Signaling pathways mediating cardiac myocyte gene expression in physiological and stress responses

The contractile cells in the heart (the cardiac myocytes) are terminally differentiated. In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis, responses associated with the development of cardiac pathologies. There has been much effort expended in gaining an understanding of the stimuli which promote these responses, and in identifying the intracellular signaling pathways which are activated and potentially involved. These signaling pathways presumably modulate gene and protein expression to elicit the end-stage response. For the regulation of gene expression, the signal may traverse the cytoplasm to modulate nuclear-localized transcription factors as occurs with the mitogen-activated protein kinase or protein kinase B/Akt cascades. Alternatively, the signal may promote translocation of transcription factors from the cytoplasm to the nucleus as is seen with the calcineurin/NFAT and JAK/STAT systems. We present an overview of the principal signaling pathways implicated in the regulation of gene expression in cardiac myocyte pathophysiology, and summarize the current understanding of these pathways, the transcription factors they regulate and the changes in gene expression associated with the development of cardiac pathologies. Finally, we discuss how intracellular signaling and gene expression may be integrated to elicit the overall change in cellular phenotype.

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely. E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Peptidomic Analysis of Human Cell Lines

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEIC293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the PI position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells.

High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Diet-induced obesity impairs AKT signalling in the retina and causes retinal degeneration

Retinopathy, a common complication of diabetes, is characterized by an unbalanced production of nitric oxide (NO), a process regulated by nitric oxide synthase (NOS). We hypothesized that retinopathy might stem from changes in the insulin receptor substrate (IRS)/PI3K/AKT pathway and/or expression of NOS isoforms. Thus, we analysed the morphology and apoptosis index in retinas of obese rats in whom insulin resistance had been induced by a high-fat diet (HFD). Immunoblotting analysis revealed that the retinal tissue of HFD rats had lower levels of AKT1, eNOS and nNOS protein than those of samples taken from control animals. Furthermore, immunohistochemical analyses indicated higher levels of iNOS and 4-hydroxynonenal and a larger number of apoptotic nuclei in HFD rats. Finally, both the inner and outer retinal layers of HFD rats were thinner than those in their control counterparts. When considered alongside previous results, these patterns suggest two major ways in which HFD might impact animals: direct activity of ingested fatty acids and/or via insulin-resistance-induced changes in intracellular pathways. We discuss these possibilities in further detail and advocate the use of this animal model for further understanding relationships between retinopathy, metabolic syndrome and type 2 diabetes. © 2012 John Wiley & Sons, Ltd.

Acute exercise decreases PTP-1B protein level and improves insulin signaling in the liver of old rats

It is now commonly accepted that chronic inflammation associated with obesity during aging induces insulin resistance in the liver. In the present study, we investigated whether the improvement in insulin sensitivity and insulin signaling, mediated by acute exercise, could be associated with modulation of protein-tyrosine phosphatase 1B (PTP-1B) in the liver of old rats. Aging rats were subjected to swimming for two 1.5-h long bouts, separated by a 45 min rest period. Sixteen hours after the exercise, the rats were sacrificed and proteins from the insulin signaling pathway were analyzed by immunoblotting. Our results show that the fat mass was increased in old rats. The reduction in glucose disappearance rate (Kitt) observed in aged rats was restored 16 h after exercise. Aging increased the content of PTP-1B and attenuated insulin signaling in the liver of rats, a phenomenon that was reversed by exercise. Aging rats also increased the IRβ/PTP-1B and IRS-1/PTP-1B association in the liver when compared with young rats. Conversely, in the liver of exercised old rats, IRβ/PTP-1B and IRS-1/PTP-1B association was markedly decreased. Moreover, in the hepatic tissue of old rats, the insulin signalling was decreased and PEPCK and G6Pase levels were increased when compared with young rats. Interestingly, 16 h after acute exercise, the PEPCK and G6Pase protein level were decreased in the old exercised group. These results provide new insights into the mechanisms by which exercise restores insulin signalling in liver during aging. © 2013 Moura et al; licensee BioMed Central Ltd.

Angiotensin II AT(1) receptor mutants expressed in CHO cells caused morphological change and inhibition of cell growth

To assess the importance of the leucine residues in positions 262 and 265 of the angiotensin AT, receptor for signaling pathways and receptor expression and regulation, we compared the properties of CHO cells transfected with the wild type or the L262D or L265D receptor point mutants. It was found that the two mutants significantly increased the basal intracellular cyclic AMP (cAMP) formation in an agonist-independent mode. The morphology transformation of CHO cells was correlated with the increased cAMP formation, since forskolin, a direct activator of adenylate cyclase mimicked this effect on WT-expressing CHO cells. DNA synthesis was found to be inhibited in these cell lines, indicating that cAMP may also have determined the inhibitory effect on cell growth, in addition to the cell transformation from a tumorigenic to a non-tumorigenic phenotype. However a role for an increased Ca2(+) influx induced by the mutants in non-stimulated cells cannot be ruled out since this ion also was shown to cause transformed cells to regain the morphology and growth regulation. (c) 2005 Elsevier B.V. All rights reserved.

Biology and oncogenicity of the Kaposi sarcoma herpesvirus K1 protein

The Kaposi sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is a gammaherpesvirus etiologically linked to the development of Kaposi sarcoma, primary effusion lymphomas, and multicentric Castleman disease in humans. KSHV is unique among other human herpesviruses because of the elevated number of viral products that mimic human cellular proteins, such as a viral cyclin, a viral G protein-coupled receptor, anti-apoptotic proteins (e.g. v-bcl2 and v-FLIP), viral interferon regulatory factors, and CC chemokine viral homologues. Several KSHV products have oncogenic properties, including the transmembrane K1 glycoprotein. KSHV K1 is encoded in the viral ORFK1, which is the most variable portion of the viral genome, commonly used to discriminate among viral genotypes. The extracellular region of K1 has homology with the light chain of lambda immunoglobulin, and its cytoplasmic region contains an immunoreceptor tyrosine-based activation motif (ITAM). KSHV K1 ITAM activates several intracellular signaling pathways, notably PI3K/AKT. Consequently, K1 expression inhibits proapoptotic proteins and increases the life-span of KSHV-infected cells. Another remarkable effect of K1 activity is the production of inflammatory cytokines and proangiogenic factors, such as vascular endothelial growth factor. KSHV K1 immortalizes primary human endothelial cells and transforms rodent fibroblasts in vitro; moreover, K1 induces tumors in vivo in transgenic mice expressing this viral protein. This review aims to consolidate and discuss the current knowledge on this intriguing KSHV protein, focusing on activities of K1 that can contribute to the pathogenesis of KSHV-associated human cancers. Copyright © 2015 John Wiley & Sons, Ltd.

High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

In this study, we evaluated the effects of obesity and insulin resistance induced by a high-fat diet on prostate morphophysiology, focusing on cell proliferation, expression of androgen (AR) and estrogen receptors (ER) and proteins of the insulin signaling pathway. Adult male Wistar rats were fed a high-fat diet (20% fat) for 15 weeks, whereas control animals received a balanced diet (4% fat). Both groups were then divided and treated for 2 weeks with 1 mg/kg body weight/day of the aromatase inhibitor letrozole or vehicle only. The ventral prostate was analyzed with immunohistochemical, histopathological, stereological, and Western blotting methods. Obese rats showed insulin resistance, hyperinsulinemia, and reduced plasma testosterone levels. The incidence of prostatic intraepithelial neoplasia (PIN) was 2.7 times higher in obese rats and affected 0.4% of the gland compared with 0.1% PIN areas found in control rats. Obesity doubled cell proliferation in both prostate epithelium and stroma. AR content decreased in the prostate of obese rats and estrogen receptor beta (ER beta) increased in this group. Protein levels of insulin receptor substrate 1 and protein kinase B diminished in the obese group, whereas phosphatidylinositol 3-kinase (PI3K) increased significantly. Most structural changes observed in the prostate of obese rats normalized after letrozole treatment, except for increased stromal cell proliferation and ER beta expression, which might be associated with insulin resistance. This experimental model of obesity and insulin resistance induced by a high-fat diet increases cell proliferation in rat prostate. Such alterations are associated with decreased levels of AR and increased ER beta and PI3K proteins. This change can facilitate the establishment of proliferative lesions in rat prostate.

Serum amyloid A is a growth factor for 3T3-L1 adipocytes, inhibits differentiation and promotes insulin resistance

BACKGROUND/OBJECTIVES: Serum amyloid A (SAA) is an acute-phase protein that has been recently correlated with obesity and insulin resistance. Therefore, we first examined whether human recombinant SAA (rSAA) could affect the proliferation, differentiation and metabolism of 3T3-L1 preadipocytes. DESIGN: Preadipocytes were treated with rSAA and analyzed for changes in viability and [H-3-methyl]-thymidine incorporation as well as cell cycle perturbations using flow cytometry analysis. The mRNA expression profiles of adipogenic factors during the differentiation protocol were also analyzed using real-time PCR. After differentiation, 2-deoxy-[1,2-H-3]-glucose uptake and glycerol release were evaluated. RESULTS: rSAA treatment caused a 2.6-fold increase in cell proliferation, which was consistent with the results from flow cytometry showing that rSAA treatment augmented the percentage of cells in the S phase (60.9 +/- 0.54%) compared with the control cells (39.8 +/- 2.2%, ***P<0.001). The rSAA-induced cell proliferation was mediated by the ERK1/2 signaling pathway, which was assessed by pretreatment with the inhibitor PD98059. However, the exposure of 3T3-L1 cells to rSAA during the differentiation process resulted in attenuated adipogenesis and decreased expression of adipogenesis-related factors. During the first 72 h of differentiation, rSAA inhibited the differentiation process by altering the mRNA expression kinetics of adipogenic transcription factors and proteins, such as PPAR gamma 2 (peroxisome proliferator-activated receptor gamma 2), C/EBP beta (CCAAT/enhancer-binding protein beta) and GLUT4. rSAA prevented the intracellular accumulation of lipids and, in fully differentiated cells, increased lipolysis and prevented 2-deoxy-[1,2-H-3]-glucose uptake, which favors insulin resistance. Additionally, rSAA stimulated the secretion of proinflammatory cytokines interleukin 6 and tumor necrosis factor alpha, and upregulated SAA3 mRNA expression during adipogenesis. CONCLUSIONS: We showed that rSAA enhanced proliferation and inhibited differentiation in 3T3-L1 preadipocytes and altered insulin sensitivity in differentiated cells. These results highlight the complex role of SAA in the adipogenic process and support a direct link between obesity and its co-morbidities such as type II diabetes.

Production of bioactive peptides through sequencial action of Yarrowia lipolytica proteases and chemical glycation

This PhD thesis is aimed at studying the suitability of proteases realised by Yarrowia lipolytica to hydrolyse proteins of different origins available as industrial food by-products. Several strains of Y. lipolytica have been screened for the production of extracellular proteases by zymography. On the basis of the results some strains released only a protease having a MW of 37 kDa, which corresponds to the already reported acidic protease, while other produced prevalently or only a protease with a MW higher than 200 kDa. The proteases have been screened for their "cold attitude" on gelatin, gluten and skim milk. This property can be relevant from a biotechnological point of view in order to save energy consumption during industrial processes. Most of the strains used were endowed with proteolytic activity at 6 °C on all the three proteins. The proteolytic breakdown profiles of the proteins, detected at 27 °C, were different related to the specific strains of Y. lipolytica. The time course of the hydrolysis, tested on gelatin, affected the final bioactivities of the peptide mixtures produced. In particular, an increase in both the antioxidant and antimicrobial activities was detected when the protease of the strain Y. lipolytica 1IIYL4A was used. The final part of this work was focused on the improvement of the peptides bioactivities through a novel process based on the production of glycopeptides. Firstly, the main reaction parameters were optimized in a model system, secondly a more complex system, based on gluten hydrolysates, was taken into consideration to produce glycopeptides. The presence of the sugar moiety reduced the hydrophobicity of the glycopeptides, thus affecting the final antimicrobial activity which was significantly improved. The use of this procedure could be highly effective to modify peptides and can be employed to create innovative functional peptides using a mild temperature process.

Functional characterization of the placenta specific protein PLAC1 and its use for cancer immunotherapy

Summary Antibody-based cancer therapies have been successfully introduced into the clinic and have emerged as the most promising therapeutics in oncology. The limiting factor regarding the development of therapeutical antibody vaccines is the identification of tumor-associated antigens. PLAC1, the placenta-specific protein 1, was categorized for the first time by the group of Prof. Sahin as such a tumor-specific antigen. Within this work PLAC1 was characterized using a variety of biochemical methods. The protein expression profile, the cellular localization, the conformational state and especially the interacting partners of PLAC1 and its functionality in cancer were analyzed. Analysis of the protein expression profile of PLAC1 in normal human tissue confirms the published RT-PCR data. Except for placenta no PLAC1 expression was detectable in any other normal human tissue. Beyond, an increased PLAC1 expression was detected in several cancer cell lines derived of trophoblastic, breast and pancreatic lineage emphasizing its properties as tumor-specific antigen. rnThe cellular localization of PLAC1 revealed that PLAC1 contains a functional signal peptide which conducts the propeptide to the endoplasmic reticulum (ER) and results in the secretion of PLAC1 by the secretory pathway. Although PLAC1 did not exhibit a distinct transmembrane domain, no unbound protein was detectable in the cell culture supernatant of overexpressing cells. But by selective isolation of different cellular compartments PLAC1 was clearly enriched within the membrane fraction. Using size exclusion chromatography PLAC1 was characterized as a highly aggregating protein that forms a network of high molecular multimers, consisting of a mixture of non-covalent as well as covalent interactions. Those interactions were formed by PLAC1 with itself and probably other cellular components and proteins. Consequently, PLAC1 localize outside the cell, where it is associated to the membrane forming a stable extracellular coat-like structure.rnThe first mechanistic hint how PLAC1 promote cancer cell proliferation was achieved identifying the fibroblast growth factor FGF7 as a specific interacting partner of PLAC1. Moreover, it was clearly shown that PLAC1 as well as FGF7 bind to heparin, a glycosaminoglycan of the ECM that is also involved in FGF-signaling. The participation of PLAC1 within this pathway was approved after co-localizing PLAC1, FGF7 and the FGF7 specific receptor (FGFR2IIIb) and identifying the formation of a trimeric complex (PLAC1, FGF7 and the specific receptor FGFR2IIIb). Especially this trimeric complex revealed the role of PLAC1. Binding of PLAC1 together with FGF7 leads to the activation of the intracellular tyrosine kinase of the FGFR2IIIb-receptor and mediate the direct phosphorylation of the AKT-kinase. In the absence of PLAC1, no FGF7 mediated phosphorylation of AKT was observed. Consequently the function of PLAC1 was clarified: PLAC1 acts as a co-factor by stimulating proliferation by of the FGF7-FGFR2 signaling pathway.rnAll together, these novel biochemical findings underline that the placenta specific protein PLAC1 could be a new target for cancer immunotherapy, especially considering its potential applicability for antibody therapy in tumor patients.

Agonist-biased signaling at the sst2A receptor: the multi-somatostatin analogs KE108 and SOM230 activate and antagonize distinct signaling pathways

Somatostatin analogs that activate the somatostatin subtype 2A (sst2A) receptor are used to treat neuroendocrine cancers because they inhibit tumor secretion and growth. Recently, new analogs capable of activating multiple somatostatin receptor subtypes have been developed to increase tumor responsiveness. We tested two such multi-somatostatin analogs for functional selectivity at the sst2A receptor: SOM230, which activates sst1, sst2, sst3, and sst5 receptors, and KE108, which activates all sst receptor subtypes. Both compounds are reported to act as full agonists at their target sst receptors. In sst2A-expressing HEK293 cells, somatostatin inhibited cAMP production, stimulated intracellular calcium accumulation, and increased ERK phosphorylation. SOM230 and KE108 were also potent inhibitors of cAMP accumulation, as expected. However, they antagonized somatostatin stimulation of intracellular calcium and behaved as partial agonists/antagonists for ERK phosphorylation. In pancreatic AR42J cells, which express sst2A receptors endogenously, SOM230 and KE108 were both full agonists for cAMP inhibition. However, although somatostatin increased intracellular calcium and ERK phosphorylation, SOM230 and KE108 again antagonized these effects. Distinct mechanisms were involved in sst2A receptor signaling in AR42J cells; pertussis toxin pretreatment blocked somatostatin inhibition of cAMP accumulation but not the stimulation of intracellular calcium and ERK phosphorylation. Our results demonstrate that SOM230 and KE108 behave as agonists for inhibition of adenylyl cyclase but antagonize somatostatin's actions on intracellular calcium and ERK phosphorylation. Thus, SOM230 and KE108 are not somatostatin mimics, and their functional selectivity at sst2A receptors must be considered in clinical applications where it may have important consequences for therapy.

Cardiac tissue-restricted deletion of plakoglobin results in progressive cardiomyopathy and activation of {beta}-catenin signaling

Mutations in the plakoglobin (JUP) gene have been identified in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients. However, the mechanisms underlying plakoglobin dysfunction involved in the pathogenesis of ARVC remain poorly understood. Plakoglobin is a component of both desmosomes and adherens junctions located at the intercalated disc (ICD) of cardiomyocytes, where it functions to link cadherins to the cytoskeleton. In addition, plakoglobin functions as a signaling protein via its ability to modulate the Wnt/beta-catenin signaling pathway. To investigate the role of plakoglobin in ARVC, we generated an inducible cardiorestricted knockout (CKO) of the plakoglobin gene in mice. Plakoglobin CKO mice exhibited progressive loss of cardiac myocytes, extensive inflammatory infiltration, fibrous tissue replacement, and cardiac dysfunction similar to those of ARVC patients. Desmosomal proteins from the ICD were decreased, consistent with altered desmosome ultrastructure in plakoglobin CKO hearts. Despite gap junction remodeling, plakoglobin CKO hearts were refractory to induced arrhythmias. Ablation of plakoglobin caused increase beta-catenin stabilization associated with activated AKT and inhibition of glycogen synthase kinase 3beta. Finally, beta-catenin/TCF transcriptional activity may contribute to the cardiac hypertrophy response in plakoglobin CKO mice. This novel model of ARVC demonstrates for the first time how plakoglobin affects beta-catenin activity in the heart and its implications for disease pathogenesis.

Comparison of genomic and proteomic data in recurrent airway obstruction affected horses using Ingenuity Pathway Analysis(R)

BACKGROUND: Recurrent airway obstruction (RAO) is a severe chronic respiratory disease affecting horses worldwide, though mostly in the Northern hemisphere. Environmental as well as genetic factors strongly influence the course and prognosis of the disease. Research has been focused on characterization of immunologic factors contributing to inflammatory responses, on genetic linkage analysis, and, more recently, on proteomic analysis of airway secretions from affected horses. The goal of this study was to investigate the interactions between eight candidate genes previously identified in a genetic linkage study and proteins expressed in bronchoalveolar lavage fluid (BALF) collected from healthy and RAO-affected horses. The analysis was carried out with Ingenuity Pathway Analysis(R) bioinformatics software. RESULTS: The gene with the greatest number of indirect interactions with the set of proteins identified is Interleukin 4 Receptor (IL-4R), whose protein has also been detected in BALF. Interleukin 21 receptor and chemokine (C-C motif) ligand 24 also showed a large number of interactions with the group of detected proteins. Protein products of other genes like that of SOCS5, revealed direct interactions with the IL-4R protein. The interacting proteins NOD2, RPS6KA5 and FOXP3 found in several pathways are reported regulators of the NFkappaB pathway. CONCLUSIONS: The pathways generated with IL-4R highlight possible important intracellular signaling cascades implicating, for instance, NFkappaB. Furthermore, the proposed interaction between SOCS5 and IL-4R could explain how different genes can lead to identical clinical RAO phenotypes, as observed in two Swiss Warmblood half sibling families because these proteins interact upstream of an important cascade where they may act as a functional unit.