Synthesis and in vitro evaluation of a novel radioligand for αvβ3 integrin receptor imaging: [(18)F]FPPA-c(RGDfK).

The development of RGD-based antagonist of αvβ3 integrin receptor has enhanced the interest in PET probes to image this receptor for the early detection of cancer, to monitor the disease progression and the response to therapy. In this work, a novel prosthetic group (N-(4-fluorophenyl)pent-4-ynamide or FPPA) for the (18)F-labeling of an αvβ3 selective RGD-peptide was successfully prepared. [(18)F]FPPA was obtained in three steps with a radiochemical yield of 44% (decay corrected). Conjugation to c(RGDfK(N3)) by the Cu(II) catalyzed Huisgen azido alkyne cycloaddition provided the [(18)F]FPPA-c(RGDfK) with a radiochemical yield of 29% (decay corrected), in an overall synthesis time of 140min.

LDLs induce fibroblast spreading independently of the LDL receptor via activation of the p38 MAPK pathway.

Because adventitial fibroblasts play an important role in the repair of blood vessels, we assessed whether elevation in LDL concentrations would affect fibroblast function and whether this depended on activation of intracellular signaling pathways. We show here that in primary human fibroblasts, LDLs induced transient activation of the p38 mitogen-activated protein kinase (MAPK) pathway, but not the c-Jun N-terminal kinase MAPK pathway. This activation did not require the recruitment of the LDL receptor (LDLR), because LDLs efficiently stimulated the p38 MAPK pathway in human and mouse fibroblasts lacking functional LDLR, and because receptor-associated protein, an LDLR family antagonist, did not block the LDL-induced p38 activation. LDL particles also induced lamellipodia formation and cell spreading. These effects were blocked by SB203580, a specific p38 inhibitor. Our data demonstrate that LDLs can regulate the shape of fibroblasts in a p38 MAPK-dependent manner, a mechanism that may participate in wound healing or vessel remodeling as in atherosclerosis.

Peroxisome proliferator-activated receptor beta/delta as a therapeutic target for metabolic diseases.

The peroxisome proliferator-activated receptor (PPAR) family comprises three distinct isotypes: PPARalpha, PPARbeta/delta and PPARgamma. PPARs are nuclear hormone receptors that mediate the effects of fatty acids and their derivatives at the transcriptional level. Until recently, the characterisation of the important role of PPARalpha in fatty acid oxidation and of PPARgamma in lipid storage contrasted with the sparse information concerning PPARbeta/delta. However, evidence is now emerging for a role of PPARbeta/delta in tissue repair and energy homeostasis. Experiments with tissue-specific overexpression of PPARbeta/delta or treatment of mice with selective PPARbeta/delta agonists demonstrated that activation of PPARbeta/delta in vivo increases lipid catabolism in skeletal muscle, heart and adipose tissue and improves the serum lipid profile and insulin sensitivity in several animal models. PPARbeta/delta activation also prevents the development of obesity and improves cholesterol homeostasis in obesity-prone mouse models. These new insights into PPARbeta/delta functions suggest that targeting PPARbeta/delta may be helpful for treating disorders associated with the metabolic syndrome. Although these perspectives are promising, several independent and contradictory reports raise concerns about the safety of PPARbeta/delta ligands with respect to tumourigenic activity in the gut. Thus, it appears that further exploration of PPARbeta/delta functions is necessary to better define its potential as a therapeutic target.

Glucagon-like peptide-1 protects beta-cells against apoptosis by increasing the activity of an IGF-2/IGF-1 receptor autocrine loop.

OBJECTIVE: The gluco-incretin hormones glucagon-like peptide (GLP)-1 and gastric inhibitory peptide (GIP) protect beta-cells against cytokine-induced apoptosis. Their action is initiated by binding to specific receptors that activate the cAMP signaling pathway, but the downstream events are not fully elucidated. Here we searched for mechanisms that may underlie this protective effect. RESEARCH DESIGN AND METHODS: We performed comparative transcriptomic analysis of islets from control and GipR(-/-);Glp-1-R(-/-) mice, which have increased sensitivity to cytokine-induced apoptosis. We found that IGF-1 receptor expression was markedly reduced in the mutant islets. Because the IGF-1 receptor signaling pathway is known for its antiapoptotic effect, we explored the relationship between gluco-incretin action, IGF-1 receptor expression and signaling, and apoptosis. RESULTS: We found that GLP-1 robustly stimulated IGF-1 receptor expression and Akt phosphorylation and that increased Akt phosphorylation was dependent on IGF-1 but not insulin receptor expression. We demonstrated that GLP-1-induced Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism; we showed that activation of IGF-1 receptor signaling was dependent on the secretion of IGF-2. We demonstrated, both in MIN6 cell line and primary beta-cells, that reducing IGF-1 receptor or IGF-2 expression or neutralizing secreted IGF-2 suppressed GLP-1-induced protection against apoptosis. CONCLUSIONS: An IGF-2/IGF-1 receptor autocrine loop operates in beta-cells. GLP-1 increases its activity by augmenting IGF-1 receptor expression and by stimulating secretion; this mechanism is required for GLP-1-induced protection against apoptosis. These findings may lead to novel ways of preventing beta-cell loss in the pathogenesis of diabetes.

Desensitization and phosphorylation of the glucagon-like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol 12-myristate 13-acetate.

Glucagon-like peptide-1 (GLP-1) stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor linked to activation of the adenylyl cyclase pathway. Here, using insulinoma cell lines, we studied homologous and heterologous desensitization of GLP-1-induced cAMP production. Preexposure of the cells to GLP-1 induced a decrease in GLP-1-mediated cAMP production, as assessed by a 3- to 5-fold rightward shift of the dose-response curve and an approximately 20 percent decrease in the maximal production of cAMP. Activation of protein kinase C by the phorbol ester phorbol 12-myristate 13-acetate (PMA) also induced desensitization of the GLP-1-mediated response, leading to a 6- to 9-fold shift in the EC50 and a 30% decrease in the maximal production of cAMP. Both forms of desensitization were additive, and the protein kinase C inhibitor RO-318220 inhibited PMA-induced desensitization, but not agonist-induced desensitization. GLP-1- and PMA-dependent desensitization correlated with receptor phosphorylation, and the levels of phosphorylation induced by the two agents were additive. Furthermore, PMA-induced, but not GLP-1-induced, phosphorylation was totally inhibited by RO-318220. Internalization of the GLP-1 receptor did not participate in the desensitization induced by PMA, as a mutant GLP-1 receptor lacking the last 20 amino acids of the cytoplasmic tail was found to be totally resistant to the internalization process, but was still desensitized after PMA preexposure. PMA and GLP-1 were not able to induce the phosphorylation of a receptor deletion mutant lacking the last 33 amino acids of the cytoplasmic tail, indicating that the phosphorylation sites were located within the deleted region. The cAMP production mediated by this deletion mutant was not desensitized by PMA and was only poorly desensitized by GLP-1. Together, our results indicate that the production of cAMP and, hence, the stimulation of insulin secretion induced by GLP-1 can be negatively modulated by homologous and heterologous desensitization, mechanisms that involve receptor phosphorylation.

Toll-like receptor 2 (TLR2) polymorphisms are associated with reversal reaction in leprosy.

BACKGROUND: Leprosy is characterized by a spectrum of clinical manifestations that depend on the type of immune response against the pathogen. Patients may undergo immunological changes known as "reactional states" (reversal reaction and erythema nodosum leprosum) that result in major clinical deterioration. The goal of the present study was to assess the effect of Toll-like receptor 2 (TLR2) polymorphisms on susceptibility to and clinical presentation of leprosy. METHODS: Three polymorphisms in TLR2 (597C-->T, 1350T-->C, and a microsatellite marker) were analyzed in 431 Ethiopian patients with leprosy and 187 control subjects. The polymorphism-associated risk of developing leprosy, lepromatous (vs. tuberculoid) leprosy, and leprosy reactions was assessed by multivariate logistic regression models. RESULTS: The microsatellite and the 597C-->T polymorphisms both influenced susceptibility to reversal reaction. Although the 597T allele had a protective effect (odds ratio [OR], 0.34 [95% confidence interval {CI}, 0.17-0.68]; P= .002 under the dominant model), homozygosity for the 280-bp allelic length of the microsatellite strongly increased the risk of reversal reaction (OR, 5.83 [95% CI, 1.98-17.15]; P= .001 under the recessive model). These associations were consistent among 3 different ethnic groups. CONCLUSIONS: These data suggest a significant role for TLR-2 in the occurrence of leprosy reversal reaction and provide new insights into the immunogenetics of the disease.

Pharmacological inhibition of fibroblast growth factor (FGF) receptor signaling ameliorates FGF23-mediated hypophosphatemic rickets.

Fibroblast growth factor 23 (FGF23) is a circulating factor secreted by osteocytes that is essential for phosphate homeostasis. In kidney proximal tubular cells FGF23 inhibits phosphate reabsorption and leads to decreased synthesis and enhanced catabolism of 1,25-dihydroxyvitamin D3 (1,25[OH]2 D3 ). Excess levels of FGF23 cause renal phosphate wasting and suppression of circulating 1,25(OH)2 D3 levels and are associated with several hereditary hypophosphatemic disorders with skeletal abnormalities, including X-linked hypophosphatemic rickets (XLH) and autosomal recessive hypophosphatemic rickets (ARHR). Currently, therapeutic approaches to these diseases are limited to treatment with activated vitamin D analogues and phosphate supplementation, often merely resulting in partial correction of the skeletal aberrations. In this study, we evaluate the use of FGFR inhibitors for the treatment of FGF23-mediated hypophosphatemic disorders using NVP-BGJ398, a novel selective, pan-specific FGFR inhibitor currently in Phase I clinical trials for cancer therapy. In two different hypophosphatemic mouse models, Hyp and Dmp1-null mice, resembling the human diseases XLH and ARHR, we find that pharmacological inhibition of FGFRs efficiently abrogates aberrant FGF23 signaling and normalizes the hypophosphatemic and hypocalcemic conditions of these mice. Correspondingly, long-term FGFR inhibition in Hyp mice leads to enhanced bone growth, increased mineralization, and reorganization of the disturbed growth plate structure. We therefore propose NVP-BGJ398 treatment as a novel approach for the therapy of FGF23-mediated hypophosphatemic diseases.

Tools and techniques to study ligand-receptor interactions and receptor activation by TNF superfamily members.

Ligands and receptors of the TNF superfamily are therapeutically relevant targets in a wide range of human diseases. This chapter describes assays based on ELISA, immunoprecipitation, FACS, and reporter cell lines to monitor interactions of tagged receptors and ligands in both soluble and membrane-bound forms using unified detection techniques. A reporter cell assay that is sensitive to ligand oligomerization can identify ligands with high probability of being active on endogenous receptors. Several assays are also suitable to measure the activity of agonist or antagonist antibodies, or to detect interactions with proteoglycans. Finally, self-interaction of membrane-bound receptors can be evidenced using a FRET-based assay. This panel of methods provides a large degree of flexibility to address questions related to the specificity, activation, or inhibition of TNF-TNF receptor interactions in independent assay systems, but does not substitute for further tests in physiologically relevant conditions.

The peroxisome proliferator-activated receptor alpha regulates amino acid metabolism.

The peroxisome proliferator-activated receptor alpha is a ligand-activated transcription factor that plays an important role in the regulation of lipid homeostasis. PPARalpha mediates the effects of fibrates, which are potent hypolipidemic drugs, on gene expression. To better understand the biological effects of fibrates and PPARalpha, we searched for genes regulated by PPARalpha using oligonucleotide microarray and subtractive hybridization. By comparing liver RNA from wild-type and PPARalpha null mice, it was found that PPARalpha decreases the mRNA expression of enzymes involved in the metabolism of amino acids. Further analysis by Northern blot revealed that PPARalpha influences the expression of several genes involved in trans- and deamination of amino acids, and urea synthesis. Direct activation of PPARalpha using the synthetic PPARalpha ligand WY14643 decreased mRNA levels of these genes, suggesting that PPARalpha is directly implicated in the regulation of their expression. Consistent with these data, plasma urea concentrations are modulated by PPARalpha in vivo. It is concluded that in addition to oxidation of fatty acids, PPARalpha also regulates metabolism of amino acids in liver, indicating that PPARalpha is a key controller of intermediary metabolism during fasting.

Essential role of platelet activation via protease activated receptor 4 in tissue factor-initiated inflammation.

INTRODUCTION: Tissue factor (TF) activation of the coagulation proteases enhances inflammation in animal models of arthritis and endotoxemia, but the mechanism of this effect is not yet fully understood - in particular, whether this is primarily due to fibrin formation or through activation of protease activated receptors (PARs). METHODS: We induced extravascular inflammation by injection of recombinant soluble murine TF (sTF1-219) in the hind paw. The effects of thrombin inhibition, fibrinogen and platelet depletion were evaluated, as well as the effects of PAR deficiency using knockout mice deficient for each of the PARs. RESULTS: Injection of soluble TF provoked a rapid onset of paw swelling. Inflammation was confirmed histologically and by increased serum IL-6 levels. Inflammation was significantly reduced by depletion of fibrinogen (P < 0.05) or platelets (P = 0.015), and by treatment with hirudin (P = 0.04) or an inhibitor of activated factor VII (P < 0.001) compared with controls. PAR-4-deficient mice exhibited significantly reduced paw swelling (P = 0.003). In contrast, a deficiency in either PAR-1, PAR-2 or PAR-3 did not affect the inflammatory response to soluble TF injection. CONCLUSION: Our results show that soluble TF induces acute inflammation through a thrombin-dependent pathway and both fibrin deposition and platelet activation are essential steps in this process. The activation of PAR-4 on platelets is crucial and the other PARs do not play a major role in soluble TF-induced inflammation.

Peroxisome-proliferator-activated receptor (PPAR)-gamma activation stimulates keratinocyte differentiation.

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.

GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling.

Wounded leaves communicate their damage status to one another through a poorly understood process of long-distance signalling. This stimulates the distal production of jasmonates, potent regulators of defence responses. Using non-invasive electrodes we mapped surface potential changes in Arabidopsis thaliana after wounding leaf eight and found that membrane depolarizations correlated with jasmonate signalling domains in undamaged leaves. Furthermore, current injection elicited jasmonoyl-isoleucine accumulation, resulting in a transcriptome enriched in RNAs encoding key jasmonate signalling regulators. From among 34 screened membrane protein mutant lines, mutations in several clade 3 GLUTAMATE RECEPTOR-LIKE genes (GLRs 3.2, 3.3 and 3.6) attenuated wound-induced surface potential changes. Jasmonate-response gene expression in leaves distal to wounds was reduced in a glr3.3 glr3.6 double mutant. This work provides a genetic basis for investigating mechanisms of long-distance wound signalling in plants and indicates that plant genes related to those important for synaptic activity in animals function in organ-to-organ wound signalling.

Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation.

Site-directed mutagenesis and molecular dynamics simulations of the alpha 1B-adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B-AR to build a theoretical model which defines the essential features of R and R. The results of site-directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B-AR. (ii) The shift of R143 of the DRY sequence out of a conserved 'polar pocket' formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B-AR. Our findings might provide interesting generalities about the activation process of G protein-coupled receptors.

Polymorphisms in Toll-like receptor 4 (TLR4) are associated with protection against leprosy.

Accumulating evidence suggests that polymorphisms in Toll-like receptors (TLRs) influence the pathogenesis of mycobacterial infections, including leprosy, a disease whose manifestations depend on host immune responses. Polymorphisms in TLR2 are associated with an increased risk of reversal reaction, but not susceptibility to leprosy itself. We examined whether polymorphisms in TLR4 are associated with susceptibility to leprosy in a cohort of 441 Ethiopian leprosy patients and 197 healthy controls. We found that two single nucleotide polymorphisms (SNPs) in TLR4 (896G>A [D299G] and 1196C>T [T399I]) were associated with a protective effect against the disease. The 896GG, GA and AA genotypes were found in 91.7, 7.8 and 0.5% of leprosy cases versus 79.9, 19.1 and 1.0% of controls, respectively (odds ratio [OR] = 0.34, 95% confidence interval [CI] 0.20-0.57, P < 0.001, additive model). Similarly, the 1196CC, CT and TT genotypes were found in 98.1, 1.9 and 0% of leprosy cases versus 91.8, 7.7 and 0.5% of controls, respectively (OR = 0.16, 95% CI 0.06--.40, P < 0.001, dominant model). We found that Mycobacterium leprae stimulation of monocytes partially inhibited their subsequent response to lipopolysaccharide (LPS) stimulation. Our data suggest that TLR4 polymorphisms are associated with susceptibility to leprosy and that this effect may be mediated at the cellular level by the modulation of TLR4 signalling by M. leprae.