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AIMS We evaluate whether the serum and aqueous humour (AH) level of IgG anti-Hsp70.1 antibodies improved the biological diagnosis of ocular toxoplasmosis. METHODS AND RESULTS In this prospective cross-sectional and multicentre study, serum and AH were collected at the time of active uveitis. Anti-Hsp70.1-antibody levels were determined by ELISA. Patients with confirmed (Group A1, n = 21) or suspected ocular toxoplasmosis (group A2, n = 30) were enrolled, as well as a control group of patients with cataract (group B, n = 42). Serum IgG anti-Hsp70.1 antibody levels were not significantly different within the group of uveitis patients (A1, n = 21 vs A2, n = 30, P = .8) and were significantly associated with the affected retinal zone (P = .006) and with the size of the retinal lesion (P = .03). Serum anti-Hsp70.1 antibody level was positive in 10 out of the 18 patients of group A2. Significant anti-Hsp-70.1 antibody level in AH was reported in only three patients (3 eyes) with confirmed ocular toxoplasmosis. CONCLUSION While the level of IgG anti-Hsp-70.1 antibody in AH did not improve the laboratory diagnosis of ocular toxoplasmosis, its level in serum was of major significance for retinal damage diagnosis.

A major problem for the detection of cancer biomarkers in plasma or serum is that common clinical practice does not require the patient to be in a fasting state. Considering that lipoproteins are the main population affected by food intake, the authors hypothesized that biomarkers could be embedded in lipid particles and thereby opens a new avenue for detection. Using the recently published biomarker, soluble VE-cadherin (sVE), the authors tested our hypothesis using techniques of biophysics, biochemistry and the tools of nanobiotechnology on serum samples from kidney cancer patients (n = 106). Optical density as well as contact angle measurements of serum revealed heterogeneity in the particle content of the serum samples. Isolation of the lipidic moieties by ultracentrifugation showed that sVE was detected in this compartment. Further, isolation of lipoprotein subclasses by precipitation with sodium phosphotungstate and MgCl2 , showed that HDL carried the majority of sVE. Immunoprecipitation of sVE confirmed that it was associated with Apolipoprotein A1, a major compound of HDL. Using a biomimetic lipid bilayer membrane coupled with impedance spectroscopy the authors quantified, in real-time, that the sVE adsorbed to the lipid bilayer membrane without altering its structure. Taken together, these results show for the first time a direct interaction of a cancer biomarker with lipids. The authors anticipate these results to prompt fasting for future blood tests for large-scale studies in the biomarkers research field.

Background: Bradykinin-mediated angioedema (AE) is a rare disease characterised by recurrent angioedema linked to acquired (e.g. angiotensin converting enzyme inhibitor induced AE) or hereditary disorders (e.g. AE type I or II). As the clinical picture can be misleading, diagnosis of this disease is sometimes difficult. A bradykinin-mediated AE attack may be a therapeutic emergency which requires access to effective, but expensive, treatments. Their prescription must therefore be justified. No specific marker of acute bradykinin-mediated AE attacks has yet been identified to facilitate the therapeutic decision but it has been sought in many studies. Purpose: This article reviews the literature on this type of biomarker, comparing candidate bradykinin-mediated AE markers to candidate markers of mast cell activation. Conclusion: The most interesting biomarkers are those linked to endothelial stress (VE cadherin, E-selectin, endothelin-1, von Willebrand factor and its activity) which is significantly increased during an AE attack. All these markers must now be validated by prospective studies to determine their specificity and utility in diagnosis.

International audience; Because angiogenesis is a key step in a number of pathologic processes, including tumor growth and atherosclerosis, many research studies have investigated the regulatory signals active at various stages of vascular invasion. The differential activities of the endothelial junction protein vascular endothelial (VE)-cadherin reflect the versatile behavior of endothelial cells between vascular quiescence and angiogenesis. VE-cadherin function and signaling are deeply modified in proliferating cells, and this conversion is accompanied by phosphorylation of the protein on tyrosine residues and enhanced transcription of its gene. Recent advances in the complex interplay between protein tyrosine kinases and phosphatases regulating VE-cadherin phosphorylation and function are discussed in this review.

Vascular endothelial-cadherin (VE-cadherin) is an endothelial cell-specific adhesion protein that is localised at cell–cell contacts. This molecule is an important determinant of vascular architecture and endothelial cell survival. In the adrenal cortex, steroidogenic and endothelial cells form a complex architecture. The adrenocorticotrophin hormone (ACTH) regulates gland homeostasis whose secretion is subjected to a negative feedback by adrenocorticosteroids. The aim of the present study was to determine whether VE-cadherin expression in the adrenal gland was regulated by hormonal challenge. We demonstrated that VE-cadherin protein levels were dramatically decreased (23.5 ± 3.7%) by dexamethasone injections in the mouse and were restored by ACTH within 7 days (94.9 ± 18.6%). Flow cytometry analysis of adrenal cells showed that the ratios of endothelial versus total adrenal cells were identical (35%) in dexamethasone- or ACTH-treated or untreated mice, suggesting that VE-cadherin expression could be regulated by ACTH. We demonstrate the existence of a transcriptional regulation of the VE-cadherin gene using transgenic mice carrying the chloramphenicol acetyl transferase gene under the control of the VE-cadherin promoter. Indeed, the promoter activity in the adrenals, but not in the lung or liver, was decreased in response to dexamethasone treatment (40 ± 1.3%) and was partially restored after gland regeneration by ACTH injection (82 ± 3%). In conclusion, our results show that transcription of a specific endothelial gene is controlled by the hypothalamo–pituitary axis and the data expand the knowledge regarding the role of ACTH in the regulation of the adrenal vascular network.

Although ACTH is important to adrenal growth and steroidogenesis, its role in vascular development and function has not been established in vivo. In the present study, we demonstrate the expression of mRNA for all four VEGF isoforms (mVEGF120,144,164,188) and for Flk-1/KDR and Flt-1 receptors in the mouse adrenal in vivo. Suppression of the pituitary adrenocortical axis by dexamethasone (0.5 mg · 100 g body wt−1· day−1during 6 days) induced a decrease in corticosterone levels, adrenal weights by 50% ( P < 0.001), VEGF188mRNA, and Flk-1/KDR mRNA, whereas Flt-1 remained consistent during steroid treatment. A daily injection of ACTH-(1–39) restored the transcript for Flk-1/KDR and both VEGF188and plasma corticosterone to control levels. To gain further insights into the effects of ACTH, cultured endothelial cells (ECs) were treated with forskolin, which increases cAMP, the second messenger in ACTH action. We demonstrate that Flk-1/KDR protein expression was markedly increased by forskolin within 24–48 h of treatment in a dose-dependent manner (0.1–10 μM). The biological effect of ACTH on ECs was then tested by use of coincubations of fasciculata cells and ECs in 3D-collagen assay. Within 5–7 days of culture, ECs organized into multicellular structures that resemble networks of microvasculature, which characterize angiogenesis in vitro.

We previously reported that vascular endothelial growth factor induced vascular endothelial (VE)-cadherin tyrosine phosphorylation at Y685 in a Src-dependent manner in vitro. Here, we studied the occurrence of Y685 phosphorylation in vivo in the female reproductive tract because it is a unique model of physiological vascular remodeling dependent on vascular endothelial growth factor. We first developed and characterized an anti-phospho-specific antibody against the site Y685 of VE-cadherin to monitor VE-cadherin phosphorylation along the four phases of mouse estrous cycle, termed proestrus, estrus, metestrus, and diestrus. A dynamic profile of tyrosine phosphorylated proteins was observed in both uterus and ovary throughout mouse estrous cycle, including kinase Src, which was found highly active at the estrus phase. The extent of tyrosine phosphorylated VE-cadherin was low at proestrus but strongly increased at estrus and returned to baseline at metestrus and diestrus, suggesting a potent hormonal regulation of this specific process. Indeed, C57Bl/6 female mice treatment with pregnant mare serum gonadotropin and human chorionic gonadotropin confirmed a significant increase in phosphoY685-VE-cadherin compared with that in untreated mice. These results demonstrate that VE-cadherin tyrosine phosphorylation at Y685 is a physiological and hormonally regulated process in female reproductive organs. In addition, this process was concomitant with the early steps of vascular remodeling taking place at estrus stage, suggesting that phosphoY685-VE-cadherin is a biomarker of endothelial cell activation in vivo.

Patients with metastatic neuroblastoma are rarely curable with currently available therapy, and the search for new treatment options, which include the use of inhibitors of tumor angiogenesis, is warranted. Here, we have evaluated the efficacy of one of the most promising natural inhibitors of angiogenesis described to date, endostatin, in a human neuroblastoma xenograft model in nude mice. Murine endostatin cDNA was cloned in a bacterial expression vector, expressed as a polyHis-Endostatin fusion protein and purified on Ni2+-NTA beads. The in vitro activity of soluble endostatin was confirmed on bovine capillary endothelial cells and human umbilical vein endothelial cells. The human neuroblastoma cell line SKNAS was injected subcutaneously in the flank of nude mice and administration of the recombinant angiogenesis inhibitor started when tumors reached the size of 100 μm3. Twenty mg/kg of recombinant precipitated endostatin or PBS was subcutaneously injected daily for 12 days. Serum endostatin levels were measured using a competitive enzyme immunoassay. Tumor growth was only slowed down in endostatin-treated mice when compared to control mice, and no statistically significant difference in serum levels of endostatin was observed between endostatin-treated and control groups. The lack of correlation between serum concentration and tumor response raises concern regarding the mechanism of action of endostatin.

Endothelial cells lining vessels of endocrine tissues are fenestrated. Interactions with the local environment via either soluble factors or cell-cell interactions appear to govern this terminal endothelial differentiation. Adrenocorticotropin (ACTH) has previously been reported to modulate endothelial fenestration in the rat adrenal cortex. Since vascular endothelial growth factor (VEGF) has been characterized as a potent inducer of endothelial fenestration, we aimed to characterize the status of VEGF expression in the bovine adult adrenal cortex and asked whether ACTH may regulate VEGF expression. By immunohistochemical analysis, we observed VEGF expression in steroidogenic cells from both zona glomerulosa and zona fasciculata of the bovine adrenal cortex. Double-labeling experiments performed on isolated cells in primary culture revealed VEGF immunoreactivity, essentially colocalized with the Golgi apparatus. The expression of two predominant VEGF isoforms, VEGF(121) and VEGF(165), was observed by RT-PCR analysis. ACTH (10 nM) was found to rapidly (within 2-4 h) increase the abundance of these VEGF transcripts, as assessed by both RT-PCR and Northern blot analysis. In parallel, ACTH significantly induced VEGF secretion into the medium of fasciculata cells in primary culture. Thus, our data are consistent with the involvement of ACTH, through its regulation of VEGF expression, in the maintenance of the adult adrenal cortex endothelium.

Adult adrenal cortex is a differentiated endocrine tissue that undergoes permanent regeneration. Proliferation of the progenitor cells is restricted to the outer quarter of the cortex (Zajicek et al. 1986, Mc Nicol & Duffy 1987). After this initial proliferative step, adrenocytes are displaced centripetally, until they reach the junction to the medulla (the whole process takes about 100 days in the rat) (Zajicek et al. 1986). Half of the cells die on the way and the remainder are eliminated by apoptosis in the reticularis zone (Wyllie et al. 1973). During this displacement, adrenocytes acquire three distinct functional and morphological phenotypes characterized by different patterns of steroidogenic enzyme gene expression. Younger cells differentiate into glomerulosa cells, acquiring the capacity to synthesize aldosterone. Further along the way, adrenocytes become fasciculata cells, expressing P450c17 (steroid 17a-hydroxylase) and synthesizing cortisol and/or corticosterone. In the innermost zone, the oldest adrenocytes acquire the reticularis phenotype, characterized in primates by the ability to synthesize adrenal androgens (dehydroepiandrosterone). Subcapsular remnants obtained after adrenal enucleation are able to regenerate differentiated cortical tissue with functional fasciculata and reticularis zones, even when grafted in ectopic locations (Skelton 1959, Belloni et al. 1990). The subcapsular glomerulosa zone thus probably contains what may be considered the adrenocyte stem cells. Two fairly recent reports suggest, however, that this may not be the only location of stem cells. Mitani et al. (1994) identified a layer of cells between the zona glomerulosa and the zona fasciculata that do not express aldosterone synthase and 11â-hydroxylase and proposed that it represents the layer of adrenocyte stem cells. These cells were not isolated and therefore their potential for conversion into differentiated adrenocytes with a glomerulosa or fasciculata phenotype has not been proven. More recently, Thomas et al. (1997) succeeded in reconstituting a fully differentiated and vascularized adrenocortical organoid by transplanting clones of cells derived from a single bovine fasciculata cell, in the renal capsule of scid mice. Some 25% of the clones transplanted were able to successfully form cortisol-secreting tissue, suggesting that some but not all of the fasciculata cells that possess the capacity to form a cell clone in vitro can be considered stem cells. Several experimental approaches over the last few decades have clearly established that the pituitary hormone adrenocorticotrophin (ACTH) is the primary regulator of both fetal adrenal development and adult adrenal cortex homoeostasis. Hypophysectomy of adult animals results in a dramatic regression of the adrenocortical tissue caused by a massive deletion of the fasciculata and reticularis zones through apoptosis while the glomerulosa zone remains intact and functional (Nussdorfer & Mazzochi 1972, Carsia et al. 1996). More delicate removal of the sole corticotrope and melanotrope cell populations from the pituitary gland, induced by ganciclovir treatment of transgenic mice expressing the herpes simplex virus thymidine kinase transgene under the control of the pro-opiomelanocortin promoter, results in a similar regression of the adrenal cortex tissue (Allen et al. 1995). Besides its trophic action, ACTH is also a major regulator of adrenal cortex function, i.e. steroidogenesis. It stimulates the synthesis of cortisol by two distinct mechanisms. ACTH binding to its adenylate cyclase-coupled receptor MC2-R acutely increases steroid biosynthesis by stimulating cholesterol esterase, thereby releasing free cholesterol from stored cholesterol ester pools and making substrate available to the steroidogenic enzymes in the cell (Stocco & Clark 1996). ACTH is also able to chronically increase steroidogenesis by stimulating the transcription of several key cAMP-responsive genes that encode steroidogenic enzymes (Miller 1988, Simpson & Waterman 1988) and of the mitochondrial protein StAR (Steroidogenic acute regulatory protein), an essential component of the intramitochondrial cholesterol-transfer machinery (Clark et al. 1995). In vitro studies of the proliferative action of ACTH using adrenocortical cell subpopulations from the glomerulosa or the fasciculata zones have generated controversial results. Some authors report a decrease in cell number and 7

International audience; To identify patients with autoimmune diseases who are at high risk of developing vascular cell dysfunction, early biomarkers must be identified. This study was designed to detect and characterize circulating autoantibodies to VE-cadherin (AAVEs) in patients with early-stage autoimmune diseases. An enzyme-linked immunosorbent assay (ELISA) was developed to capture autoantibodies, using a recombinant human VE-cadherin fragment covering the extracellular domains as a target antigen. AAVEs specificity for the target antigen was confirmed by western blotting. Basal AAVEs levels were determined for healthy donors (n=75). Sera from patients (n=100) with various autoimmune diseases, including rheumatoid arthritis (n=23), systemic lupus erythematosus (SLE, n=31), systemic sclerosis (n=30), and Behçet's disease (BD, n=16) were also tested. Levels of AAVEs were significantly higher in rheumatoid arthritis (P

Angiotensin-II (AII), which stimulates steroidogenesis in bovine adrenocortical (BAC) cells through the phosphoinositides pathway, activates p42-p44 mitogen-activated protein kinases (MAPKs) after 5 min of treatment (EC50 = 0.1 nM). This activation is 1) completely inhibited by the AII receptor AT1 subtype antagonist Dup 753 (10 microM), but unaffected by the AT2 antagonist PD 123177; 2) not reproduced by the AT2 agonist CGP 42112A; 3) insensitive to pretreatment with pertussis toxin; and 4) abolished by a 48-h preexposure of the cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 microM), which down-regulates protein kinase-C activity. Fibroblast growth factor-2, a potent mitogen for BAC cells, which acts through its tyrosine kinase receptor, also activates MAPK (EC50 = 0.3 in a TPA-insensitive manner, while exhibiting no detectable effect on BAC cell steroidogenesis. In contrast, ACTH, which stimulates steroidogenesis via cAMP and inhibits BAC cell proliferation, does not stimulate MAPK. Indeed, ACTH completely blocks (IC50 = 0.01 nM) the stimulation of MAPK by AII, fibroblast growth factor-2, or TPA. Therefore, bovine adrenocortical cells provide an example of positive and negative hormonal regulation of MAPK activity through a cross-talk between the inositide-, cAMP-, and growth factor-activated tyrosine kinase pathways.

The subcellular fractions containing protein kinases capable of phosphorylating basic fibroblast growth factor (FGF-2) are unknown, but having previously characterized one that is associated with the plasma membrane [1991, Mol. Endocrinol. 5, 1003310121 we evaluated the catalytic properties of another in the nucleus. The reaction is time (linear up to 15 min), enzyme (2,000-25,000 nuclei/ml), and substrate (X, 0.18 ,uM) dependent, and the targets serine. DNase pretreatment of nuclei decreases the incorporation of phosphate into FGF-2 by 50% and the reaction. It is also inhibited by heparin (EC, 1 pug/ml) and spermidine (EC,, 3 PM). Calcium and CAMP have no effect. We conclude that the kinase is distinct from PKA, and PKC, and suggest that changes in glycosaminoglycan and polyamine concentrations during the cell cycle may modulate FGF-2 phosphorylation in the nucleus, or as it is translocated to the nucleus. Growth factor; Nucleus; Kinase; Phosphorylation; FGF; SK-Hep cell

Summary It is frequent to oppose differentiaton and proliferation processes. For example, a non-transformed cell stops proliferating when expressing its differentiated phenotype and loses its differentiated functions when overproliferating because of transformation. It is, however, very exciting to observe that several of the responses to growth and differentiation factors are similar. Both types of factors modify phosphoinositide metabolism, induce specific protein phosphorylations or act at the nuclear level through dimeric transcriptional regulators. Despite the rapidly increasing amount of information collected about the early biochemical responses induced at the membrane level and the distal gene control mechanisms at the nuclear level, much more work is going to be needed to complete the picture of the mechanism of action of hormones and growth factors.

A protein kinase capable of phosphorylating basic fibroblast growth factor (FGF) can be localized on the outer cell surface of human hepatoma cells (SK-Hep cells). The addition of [gamma-32P]ATP, but not H3(32)PO4, results in a rapid (less than 10 min) incorporation of 32P into exogenously added basic FGF. The reaction is time and concentration dependent (apparent Km, 170 nM) and is stimulated by the addition of cAMP (EC50, 0.5 microM), but not the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate. There is also no tyrosine protein kinase detected on the cell surface. The inhibition of basic FGF binding to its low and/or high affinity sites decreases the phosphorylation of basic FGF by the ecto-protein kinase. Accordingly, pretreatment of cells with heparinase for 30 min or coincubation with heparin (0.1-10 micrograms/ml) decreases phosphorylation in a dose-dependent manner. Furthermore, the addition of a nonphosphorylatable peptide analog of basic FGF ([Val112] basic FGF-(106-146)NH2) that can compete with basic FGF binding to cells prevents the phosphorylation of basic FGF. Together, these observations suggest that 1) exogenous basic FGF must associate with its low and/or high affinity binding sites to be phosphorylated, and 2) the kinase is cAMP dependent and associated with the outer cell surface, and support the hypothesis that phosphorylation may regulate the activity and/or bioavailability of the growth factor.

International audience; Platelet-activating factor (PAF), a potent inflammatory mediator, is involved in endothelial permeability. This study was designed to characterize PAF receptor (PAF-R) expression and its specific contribution to the modifications of adherens junctions in mouse endothelial cells. We demonstrated that PAF-R was expressed in mouse endothelial cells and was functionally active in stimulating p42/p44 MAPK and phosphatidylinositol 3-kinase (PtdIns3'-kinase)/Akt activities. Treatment of cells with PAF induced a rapid time- and dose-dependent (10(-7) to 10(-10) M) increase in tyrosine phosphorylation of a subset of proteins ranging from 90 to 220 kDa, including the VE-cadherin, the latter effect being prevented by the tyrosine kinase inhibitors herbimycin A and bis-tyrphostin. We demonstrated that PAF promoted formation of multimeric aggregates of VE-cadherin with PtdIns3'-kinase, which was also inhibited by herbimycin and bis-tyrphostin. Finally, we show by immunostaining of endothelial cells VE-cadherin that PAF dissociated adherens junctions. The present data provide the first evidence that treatment of endothelial cells with PAF promoted activation of tyrosine kinases and the VE-cadherin tyrosine phosphorylation and PtdIns3'-kinase association, which ultimately lead to the dissociation of adherens junctions. Physical association between PtdIns3'-kinase, serving as a docking protein, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of PAF-induced cellular activation.

International audience; Vascular endothelial-cadherin (VE-cadherin) plays a key role in angiogenesis and in vascular permeability. The regulation of its biological activity may be a central mechanism in normal or pathological angiogenesis. VE-cadherin has been shown to be phosphorylated on tyrosine in vitro under various conditions, including stimulation by VEGF. In the present study, we addressed the question of the existence of a tyrosine phosphorylated form of VE-cadherin in vivo, in correlation with the quiescent versus angiogenic state of adult tissues. Phosphorylated VE-cadherin was detected in mouse lung, uterus, and ovary but not in other tissues unless mice were injected with peroxovanadate to block protein phosphatases. Remarkably, VE-cadherin tyrosine phosphorylation was dramatically increased in uterus and ovary, and not in other organs, during PMSG/hCG-induced angiogenesis. In parallel, we observed an increased association of VE-cadherin with Flk1 (VEGF receptor 2) during hormonal angiogenesis. Additionally, Src kinase was constitutively associated with VE-cadherin in both quiescent and angiogenic tissues and increased phosphorylation of VE-cadherin-associated Src was detected in uterus and ovary after hormonal treatment. Src-VE-cadherin association was detected in cultured endothelial cells, independent of VE-cadherin phosphorylation state and Src activation level. In this model, Src inhibition impaired VEGF-induced VE-cadherin phosphorylation, indicating that VE-cadherin phosphorylation was dependent on Src activation. We conclude that VE-cadherin is a substrate for tyrosine kinases in vivo and that its phosphorylation, together with that of associated Src, is increased by angiogenic stimulation. Physical association between Flk1, Src, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of VEGF-stimulated angiogenic processes.

Angiopoietin-1 (Ang-1), a newly discovered ligand of the endothelial-specific tyrosine kinase receptor Tie-2, has been found to promote cell survival, vascular maturation, and stabilization, and to function in concert with vascular endothelial growth factor. Adrenal gland has an intense capillary network that regulation remains to be documented. Recently, we demonstrated that vascular endothelial growth factor, and its receptors are expressed in mouse adrenal in vivo, but no detailed study on Ang expression in the adrenal has been reported. The present study shows the expression of Tie2 receptors, Ang-1, and its endogenous antagonist, Ang-2 in mouse adrenal in vivo. Immunohistochemistry disclosed that Tie2 colocalized with platelet-endothelial-cell-adhesion-molecule in endothelial cells from normal mouse adrenal. Daily administration of dexamethasone (DEX) (0.5 mg/100 g body weight.d) for 6 d in mice, decreased steroidogenic function of adrenal as shown by inhibition of the 36-kDa ACTH receptor protein expression, and decreased plasma corticosterone level [control from 465 +/- 35 ng/ml to 114 +/- 18 ng/ml in DEX group (P0.001)]. Using semiquantitative RT-PCR, we demonstrate that DEX treatment down regulates Ang-1 mRNA levels by 3- to 4-fold. No significant changes in Ang-2 were detected between control and DEX groups, resulting in an altered Ang-2 to Ang-1 relative ratio. The Tie2 receptor was also found to be down-regulated in DEX group at both mRNA and protein level. ACTH was found to play a causal role in DEX-induced decrease in Ang-1/Tie2 system, because 7 d treatment with long acting 1-39 ACTH (30 IU/kg x d) increased Ang-1, Tie2 expression, and plasma corticosterone back to control levels. These results reinforce the role of ACTH in the regulation of angiogenic factors in adrenal gland and suggest that the Ang/Tie2 system might represent a key player for stabilization of adrenal endothelium.

During activation of adrenocortical cells by adrenocorticotrophic hormone (ACTH), tyrosine dephosphorylation of paxillin is stimulated and this correlates with protrusion of filopodial structures and a decreased number of focal adhesions. These effects are inhibited by Na3VO4, a phosphotyrosine phosphatase inhibitor [Vilgrain, Chinn, Gaillard, Chambaz and Feige (1998) Biochem. J. 332, 533–540]. However, the tyrosine phosphatases involved in these processes remain to be identified. In this study, we provide evidence that the Src homology domain (SH)2-containing phosphotyrosine phosphatase (SHP)2, but not SHP1, is expressed in adrenocortical cells and is phosphorylated upon ACTH challenge. ACTH (10-8M) treatment of 32P-labelled adrenocortical cells resulted in an increase in phosphorylated SHP2. By probing SHP2-containing immunoprecipitates with an antibody to phosphoserine we found that SHP2 was phosphorylated on serine in ACTH-treated cells in a dose- and time-dependent manner. Furthermore, using an in vitro kinase assay, we showed that SHP2 was a target for cAMP-dependent protein kinase (PKA). Serine was identified as the only target amino acid phosphorylated in SHP2. Phosphorylation of SHP2 by PKA resulted in a dramatic stimulation of phosphatase activity measured either with insulin receptor substrate-1 or with the synthetic peptide [32P]poly(Glu/Tyr) as substrate. In an in-gel assay of SHP2-containing immunoprecipitates, phosphorylated in vitro by PKA or isolated from adrenocortical cells treated with 10nM ACTH, a pronounced activation of SHP2 activity was shown. These observations clearly support the idea that a PKA-mediated signal transduction pathway contributes to SHP2 regulation in adrenocortical cells and point to SHP2 as a possible mediator of the effects of ACTH.

A study of bovine adrenocortical cell shape on adrenocorticotropic hormone (ACTH) challenge showed that the cells round up and develop arborized processes. This effect was found to be (1) specific for ACTH because angiotensin II and basic fibroblast growth factor have no effect; (2) mediated by a cAMP-dependent pathway because forskolin reproduces the effect of the hormone; (3) inhibited by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, but unchanged by okadaic acid, a serine/threonine phosphatase inhibitor; and (4) correlated with a complete loss of focal adhesions. Biochemical studies of the focal-adhesion-associated proteins showed that pp125fak, vinculin (110 kDa) and paxillin (70 kDa) were detected in the Triton X-100-insoluble fraction from adrenocortical cells. During cell adhesion on fibronectin as substratum, two major phosphotyrosine-containing proteins of molecular masses 125 and 68 kDa were immunodetected in the same fraction. A dramatic decrease in the extent of tyrosine phosphorylation of these proteins was observed within 60 min after treatment with ACTH. No change in pp125fak tyrosine phosphorylation nor in Src activity was detected. In contrast, paxillin was found to be tyrosine-dephosphorylated in a time-dependent manner in ACTH-treated cells. Sodium orthovanadate completely prevented the effect of ACTH. These observations suggest a possible role for phosphotyrosine phosphatases in hormone-dependent cellular regulatory processes.

BACKGROUND: Vascular endothelial (VE)-cadherin is an endothelial specific adhesion molecule of vital importance for the maintenance of endothelium integrity. Because gliomas are hypervascular tumors, the present study was designed to address the question whether Vascular endothelial-cadherin (VE-cadherin), a protein exclusively expressed in endothelial cells, could be a potential candidate biomarker for clinical use in the setting of gliomas. METHODS: Fifty-six patients newly diagnosed glioma who underwent radiochemotherapy were enrolled in this retrospective study. Serum samples were collected prior treatment. Therapeutic response to Temodal was observed by RMI in 24 patients (25 oligodendroglioma, 2 glioblastoma multiform) median age 42.15 (range 23-65) while 32 patients were resistant (5 anaplastic oligodendroglioma, 9 anaplastic astrocytomas and 17 glioblastomas multiforme), median age 51.9 (range 19-84). sVE-cadherin levels was evaluated by westernblotting and sandwich ELISA. Data were analyzed using logistic regression and receiver operating characteristics (ROC) curve analysis. RESULTS: VE-cadherin 90 kDa extracellular domain was detected in human serum by western blot and its accurate detection was dependent upon a pre-dilution in a detergent-containing buffer (Triton X-100). Of importance, we found that sVE-cadherin was carried by human lipoproteins and that the detergent allowed to separate both entities to increase protein detectability. Interestingly, we found that sVE cadherin was significantly lower in the non responders group as compared to responders. We developped an ELISA that clearly discriminate patients NR (0.669+/-0.104 AU) from patients R (0.975+/-0.098 AU (p=0.0024). Receiver-operating characteristic (ROC) curve analysis revealed that the predictive cut-off value of serum VE-cadherin for chemoresistance was 0.735 AU (area-under-the-curve 0.82; 95% confidence interval 0.71-0.87; maximal accuracy 0.875) Patients lipid profile (total cholesterol, triglycerides, LDL and HDL) were not significantly different between the two groups and could not account for the differences observed in serum VE-cadherin levels. CONCLUSIONS: We show for the first time that the lipoprotein component of serum — which is most often ignored in protein diagnostics — contains proteins that can unexpectedly serve as biomarkers for disease. This finding may open completely new avenues of biomarker research. Second, we demonstrate, one candidate marker (soluble VE-cadherin) to follow glioma therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2724.

The calcium-activated, phospholipid-dependent protein kinase (C kinase) and its proteolytic product (M kinase), originally discovered in central nervous tissue (Takai, Y., Kishimoto, A., Inoue, M., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7603-7610) were characterized in bovine adrenal cortex cytosol. An endogenous calcium-dependent protease able to generate M kinase from the isolated C kinase in vitro was also present in adrenocortical extracts. Bovine adrenocortical cells in suspension as well as in primary culture contain the C and the M kinase activities. Treatment of these cells by steroidogenic concentrations (nM to microM) of ACTH resulted in a time and dose-dependent increase of cytosolic C kinase activity, whereas no change in M kinase activity was detected. This apparent activation appears to result mostly from an intracellular shift of the membrane-associated C kinase to a soluble cytosolic form of the enzyme. These observations open the question of the possible implication of the calcium, phospholipid-dependent protein phosphorylation system in hormone-dependent cellular regulatory processes.

Two 67 kDa proteins adsorbed to membranes in the presence of Ca2+ have been purified to homogeneity from pig lung using conventional procedures, followed by calcium-dependent affinity chromatography on polyacrylamide-immobilized phosphatidylserine. The two proteins were, respectively, excluded (67E) and retained (67R) on the column in the presence of Ca2+. On the basis of amino acid composition and isoelectric point, 67R was identified as 67 kDa calelectrin/calcimedin, whereas 67E could be differentiated from albumin, calregulin, 67 kDa fragment of protein kinase C and surfactant-associated proteins. Only 67R was slightly phosphorylated by protein kinase C, reacted with an antibody raised against 32.5 kDa endonexin and inhibited pig pancreas phospholipase A2 in a way similar to that of lipocortin or endonexin. These data bring further support to the view that inhibition of phospholiphase A2 by lipocortin or other related proteins involves interaction with the lipid/water interface. They also provide evidence for a new kind of Ca2+-binding protein (67E), whose role still remains to be determined.

Four calcium and phospholipid binding proteins purified from mononuclear cells were characterized for PKC and EGF phosphorylation, actin binding capacity, and partial tissue distribution. Those named 35K, 32K, and 73K are equivalent, respectively, to lipocortin III, endonexin II and the 67 kDa calelectrin; 36K is a fragment of 73K. After purification, 35K and 73K were phosphorylated by protein kinase C in vitro but 36K nor 32K were not. None were phosphorylated by the epidermal growth factor receptor kinase in vitro; 73K bound F-actin in a calcium-dependent manner, whereas 35K, 36K, and 32K did not. Using Western blotting analysis, 32K and 73K were detected in high amounts in human lymphocytes, monocytes, liver, and placenta and in rat adrenal medulla; but 32K was not detected in polymorphonuclear cells, and 36K and 35K were detected in high amounts only, respectively, in human blood lymphocytes and polymorphonuclear cells. Thus, 32K and 73K appear to have a wide tissue distribution, whereas 35K has a much more restricted distribution.

Purified bovine adrenocortical cytochrome P-450scc (specific for cholesterol side chain cleavage in the inner mitochondrial membrane) was selectively phosphorylated in vitro by a Ca2+-activated, phospholipid-sensitive protein kinase (protein kinase C) preparation, whereas cyclic AMP dependent and two cyclic nucleotide independent kinases were ineffective. Cytochrome P-450scc incorporated a maximum of 4 mol of phosphate in the presence of protein kinase C within 15 min at 30 degrees C, with apparent Km and Vmax of 0.14 mumol and 0.76 pmol/min, respectively. Serine and threonine were the two target aminoacids phosphorylated in a ratio of about 1:1. In the presence of 1 microM Ca2+, a mixture of phosphatidylserine and diolein (or a potent tumor promoter phorbol ester) was required for optimal cytochrome P-450scc phosphorylation. In addition, purified inner mitochondrial membrane preparations from adrenocortical mitochondria were found to contain protein kinase C activity. These findings, together with the previous demonstration that activators of protein kinase C such as a potent phorbol ester activates steroidogenesis of intact adrenocortical cells, suggest that phosphorylation of P-450scc should be examined for its possible role in the regulation of adrenocortical functions.

Exposure of bovine adrenocortical cells to optimal concentrations of angiotensin II (A II) resulted in an almost 2-fold enhancement of cellular cAMP accumulation in response to steroidogenic concentrations of ACTH. This effect was dose-dependent and transient, with a maximum after 4–6 min of treatment with A II. Activators of protein kinase C such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol mimicked that effect in a sustained fashion. The ACTH-sensitized state of the adreno-cortical adenylate cyclase system induced by TPA exhibited also an enhanced response to forskolin. On the other hand, previous treatment of the cells by pertussis toxin suppressed any further effect of TPA. It is suggested that, following A II exposure, the Gi inhibitory components of the adrenocortical cell adenylate cyclase system may be inactivated, leading to increased response to ACTH. This process may involve protein kinase C activation, subsequent to intracellular generation of lipidic messengers resulting from accelerated phosphoinositide breakdown induced by angiotensin.

Y1 adrenal tumor cells are resistant to the steroidogenic effect of A-II though they possess specific A-II binding sites. The number of these binding sites is lower in Y1 cells than in bovine adrenal cells, but the affinity is similar in the two models. Moreover, Y1 cells are shown to contain a high level of cytosolic protein kinase C whose properties appear similar to those observed in bovine adrenal cells. However, the activation of protein kinase C by a phorbol ester (PMA) or diacylglycerol (OAG) does not induce steroidogenesis in Y1 cells. On the other hand, A-II, without any effect on adenylate cyclase in basal conditions, reduces the ACTH-induced cAMP production in Y1 cells. This inhibitory effect of A-II is not blocked by phosphodiesterase inhibitor but is completely abolished after 24 hours of pretreatment of intact cells with pertussis toxin. This inhibition is probably mediated by the inhibitory guanine nucleotide regulatory protein (Gi) since the labeled 41 KD-ADP ribosylated protein disappeared after 24 hours of pretreatment of intact cells with pertussis toxin. Moreover, the accumulation of inositol phosphates under A-II stimulation was low, which suggests that the coupling of A-II receptors with phospholipase C is reduced in Y1 cells. The Y1 cell line is probably a good model to study the post membrane events in A-II action.

Protein kinase C purified to apparent homogeneity from rat brain was resolved into four active moieties following chromatography over a hydroxyapatite high resolution system. By contrast, the same procedure applied to bovine adrenocortical protein kinase C revealed that a single protein kinase C isoform could be detected in this tissue, with a chromatographic behavior identical to that of one of the brain isoenzymes. Although the isolated protein kinase C isozymes were all activated to various degrees in the presence of phospholipids and calcium, quantitative differences were observed in their catalytic properties, especially with regard to their sensitivity to diacylglycerol and TPA and to their relative affinity for different protein substrates. These observations confirmed at the protein level the heterogeneity of protein kinase C predicted on the basis of cDNA cloning studies. They also suggest that the expression of a specific set of protein kinase C isoenzyme(s) in a given cell type deserves further attention, since it may reflect a functionnal significance with regard to the regulation of specific cellular processes.

Cytosols prepared from adrenal glands of ovine fetuses (110–144 days of gestation) and of newborn lambs (1–6 days post-partum) were analysed for their protein kinase activities. Two major peaks of casein kinase activities and two major peaks of histone kinase activities were observed in all cytosols of both fetal and neonatal adrenal glands. They were characterized as cAMP-independent casein kinases of type A and type G, and as cAMP-dependent histone kinase isoenzymes of type I and type II. The specific activity of each enzyme increased 2-fold between 118 days of gestation and 6 days post-partum. Casein kinase of the G type was 4-fold higher than casein kinase of the A type; in contrast, the mean ratio of type II to type I histone kinase activity varied between 5- and 12-fold. Infusion of ACTH 1–24 (100 μg/day) for 5 days to 118- to 128-day-old ovine fetuses in utero increased their plasma corticosteroid levels and the responsiveness of their adrenal cells to stimulation by ACTH 1–24 in vitro. In addition, such treatment doubled the specific activity of casein kinases A and G, but had no significant effect on cAMP-dependent histone kinases activities. In relation to current concepts of the role of protein kinases in adult adrenal cells, the present results suggest that casein kinase activities are involved in cell multiplication and differentiation in the fetal adrenal gland. In addition, they show that neither cytosolic histone kinase of type I nor that of type II is likely rate-limiting in the steroidogenic response to ACTH of ovine fetal adrenal cells.