T cell specific adaptor protein (TSAd) promotes interaction of Nck with Lck and SLP-76 in T cells

Background: The Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated. Results: To identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr280 (pTyr280) and pTyr305. These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr280 and pTyr305 on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck. Conclusions: TSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells. © 2015 Hem et al

Influence of birth weight on gene regulators of lipid metabolism and utilization in subcutaneous adipose tissue and skeletal muscle of neonatal pigs.

Epidemiological studies suggest that low-birth weight infants show poor neonatal growth and increased susceptibility to metabolic syndrome, in particular, obesity and diabetes. Adipose tissue development is regulated by many genes, including members of the peroxisome proliferator-activated receptor (PPAR) and the fatty acid-binding protein (FABP) families. The aim of this study was to determine the influence of birth weight on key adipose and skeletal muscle tissue regulating genes. Piglets from 11 litters were ranked according to birth weight and 3 from each litter assigned to small, normal, or large-birth weight groups. Tissue samples were collected on day 7 or 14. Plasma metabolite concentrations and the expression of PPARG2, PPARA, FABP3, and FABP4 genes were determined in subcutaneous adipose tissue and skeletal muscle. Adipocyte number and area were determined histologically. Expression of FABP3 and 4 was significantly reduced in small and large, compared with normal, piglets in adipose tissue on day 7 and in skeletal muscle on day 14. On day 7, PPARA and PPARG2 were significantly reduced in adipose tissue from small and large piglets. Adipose tissue from small piglets contained more adipocytes than normal or large piglets. Birth weight had no effect on adipose tissue and skeletal muscle lipid content. Low-birth weight is associated with tissue-specific and time-dependent effects on lipid-regulating genes as well as morphological changes in adipose tissue. It remains to be seen whether these developmental changes alter an individual's susceptibility to metabolic syndrome

Underexpression of peroxisome proliferator-activated receptor (PPAR)γ in PAX8/PPARγ-negative thyroid tumours

Funding: We gratefully acknowledge the technical assistance of Ms Teresa Pereira. Ana Rita Marques is a recipient of a PhD fellowship (PRAXIS XXI/BD/21329/99) from Fundação para a Ciência e Tecnologia. This work was supported by Núcleo Regional do Sul Liga Portuguesa Contra O Cancro (Terry Fox) and Fundação para a Ciência e Tecnologia (POCTI/CBO/48922/2002), Portugal.The expression of peroxisome proliferator-activated receptor (PPAR)γ in thyroid neoplasias and in normal thyroid (NT) tissues has not been fully investigated. The objectives of the present work were: to study and compare the relative expression of PPARγ in normal, benign and malignant thyroid tissues and to correlate PPARγ immunostaining with clinical/pathological features of patients with thyroid cancer. We analysed the expression of PPARγ in several types of thyroid tissues by reverse transcription- polymerase chain reaction (RT-PCR), interphase fluorescent in situ hybridisation, real-time RT-PCR and immunohistochemistry. We have demonstrated that NT tissues express PPARy both at mRNA and at protein level. PAX8-PPARγ fusion gene expression was found in 25% (six of 24) of follicular thyroid carcinomas (FTCs) and in 17% (six of 36) of follicular thyroid adenomas, but in none of the 10 normal tissues, 28 nodular hyperplasias, 38 papillary thyroid carcinomas (PTCs) and II poorly differentiated thyroid carcinomas (PDTCs). By real-time RT-PCR, we observed that tumours negative for the PAX8-PPARγ rearrangement expressed lower levels of PPARy mRNA than the NT. Overexpression of PPARγ transcripts was detected in 80% (four of five) of translocation-positive tumours. Diffuse nuclear staining was significantly (P < 0.05) less prevalent in FTCs (53%; 18 of 34), PTCs (49%; 19 of 39) and PDTCs (0%; zero of 13) than in normal tissue (77%; 36 of 47). Peroxisome proliferator-activated receptory-negative FTCs were more likely to be locally invasive, to persist after surgery, to metastasise and to have poorly differentiated areas. Papillary thyroid carcinomas with a predominantly follicular pattern were more often PPARy negative than classic PTCs (80% vs 28%; P = 0.01). Our results demonstrated that PPARγ is underexpressed in translocation-negative thyroid tumours of follicular origin and that a further reduction of PPARγ expression is associated with dedifferentiation at later stages of tumour development and progression.publishersversionpublishe

VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis

Vascular endothelial growth factor receptor-2 (VEGFR-2) activation by VEGF-A is essential in vasculogenesis and angiogenesis. We have generated a pan-phosphorylation site map of VEGFR-2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C-terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR-2-expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T-cell-specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF-A-induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd-deficient mice, indicating a critical role of Y951-TSAd signaling in pathological angiogenesis

Polymorphisms of peroxisome proliferator-activated receptors and survival of lung cancer and upper aero-digestive tract cancers

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) are transcriptional factors involved in several biological processes such as inflammation, cancer growth, progression and apoptosis that are important in lung and upper aero-digestive tract (UADT) cancer outcomes. Nonetheless, there are no published studies of the relationship between PPARs gene polymorphisms and survival of patients with lung cancer or UADT cancers. METHODS: 1,212 cancer patients (611 lung, 303 oral, 100 pharyngeal, 90 laryngeal, and 108 esophageal) were followed for a median duration of 11 years. We genotyped three potentially functional single nucleotide polymorphisms (SNPs) using Taqman--rs3734254 of the gene PPARD and rs10865710 and rs1801282 of the gene PPARG--and investigated their associations with lung and UADT cancer survival using Cox regression. A semi-Bayesian shrinkage approach was used to reduce the potential for false positive findings when examining multiple associations. RESULTS: The variant homozygote CC (vs. TT) of PPARD rs3734254 was inversely associated with mortality of both lung cancer (adjusted hazard ratio [aHR] = 0.63, 95% confidence interval [CI] = 0.42, 0.96) and UADT cancers (aHR = 0.51, 95% CI = 0.27, 0.99). Use of the semi-Bayesian shrinkage approach yielded a posterior aHR for lung cancer of 0.66 (95% posterior limits = 0.44, 0.98) and a posterior aHR for UADT cancers of 0.58 (95% posterior limits = 0.33, 1.03). CONCLUSION: Our findings suggest that lung-cancer patients with the CC variant of PPARD rs3734254 may have a survival advantage over lung-cancer patients with other gene variants

Xenobiotic Metabolism, Disposition, and Regulation by Receptors: From Biochemical Phenomenon to Predictors of Major Toxicities

To commemorate the 50th anniversary of the Society of Toxicology, this special edition article reviews the history and current scope of xenobiotic metabolism and transport, with special emphasis on the discoveries and impact of selected “xenobiotic receptors.” This overall research realm has witnessed dynamic development in the past 50 years, and several of the key milestone events that mark the impressive progress in these areas of toxicological sciences are highlighted. From the initial observations regarding aspects of drug metabolism dating from the mid- to late 1800’s, the area of biotransformation research witnessed seminal discoveries in the mid-1900’s and onward that are remarkable in retrospect, including the discovery and characterization of the phase I monooxygenases, the cytochrome P450s. Further research uncovered many aspects of the biochemistry of xenobiotic metabolism, expanding to phase II conjugation and phase III xenobiotic transport. This led to hallmark developments involving integration of genomic technologies to elucidate the basis for interindividual differences in response to xenobiotic exposures and discovery of nuclear and soluble receptor families that selectively “sense” the chemical milieu of the mammalian cell and orchestrate compensatory changes in gene expression programming to accommodate complex xenobiotic exposures. This review will briefly summarize these developments and investigate the expanding roles of xenobiotic receptor biology in the underlying basis of toxicological response to chemical agents