Regulation of endothelial cell integrin function and angiogenesis by COX-2, cAMP and Protein Kinase A.

Angiogenesis, the development of new blood vessels from preexisting vessels, is a key step in tumor growth, invasion and metastasis formation. Inhibition of tumor angiogenesis is considered as an attractive approach to suppress cancer progression and spreading. Adhesion receptors of the integrin family promote tumor angiogenesis by mediating cell migration, proliferation and survival of angiogenic endothelial cells. Integrins up regulated and highly expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, have been considered as relevant targets for anti-angiogenic therapies. Small molecular integrin antagonists or blocking antibodies suppress angiogenesis and tumor progression in many animal models, and some of them are currently being tested in cancer clinical trials as anti-angiogenic agents. COX-2 inhibitors exert anti-cancer effects, at least in part, by inhibiting tumor angiogenesis. We have recently shown that COX-2 inhibitors suppress endothelial cell migration and angiogenesis by preventing alphaVbeta3-mediated and cAMP/PKA-dependent activation of the small GTPases Rac and Cdc42. Here we will review the evidence for the involvement of vascular integrins in mediating angiogenesis and the role of COX-2 metabolites in modulating the cAMP/Protein Kinase A pathway and alphaVbeta3-dependent Rac activation in endothelial cells.

The C-Jun N-Terminal Kinase Inhibition in Intracerebral Hemorrhage

Background: In intracerebral hemorrhage (ICH), a subtype of stroke, the bloodentry into the brain triggers toxicity resulting in a strong loss of neurons andinflammation. Water content is also increases leading to growing intracranial pressure,which worsens neurological outcome. C-Jun N-terminal kinases (JNKs) areactivated in response to stress stimuli. Specific inhibition of JNK by a TAT-coupledpeptide (XG-102) mediates neuroprotection in several models of ischemic stroke.Recently, we have noted that the JNK pathway is also activated in a mouse modelof ICH, raising the question of the efficacy of XG-102 in this model.Method: ICH was induced in the mouse by intrastriatal injection of bacterialcollagenase (0,1U). Three hours later, animals received an i.v. injection of XG-102(100μg/kg). The neuroscore was assessed using a scale (from 0 to 9) based on 3behavioral tests performed daily. Then, mice were sacrificed at 6h, 24h, 48h and 5dafter ICH and histological studies performed.Results: XG-102 significantly improves neurological outcome at 24h (mean score:1,8±1.4 vs 3,4±1.8, p<0.01). Analysis of the lesion volume revealed a significantdecrease of the lesion area in the treated group at 48h (29±11 mm3 vs 39±5 mm3,p = 0.04). XG-102 mainly inhibits the edema component of the lesion. Indeed, asignificant decrease of the brain swelling was observed in treated animals at 48h(14±13% vs 26±9%, p=0.04) and 5d (-0,3±4.5% vs 5,1±3.6%, p=0.01).Conclusions: Inhibition of the JNK pathway by XG-102 appears to lead to asignificant decrease of the cerebral edema in the ICH model providing a furtherbeneficial effect of the XG-102 treatment. This result is of interest becausecurrently, clinical treatment for brain edema is limited. Importantly, the beneficialeffects observed with XG-102 in both stroke models open the possibility to rapidlytreat patients before identifying the stroke subtype by imaging.

Drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation.

Although Drosophila systemic immunity is extensively studied, little is known about the fly's intestine-specific responses to bacterial infection. Global gene expression analysis of Drosophila intestinal tissue to oral infection with the Gram-negative bacterium Erwinia carotovora revealed that immune responses in the gut are regulated by the Imd and JAK-STAT pathways, but not the Toll pathway. Ingestion of bacteria had a dramatic impact on the physiology of the gut that included modulation of stress response and increased stem cell proliferation and epithelial renewal. Our data suggest that gut homeostasis is maintained through a balance between cell damage due to the collateral effects of bacteria killing and epithelial repair by stem cell division. The Drosophila gut provides a powerful model to study the integration of stress and immunity with pathways associated with stem cell control, and this study should prove to be a useful resource for such further studies.

Adaptive sequence evolution in a color gene involved in the formation of the characteristic egg-dummies of male haplochromine cichlid fishes.

BACKGROUND: The exceptionally diverse species flocks of cichlid fishes in East Africa are prime examples of parallel adaptive radiations. About 80% of East Africa's more than 1 800 endemic cichlid species, and all species of the flocks of Lakes Victoria and Malawi, belong to a particularly rapidly evolving lineage, the haplochromines. One characteristic feature of the haplochromines is their possession of egg-dummies on the males' anal fins. These egg-spots mimic real eggs and play an important role in the mating system of these maternal mouthbrooding fish. RESULTS: Here, we show that the egg-spots of haplochromines are made up of yellow pigment cells, xanthophores, and that a gene coding for a type III receptor tyrosine kinase, colony-stimulating factor 1 receptor a (csf1ra), is expressed in egg-spot tissue. Molecular evolutionary analyses reveal that the extracellular ligand-binding and receptor-interacting domain of csf1ra underwent adaptive sequence evolution in the ancestral lineage of the haplochromines, coinciding with the emergence of egg-dummies. We also find that csf1ra is expressed in the egg-dummies of a distantly related cichlid species, the ectodine cichlid Ophthalmotilapia ventralis, in which markings with similar functions evolved on the pelvic fin in convergence to those of the haplochromines. CONCLUSION: We conclude that modifications of existing signal transduction mechanisms might have evolved in the haplochromine lineage in association with the origination of anal fin egg-dummies. That positive selection has acted during the evolution of a color gene that seems to be involved in the morphogenesis of a sexually selected trait, the egg-dummies, highlights the importance of further investigations of the comparative genomic basis of the phenotypic diversification of cichlid fishes.

Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

Equine herpesvirus protein E10 induces membrane recruitment and phosphorylation of its cellular homologue, bcl-10.

v-E10, a caspase recruitment domain (CARD)-containing gene product of equine herpesvirus 2, is the viral homologue of the bcl-10 protein whose gene was found to be translocated in mucosa-associated lymphoid tissue (MALT) lymphomas. v-E10 efficiently activates the c-jun NH(2)-terminal kinase (JNK), p38 stress kinase, and the nuclear factor (NF)-kappaB transcriptional pathway and interacts with its cellular homologue, bcl-10, via a CARD-mediated interaction. Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization. Expression of v-E10 induces hyperphosphorylation and redistribution of bcl-10 from the cytoplasm to the plasma membrane, a process which is dependent on the intactness of the v-E10 CARD motif. Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6. Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.

Connexin26 is regulated in rat urothelium by the scaffold protein IB1/JIP-1.

Proper function of the wall of bladder requires gap junctional communication for coordinating the responses of smooth muscle (SMC) and urothelial cells exposed to urine pressure. In the rat bladder, Cx43 is expressed by SMC and urothelial cells, whereas Cx26 expression is restricted to the epithelium. We used a model of bladder outlet obstruction, in which a ligature is placed around the urethra to increase voiding pressure. Increased fluid pressure was associated with increased Cx43 and Cx26 mRNA expression and with the activation of a signaling cascade including the transcription factor c-Jun, which is a component of the AP-1 complex. The signaling pathway of the c-Jun NH2 terminal kinase (JNK) requires the presence of the scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1). Under stress conditions resulting from urine retention, we have found a reduced content of IB1/JIP-1 in urothelial cells, which in turn induced a drastic increase of JNK and AP-1 binding activities. The stress-induced activation of JNK was prevented by overexpressing IB1/JIP-1, using a viral gene transfer approach, a condition which also resulted in a decrease in Cx26 mRNA. The data show that: 1) mechanical stress of urothelial cells activates in vivo JNK, as a consequence of a regulated expression of IB1/JIP-1 and 2) that urothelial Cx26 may be directly regulated by the AP-1 complex.

DNA structure-specific priming of ATR activation by DNA-PKcs.

Three phosphatidylinositol-3-kinase-related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)-covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs.

TAK1 is required for survival of mouse fibroblasts treated with TRAIL, and does so by NF-kappaB dependent induction of cFLIPL.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a "death ligand"-a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-kappaB and JNK signalling pathways. To determine the role of TGF-beta-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1-/- MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-kappaB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-kappaB, protected TAK1-/- MEFs against TRAIL killing, suggesting that TAK1 activation of NF-kappaB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-kappaB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1-/- MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1-NF-kappaB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.

Antiapoptotic role of PPARbeta in keratinocytes via transcriptional control of the Akt1 signaling pathway.

Apoptosis, differentiation, and proliferation are cellular responses which play a pivotal role in wound healing. During this process PPARbeta translates inflammatory signals into prompt keratinocyte responses. We show herein that PPARbeta modulates Akt1 activation via transcriptional upregulation of ILK and PDK1, revealing a mechanism for the control of Akt1 signaling. The resulting higher Akt1 activity leads to increased keratinocyte survival following growth factor deprivation or anoikis. PPARbeta also potentiates NF-kappaB activity and MMP-9 production, which can regulate keratinocyte migration. Together, these results provide a molecular mechanism by which PPARbeta protects keratinocytes against apoptosis and may contribute to the process of skin wound closure.

The gene MAPK8IP1, encoding islet-brain-1, is a candidate for type 2 diabetes.

Type 2 diabetes is a polygenic and genetically heterogeneous disease . The age of onset of the disease is usually late and environmental factors may be required to induce the complete diabetic phenotype. Susceptibility genes for diabetes have not yet been identified. Islet-brain-1 (IB1, encoded by MAPK8IP1), a novel DNA-binding transactivator of the glucose transporter GLUT2 (encoded by SLC2A2), is the homologue of the c-Jun amino-terminal kinase-interacting protein-1 (JIP-1; refs 2-5). We evaluated the role of IBi in beta-cells by expression of a MAPK8IP1 antisense RNA in a stable insulinoma beta-cell line. A 38% decrease in IB1 protein content resulted in a 49% and a 41% reduction in SLC2A2 and INS (encoding insulin) mRNA expression, respectively. In addition, we detected MAPK8IP1 transcripts and IBi protein in human pancreatic islets. These data establish MAPK8IP1 as a candidate gene for human diabetes. Sibpair analyses performed on i49 multiplex French families with type 2 diabetes excluded MAPK8IP1 as a major diabetogenic locus. We did, however, identify in one family a missense mutation located in the coding region of MAPK8IP1 (559N) that segregated with diabetes. In vitro, this mutation was associated with an inability of IB1 to prevent apoptosis induced by MAPK/ERK kinase kinase 1 (MEKK1) and a reduced ability to counteract the inhibitory action of the activated c-JUN amino-terminal kinase (JNK) pathway on INS transcriptional activity. Identification of this novel non-maturity onset diabetes of the young (MODY) form of diabetes demonstrates that IB1 is a key regulator of 3-cell function.

Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt.

Bisphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumor bone metastases. Recent evidence indicates that bisphosphonates have direct cytotoxic activity on tumor cells and suppress angiogenesis, but the associated molecular events have not been fully characterized. In this study we investigated the effects of zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen-containing bisphosphonate, on human umbilical vein endothelial cell (HUVEC) adhesion, migration, and survival, three events essential for angiogenesis. Zoledronate inhibited HUVEC adhesion mediated by integrin alphaVbeta3, but not alpha5beta1, blocked migration and disrupted established focal adhesions and actin stress fibers without modifying cell surface integrin expression level or affinity. Zoledronate treatment slightly decreased HUVEC viability and strongly enhanced tumor necrosis factor (TNF)-induced cell death. HUVEC treated with zoledronate and TNF died without evidence of enhanced annexin-V binding, chromatin condensation, or nuclear fragmentation and caspase dependence. Zoledronate inhibited sustained phosphorylation of focal adhesion kinase (FAK) and in combination with TNF, with and without interferon (IFN) gamma, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNgamma. Phosphorylation of c-Src and activation of NF-kappaB were not affected by zoledronate. Clodronate had no effect on HUVEC adhesion, migration, and survival nor did it enhanced TNF cytotoxicity. Taken together these data demonstrate that zoledronate sensitizes endothelial cells to TNF-induced, caspase-independent programmed cell death and point to the FAK-PKB/Akt pathway as a novel zoledronate target. These results have potential implications to the clinical use of zoledronate as an anti-angiogenic or anti-cancer agent.

In vitro biotransformation of imatinib by the tumor expressed CYP1A1 and CYP1B1.

The main objective of the study was to examine the biotransformation of the anticancer drug imatinib in target cells by incubating it with oxidoreductases expressed in tumor cells. The second objective was to obtain an in silico prediction of the potential activity of imatinib metabolites. An in vitro enzyme kinetic study was performed with cDNA expressed human oxidoreductases and LC-MS/MS analysis. The kinetic parameters (Km and Vmax) were determined for six metabolites. A molecular modeling approach was used to dock these metabolites to the target Abl or Bcr-Abl kinases. CYP3A4 isozyme showed the broadest metabolic capacity, whereas CYP1A1, CYP1B1 and FMO3 isozymes biotransformed imatinib with a high intrinsic clearance. The predicted binding modes for the metabolites to Abl were comparable to that of the parent drug, suggesting potential activity. These findings indicate that CYP1A1 and CYP1B1, which are known to be overexpressed in a wide range of tumors, are involved in the biotransformation of imatinib. They could play a role in imatinib disposition in the targeted stem, progenitor and differentiated cancer cells, with a possible contribution of the metabolites toward the activity of the drug.

A-Kinase Anchoring Protein (AKAP)-Lbc Anchors a PKN-based Signaling Complex Involved in α1-Adrenergic Receptor-induced p38 Activation.

The mitogen-activated protein kinases (MAPKs) pathways are highly organized signaling systems that transduce extracellular signals into a variety of intracellular responses. In this context, it is currently poorly understood how kinases constituting these signaling cascades are assembled and activated in response to receptor stimulation to generate specific cellular responses. Here, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critically involved in the activation of the p38α MAPK downstream of α(1b)-adrenergic receptors (α(1b)-ARs). Our results indicate that AKAP-Lbc can assemble a novel transduction complex containing the RhoA effector PKNα, MLTK, MKK3, and p38α, which integrates signals from α(1b)-ARs to promote RhoA-dependent activation of p38α. In particular, silencing of AKAP-Lbc expression or disrupting the formation of the AKAP-Lbc·p38α signaling complex specifically reduces α(1)-AR-mediated p38α activation without affecting receptor-mediated activation of other MAPK pathways. These findings provide a novel mechanistic hypothesis explaining how assembly of macromolecular complexes can specify MAPK signaling downstream of α(1)-ARs.

Mobile gibberellin directly stimulates Arabidopsis hypocotyl xylem expansion.

Secondary growth of the vasculature results in the thickening of plant structures and continuously produces xylem tissue, the major biological carbon sink. Little is known about the developmental control of this quantitative trait, which displays two distinct phases in Arabidopsis thaliana hypocotyls. The later phase of accelerated xylem expansion resembles the secondary growth of trees and is triggered upon flowering by an unknown, shoot-derived signal. We found that flowering-dependent hypocotyl xylem expansion is a general feature of herbaceous plants with a rosette growth habit. Flowering induction is sufficient to trigger xylem expansion in Arabidopsis. By contrast, neither flower formation nor elongation of the main inflorescence is required. Xylem expansion also does not depend on any particular flowering time pathway or absolute age. Through analyses of natural genetic variation, we found that ERECTA acts locally to restrict xylem expansion downstream of the gibberellin (GA) pathway. Investigations of mutant and transgenic plants indicate that GA and its signaling pathway are both necessary and sufficient to directly trigger enhanced xylogenesis. Impaired GA signaling did not affect xylem expansion systemically, suggesting that it acts downstream of the mobile cue. By contrast, the GA effect was graft transmissible, suggesting that GA itself is the mobile shoot-derived signal.