrarenal aortic ring segments were superimposed on control EDR curves. The EIR and the vasoconstriction with norepinephrine for all groups were similar. CONCLUSION: Endothelial cells exposed to thrombin have increased arginase I messenger RNA and protein levels. Arterial thrombosis causes endothelial dysfunction without affecting smooth muscle responsiveness. Arginase blockade can lead to normalization of arterial vasomotor function.

Topical heat shock protein 70 prevents imiquimod-induced psoriasis-like inflammation in mice.

Psoriasis is a chronic inflammatory skin disease with systemic manifestations and potential genetic etiology. The newest treatments utilize antibodies against one of several cytokines known to underlie the inflammatory signaling molecules that produce the skin and systemic symptoms. However, these agents must be regularly injected, and they may compromise the normal responses of the immune system. Furthermore, they do not address the causes of the abnormal immunoregulatory responses of the disease because the etiology is not yet completely understood. In this short-term treatment study, the potential anti-inflammatory activity of an alfalfa-derived Hsp70-containing skin cream (aHsp70) was tested on imiquimod (IMQ)-induced psoriasis-like lesions in wild-type mice. Treatment of the mice with the aHsp70 skin cream simultaneously with the imiquimod application mitigated the induction of psoriatic-like lesions and correlated with altered expression of various skin cytokines.