Diabetes is a major risk factor for ischemic stroke and is associated with increased mortality. Additionally, hyperglycemia, a common complication in acute stroke, is associated with poor outcome. In order to identify the correlation between blood glucose and early mortality, multiple logistic regression analyses were used and odds ratios calculated in a retrospective study of 447 stroke patients. Eighty-one patients (18%) had diabetes. The odds ratios for 30-day case-fatality and blood glucose were 1.9 and 1.6 in diabetic and non-diabetic patients respectively. Optimal blood glucose concentrations in respective group were 10.3 and 6.3 mmol/L, as determined by receiver operator characteristic (ROC) curves. Cerebral ischemia triggers different signaling pathways including mitogen-activated protein kinases (MAPK) which regulate fundamental cell functions. In an experimental rat model of combined hyperglycemia and transient middle cerebral artery occlusion (MCAO), the activation pattern of one such MAPK, extracellular signal-regulated kinase (ERK) was studied along with infarct volumes and neurological function. Hyperglycemia resulted in markedly increased ERK activation and approximately three-fold increase of infarcts compared with controls. Based on the increased ERK activation, further experiments were conducted to limit the hyperglycemic-ischemic damage by interfering with ERK and supposedly related mechanisms. Consequently, rats were given U0126 (inhibiting ERK activation), PBN (anti-oxidative), PP2 (inhibiting src-family kinases), or vehicle. PBN reduced infarcts and improved neurological function compared with controls while no statistically significant effects were observed for U0126 or PP2. However, when the dose was doubled, U0126 significantly reduced infarcts and improved neurological function after 1 day in hyperglycemic rats. Post-ischemic ERK activation was completely inhibited by U0126 as demonstrated with Western immunoblotting. The findings suggest that ERK is an important mediator of hyperglycemic-ischemic brain injury and possible target for future interventions.

An injured vascular system has a substantial impact on an individuals overall health, and an understanding of the mechanisms that underlie blood vessel pathophysiology is required for the development of rational and effective treatment strategies. The phenotypic modulation of smooth muscle cells (SMC) during vascular injury, characterized by altered adhesion, migration and synthetic behavior, plays an important role in the eventual outcome. Specifically, the ability of SMCs to adhere to and remodel their extracellular environment via regulation of the syndecan class of cell adhesion molecules dictates the response of the vascular wall to local injury. The effect of in vitro syndecan-4 regulation on SMC adhesion was investigated through the use of a glass microsphere centrifugation assay, and an antisense-mediated reduction in gene expression was found to correlate with decreased adhesive strength. Regulation of syndecan-1, syndecan-2, and syndecan-4 gene expression was observed experimentally by mechanical strain of SMCs. Using real-time polymerase chain reaction (PCR), the kinetics of both static and cyclic mechanical strain were found to modify the gene expression in a time and strain magnitude-dependent manner unique to each syndecan. In particular, the responses of syndecan-4 were acute, but transient, while the evolution of syndecan-1 and syndecan-2 regulation was delayed by comparison. Mechanical strain also modulated syndecan-4 protein expression and ectodomain shedding, as measured by Western immunoblotting, and this effect was found, through selective inhibition, to be at least in part dependent on mitogen-activated protein (MAP) kinase signaling. In particular, intact extracellular signal-regulated MAP kinase (ERK) 1/2 and c-Jun NH2-terminal kinase / stress-activated protein kinase (JNK/SAPK) signaling pathways were found to be required for the observed strain-induced shedding. These findings offer a better understanding of syndecan function in response to mechanical strain and suggest potential new mechanisms by which physical forces may modulate vascular SMC behavior and regulation during normal physiology, pathologic conditions, and engineered arterial substitute development.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Non-Latin script record Includes bibliographical references (leaves 112-128). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 199-250.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-04-27T15:00:50Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) mariana_aguiar_matos.pdf: 2901378 bytes, checksum: 40dbd704043d49a1eee587bb086c4eb4 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-05-16T19:24:00Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) mariana_aguiar_matos.pdf: 2901378 bytes, checksum: 40dbd704043d49a1eee587bb086c4eb4 (MD5) / Made available in DSpace on 2017-05-16T19:24:00Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) mariana_aguiar_matos.pdf: 2901378 bytes, checksum: 40dbd704043d49a1eee587bb086c4eb4 (MD5) Previous issue date: 2016 / Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O excesso de gordura corporal caracter?stico da obesidade est? relacionado a diversas altera??es metab?licas, que incluem a resist?ncia ? insulina. Dentre as medidas n?o farmacol?gicas empregadas para a melhora da sensibilidade ? insulina est? o treinamento f?sico aer?bio, como o treinamento intervalado de alta intensidade (HIIT, do ingl?s high intensity interval training). Sendo assim, esse estudo avaliou os efeitos do HIIT em componentes bioqu?micos, celulares e moleculares relacionados ? resist?ncia ? insulina em obesos. Indiv?duos obesos sens?veis (n=9) e resistentes ? insulina (n=8) foram submetidos a 8 semanas de HIIT, em cicloerg?metro, realizado 3 vezes por semana, com intensidade e volume progressivos (8 a 12 est?mulos; 80 a 110% da pot?ncia m?xima). Amostras de sangue venoso e do m?sculo vasto lateral foram obtidas antes e ap?s o programa de HIIT. Ap?s o programa de treinamento houve aumento da sensibilidade ? insulina nos obesos resistentes ? insulina, mas n?o houve redu??o da massa de gordura. A concentra??o de citocinas no soro, o estresse oxidativo sist?mico e frequ?ncia das c?lulas imunes n?o foram modificadas ap?s o treinamento. No m?sculo esquel?tico, o HIIT promoveu aumento da fosforila??o do substrato do receptor de insulina (IRS) (Tyr612), da Akt (Ser473) e da prote?na quinase dependente de c?lcio/calmodulina (CAMKII) (Thr286), e aumento do conte?do da ?-hidroxiacil-CoA desidrogenase (?-HAD) e citocromo C oxidase (COX-IV). Houve ainda, redu??o da fosforila??o da quinase regulada por sinal extracelular (ERK1/2) nos obesos resistentes ? insulina. Conclu?mos que 8 semanas de HIIT promoveram melhora da sensibilidade ? insulina, modificou componentes da via de sinaliza??o da insulina e do metabolismo oxidativo no m?sculo esquel?tico. Essas altera??es ocorreram independentes de mudan?as na gordura corporal total e de par?metros inflamat?rios sist?micos. / Tese (Doutorado) ? Programa Multic?ntrico de P?s-Gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / Obesity is characterized by excess of body fat, and its development can lead to a variety of metabolic disorders, including insulin resistance. Exercise is recognized as a non-pharmacological approach to increasing skeletal muscle insulin sensitivity, although the mechanisms are not elucidated. Additionally, the understanding of high intensity interval training (HIIT, high intensity interval training) treat insulin resistance is less understood. Therefore, this study evaluated the effects of HIIT on biochemical, molecular, and cellular markers related to insulin resistance in sedentary obese individuals. Sensitive (n=9) and insulin resistant (n=8) obese individuals (body mass index ? 30 kg/m-2) were engaged in 8 weeks of HIIT using a cycle ergometer. The HIIT was performed 3 times a week, and its intensity and volume progressively increased throughout the training period (from 8 to 12 stimuli; from 80 to 110% of the maximum power). Venous blood and the vastus lateralis muscle samples were obtained before and after the HIIT. HIIT enhanced insulin sensitivity in insulin-resistant obese individuals without changing body fat mass. Cytokine concentration in serum, blood oxidative stress, and frequency of some immune cells were not altered by HIIT. In skeletal muscle, HIIT increased the phosphorylation of insulin receptor substrate (IRS) (Tyr612), Akt (Ser473), and protein kinase dependent calcium/calmodulin (CaMKII) (Thr286). HIIT also increased the expression of ?-hydroxyacyl-CoA dehydrogenase (?-HAD) and cytochrome C oxidase (COX-IV). A reduction of the kinase phosphorylation of extracellular signal-regulated (ERK1/2) was only seen in obese insulin resistant individuals. The results show that 8 weeks of HIIT enhanced insulin sensitivity, modified components of the insulin-signaling pathway, and improved skeletal muscle oxidative metabolism. These changes were independent of alterations in body fat and inflammatory parameters.

Mammalian oocytes undergoing meiotic maturation are transcriptionally silent and gene expression is therefore regulated at the level of translation. One of the well established mechanisms employed in translational regulation of maternal mRNAs in oocytes is cytoplasmic polyadenylation. This process is generally controlled by phosphorylation and activation of cytoplasmic polyadenylation element binding protein (CPEB). The aim of this thesis is to determine the role of mitogen-activated protein kinase (MAPK) in regulation of CPEB-mediated cytoplasmic polyadenylation in maturing mouse and porcine oocytes. For this purpose, MAPK activity was inhibited using its specific inhibitor, GDC-0994 and the effect of MAPK inhibition on cyclin B1 mRNA polyadenylation was monitored. In mouse oocytes, MAPK inhibition impaired neither cyclin B1 mRNA polyadenylation nor its translation and MAPK is thus unlikely to be involved in regulation of cytoplasmic polyadenylation in this species. Based on the results of experiments performed using porcine oocytes, the possible role of MAPK in CPEB-mediated cytoplasmic polyadenylation can neither be confirmed nor ruled out. Keywords: cytoplasmic polyadenylation, mouse oocyte, porcine oocyte, mitogen-activated protein kinase (MAPK), cyclin B1, GDC-0994 inhibitor

A infecção persistente por HPV-16 está fortemente associada ao risco de desenvolvimento de neoplasias do colo do útero, vagina, vulva, pênis, canal anal e orofaringe. O estudo detalhado da variabilidade nucleotídica intra-típica de HPV-16 resultou em importantes achados no que concerne à filogenia e evolução viral, e à história natural das infecções. Variantes Asiático-Americanas (AA) e E-350G de HPV-16 foram associadas com maior risco de persistência da infecção viral e desenvolvimento de câncer de colo de útero quando comparadas à variante Européia protótipo (E-P ou E-350T), embora esta ainda apresente alto risco quando comparada aos outros tipos virais. Mais recentemente, diferenças funcionais entre as proteínas E6/E7 das distintas variantes moleculares de HPV- 16 estão sendo descritas, a fim de explicar as diferenças nas associações epidemiológicas observadas. Dados do nosso grupo apontaram para a transcrição aumentada do gene MEK2 especificamente em queratinócitos humanos primários (PHKs) transduzidos com E6/E7 da variante E-350G. Pelo exposto, objetivou-se: (1) Analisar os níveis de ativação de proteínas efetoras das vias de transdução de sinal mediadas por MAPK e PI3K/AKT em queratinócitos imortalizados por E6/E7 de três variantes moleculares de HPV-16 (AA, E-P, E-350G); (2) Analisar os efeitos das proteínas E6/E7 dessas variantes sob as vias de MAPK quanto à indução de fatores de transcrição; (3) Analisar o potencial transformante de PHKs imortalizados pelas diferentes variantes, e em cooperação com a proteína celular c-MYC; (4) Analisar o potencial de migração e invasão em PHKs imortalizados pelas diferentes variantes de HPV-16, e em cooperação com a proteína celular c-MYC. Neste estudo observou-se que a variante AA de HPV-16 induziu a maior ativação das vias de sinalização estudadas (MAPK, e PI3K/AKT). Ademais, PHKs imortalizados por esta variante apresentaram maior capacidade de migração, de invasão através de uma matriz de colágeno, além de maior potencial transformante. Adicionalmente, as células imortalizadas pela variante AA apresentaram maior expressão da proteína mesenquimal vimentina e diminuição dos níveis da proteína epitelial E-caderina, sugerindo ativação parcial de Transição Epitélio Mesênquima (EMT) nestes queratinócitos. Ademais, quando o oncogene c-MYC foi co-transduzido nas diferentes linhagens infectadas por E6/E7 de HPV-16, foi observado que em PHKs imortalizados pela variante AA também houve maior ativação da via de MAPK-ERK, maior migração, e um potencial transformante semelhante, em relação às células co-transduzidas pela variante E-350G e c-MYC. Em conjunto, estes dados sugerem que a variante AA de HPV-16 possui vantagem seletiva sob as outras variantes em promover transformação celular, migração e invasão, e isto poderia explicar, ao menos em parte, a maior prevalência desta variante no câncer cervical. Os resultados gerados neste estudo são de extrema relevância para avaliar o impacto da variabilidade intra-típica de HPV-16 sobre o potencial oncogênico observado em estudos epidemiológicos / Persistent infection with HPV-16 is strongly associated with risk of developing neoplasia in the uterine cervix, vagina, vulva, penis, anal canal and oropharynx. The detailed study of HPV-16 intra-typical nucleotide variability resulted in important findings regarding phylogeny and viral evolution, and the natural history of infections. Asian-American (AA) and E-350G variants of HPV-16 were associated with increased risk of persistent viral infection and development of cervical cancer compared to the European prototype (E-P or E-350T), although this variant still presents higher risk when compared to other viral types. More recently, functional differences between the E6/E7 proteins of distinct molecular variants of HPV-16 are being described, in order to explain the differences in the epidemiological associations observed. Data from our group pointed to increased transcription of the MEK2 gene specifically in primary human keratinocytes (PHKs) transducing E6/E7 of the E-350G variant. Consequently, the aims of this study were: 1) To examine the activation levels of effector proteins of the signal transduction pathways mediated by MAPK and PI3K/AKT in PHKs immortalized by E6/E7 of three different molecular variants of HPV-16 (AA, E-P, E-350G); (2) To analyze the effects of E6/E7 of different molecular variants of HPV-16 upon MAPK pathways concerning the induction of transcription factors; (3) To analyze the transforming potential of PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC; (4) To analyze the potential of migration and invasion in PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC. In this study we observed that the AA variant of HPV-16 induced higher activation of both signaling pathways studied (MAPK, and PI3K/AKT). Furthermore, this variant presented increased migration capacity, higher invasion through a collagen matrix, and greater transforming potential. Moreover, cells immortalized by the AA variant showed higher expression of the mesenchymal protein vimentin and a decrease of the epithelial protein E-cadherin, suggesting partial activation of Epithelial Mesenchymal Transition (EMT). In addition, when the c-MYC oncogene was co-transduced in the different cells lines infected with HPV-16 E6/E7, we observed that in PHKs immortalized by the AA variant there was also an enhanced activation of the MAPK-ERK pathway, a higher ability to migrate, and similar transformation potential in comparison with cells co-transduced with the E-350G variant and c-MYC. Taken together, this data suggest that the AA molecular variant of the HPV-16 has a selective advantage over the other variants to promote cell transformation, migration and invasion, and this could partly explain the higher prevalence of this variant in cervical cancer. The results generated in this study are very important to assess the impact of intra-typical variability of HPV-16 on the oncogenic potential observed in epidemiological studies

A subfamília p21 RAS de pequenas GTPases, incluindo KRAS, HRAS e NRAS, participa de muitas redes de sinalização, incluindo proliferação celular, organização do citoesqueleto e apoptose, e é o alvo mais freqüente de mutações ativadoras em câncer. Mutações germinativas em KRAS e HRAS causam graves anormalidades desenvolvimentais levando às síndromes de Noonan, cárdio-facial-cutânea e Costello, porem mutações ativadoras germinativas em NRAS não foram descritas até hoje. A síndrome autoimune linfoproliferativa (ALPS) é o mais comum defeito genético de apoptose linfocitária, cursando com autoimunidade e acúmulo excessivo de linfócitos, particularmente do tipo T + CD4- CD8-. As mutações causadoras de ALPS descritas até hoje afetam a apoptose mediada por Fas, uma das vias extrínsecas de apoptose. Nós demonstramos aqui que os principais achados clínicos de ALPS, bem como uma predisposição para tumores hematológicos, podem ser causados por uma mutação heterozigota ativadora G13D no oncogene NRAS, sem causar prejuízo na apoptose mediada por Fas. O aumento na quantidade intracelular de NRAS ativo, ligado a GTP, induziu a um aumento da sinalização na via RAF/MEK/ERK, o que suprimiu a expressão da proteína pró-apoptótica BIM, e atenuou a apoptose intrínseca mitocondrial. Desta forma, uma mutação germinativa ativadora em NRAS causou um fenótipo clinico diferente do visto em pacientes com mutações em outros membros da família p21 RAS, cursando com um defeito imunológico seletivo, sem distúrbios generalizados do desenvolvimento / The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4-, CD8- ab T cells. Mutations in ALPS typically affect Fas-mediated apoptosis, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair Fas-mediated apoptosis. The increase in active, GTP-bound NRAS augmented RAF/MEK/ERK signaling which markedly decreased the pro-apoptotic protein BIM and attenuated intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects

Introduction AP2α is a 52 kDa retinoic acid inducible and developmentally regulated activator of transcription, which binds to the DNA in a sequence-specific manner. Transcription factor AP-2α was isolated from HeLa cells by affinity chromatography using specific binding sites with in SV40 and human metallothionein promoters. Further screening of HeLa cDNA library with oligonucleotide probes predicted partial peptide sequence which led to the isolation of AP-2α cDNA and subsequently it was mapped to chromosome 6 near HLA locus. A differentially spliced version of AP-2α, which lacks most of the C-terminus, encodes a dominant negative protein (AP-2B). Subsequent studies led to the identification of four more isoforms: AP-2β, AP-2γ, AP-2δ and AP-2ε. AP-2 family members can form homo or hetero dimers among themselves through the unique C-terminal helix span helix motif and bind DNA through basic domain lies N-terminus of DNA binding domain. Several evidences suggest that AP-2α can act as a tumor suppressor gene. It has been shown that AP-2α can activate growth suppressor genes like p21WAF1/CIP1. Transforming viral oncogenes like adenovirus E1A and SV40 large T antigen have been shown to alter AP-2α function. In addition, reduced expression of AP-2α has been reported in human breast, ovary, colon, skin, brain and prostate cancers. Further, supporting evidences suggest that more invasiveness and tumorogenicity was observed when dominant negative mutant of AP-2α was expressed in melanoma cells. In this work, we have carried out a systematic study to find the various signal transduction pathways which regulate AP-2 activity as well as we attempted to demonstrate the importance of DNA binding domain in the growth inhibitory functions of AP-2α. HDAC inhibitors (HDIs) activate AP-2 activity through spleen tyrosine kinase (Syk) In the literature, ample evidences are available that genotoxic drugs such as adriamycin, induce tumor suppressors like p53 and p73. In this study, we have screened pharmacological drugs which damage DNA and specific inhibitors of various signal transduction pathways for their ability to activate AP-2 activity. AP-2 specific reporter, 3Χ-AP2-CAT was used in this study to measure the AP-2 activity. Of all the compounds studied, we found that Histone Deacetylase Inhibitors (HDIs) efficiently activated AP-2 activity and was found to be specific as they failed to activate 3X-AP2 mut CAT, which contains mutated AP-2 binding sites as well as pGL tk Luc, which contains thymidine kinase minimal promoter and no AP-2 binding sites. To understand the mechanism of HDI-mediated of AP-2 activation, AP-2 isoforms and its coactivators transcript and protein levels were analyzed. We found significant change in transcript levels of the some of the molecules tested. While the endogenous protein levels of various AP-2 isoforms were undetectable, we found stabilization of AP-2α protein expressed from exogenous source in cells treated with HDIs. HDI stabilized AP-2α was found to be functionally active as it showed increased sequence-specific DNA-binding as well as increased apoptosis. While HDIs known for their ability to modulate the gene activities by chromatin remodeling, it is also known that they alter various signal transduction pathways. In an effort to find pathway(s) by which HDIs activate AP-2 activity, we found that HDIs failed to activate AP-2 reporter in the presence of staurosporine suggesting the involvement a staurosporine sensitive pathway(s) in this process. Stauosporine is a non-specific kinase inhibitor of different signaling pathways. Further studies using different pathway specific inhibitors identified that spleen tyrosine kinase (Syk) is essential for HDIs mediated activation of AP-2 activity. Syk is a non receptor tyrosine kinase which is known to be activated in stress conditions. Syk is considered to be a tumor suppressor since Syk over expression leads to growth suppression of breast cancer cells and is also inactivated in a subset of breast cancers. These results suggest that HDI mediated activation of AP-2 involves AP-2α stabilization through Syk pathway. Regulation of AP-2 by MAP kinase pathway Cell growth, differentiation, and apoptosis are mediated by the activation of mitogenactivated protein kinase (MAPK) pathways. These kinases constitute MAP kinase cascades mainly regulated through phosphorylation status. In mammalian cells, at least four MAPKs, namely, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), p38 and ERK5/big MAP kinase have been identified. The ERKs are usually activated by mitogenic stimuli which in turn increase the proliferation and survival. Over expression of any activator of this signaling cascade lead to the unregulated proliferation of cells. In many cancers, ERK pathways are known to be up regulated. In this study, we found that MEK (MEK is the immediate upstream regulator of ERK) inhibitors - PD98059 and U0126 activate 3X-AP2-CAT suggesting that AP-2 activity is repressed by activated MAP kinase pathway. MEK inhibitor mediated activation was found to be specific because they failed to activate transcription from pGL tk Luc which contains thymidine kinase minimal promoter and no AP-2 binding sites. To understand the mechanism of MEK inhibitor-mediated of AP-2 activation, AP-2 isoforms and its coactivators transcript and protein levels were analyzed. We found significant change in transcript levels of the some of the molecules tested. The endogenous protein levels of various AP-2 isoforms were undetectable. When AP-2α was exogenously expressed, while no change in protein levels and DNA-binding ability was seen, we found evidence for appearance of post-ranslationally modified AP-2α protein in U0126 treated cells. We also found CITED2 (CBP/p300-interacting transactivator 2, co-activator of AP-2α) transcript levels were up regulated in UO126 treated cells. Post translational modifications of AP-2α and increased and increased CITED2 levels may be responsible for MEK inhibitor mediated AP-2 activation. Thus we conclude that ERK pathway, which is an oncogenic MAP kinase pathway, inhibits AP-2 activity thereby suggesting the importance of down regulation of AP-2 activity during transformation. Essential role of DNA-binding domain of AP-2α for its growth inhibitory functions Transcription factor AP-2α has three distinct domains, N-terminal transactivation domain (52-108 aa), C-terminal DNA binding domain (204-408 aa) and dimerization domain (277-395 aa) which lies within the DNA binding domain. AP-2α exerts its effects through binding to specific DNA sequence in the promoter of its target genes leading to either repression or activation. Recent evidences suggest that AP-2α represses many genes through its competitive binding to overlapping AP-2 and other transcription factor binding sites. This suggests an important role exclusively for the DNA binding domain in AP-2α mediated functions. To address the importance of DNA binding domain for AP-2α mediated apoptosis, we have tested the ability different deletion/point mutants of AP-2α with varying DNA binding and transactivation capability to perform growth suppressor function and ability to induce apoptosis. Replication-deficient recombinant adenoviruses expressing different mutants were used in this study. We found that an intact DNA-binding domain alone even in the absence of activation domain is sufficient for AP-2α to inhibit colony formation and to induce significant levels of apoptosis. These results suggest an important role for DNA binding domain growth inhibitory functions of AP-2α and thereby implying the importance of transcriptional repression in AP-2α functions.

Abstract Intracellular signalling cascades regulate cardiomyocyte hypertrophic response. Initially hypertrophy of individual myocytes occurs as an adaptive response to increased demands for cardiac work, e.g. during hypertension or after myocardial infarction, but a prolonged hypertrophic response, accompanied by accelerated fibrosis and apoptosis, predisposes the heart to impaired performance and the syndrome of heart failure. The goal of this work was to elucidate some of the main signalling pathways in experimental models of the cardiac hypertrophic response. Mechanical stretching of cultured neonatal rat cardiomyocytes in vitro activates the B-type natriuretic peptide (BNP) gene, a well-established marker of the hypertrophic response, through intracellular signalling cascades mitogen-activated protein kinases (MAPKs) and protein kinase A (PKA) -pathway. Further, transcription factors transcriptional enhancer factor-1 (TEF-1) and activating transcription factor 3 (ATF3) were induced during stretch, and TEF-1 activation was shown to be regulated by extracellular signal-regulated kinase (ERK), while ATF3 activation was modulated by PKA. The BNP gene was also activated by the adenoviral overexpression of the p38 MAPK isoforms p38α and p38β in vitro. Importantly, p38α–induced activation was mediated through activator protein-1 (AP-1) while p38β mediated BNP transcription through GATA-4, which suggests distinct physiological roles for different p38 isoforms. This was further confirmed by quantitative PCR, which demonstrated pro-fibrotic role for the p38α isoform and a pro-hypertrophic role for the p38β isoform. Finally, adenoviral overexpression of ATF3 in vitro and in vivo resulted in activation of cardiac survival factors nuclear factor-κВ and Nkx-2.5, and attenuation of central pro-inflammatory and pro-fibrotic mediators. Together these data suggest a protective role for ATF3 in the heart. Overall this study provides new insights into the role of several signalling molecules involved in cardiac hypertrophic process and suggests potential therapeutic strategies for the diagnosis and treatment of heart failure. / Tiivistelmä Sydämen kammioiden seinämät paksuuntuvat kuormituksen lisääntyessä mm. verenpainetaudissa tai sydäninfarktin jälkeen. Lisääntynyt kuormitus aiheuttaa sydänlihassolujen koon kasvun (hypertrofioitumisen) ohella sidekudoksen kertymistä (fibroosia) ja solukuolemaa. Nämä solutason muutokset lopulta vioittavat sydämen rakennetta niin, että sen toiminta pettää, ja sydän ajautuu vajaatoimintaan. Tätä taudin etenemistä säätelevät molekyylitasolla lukuisat solunsisäiset signaalinvälitysjärjestelmät, joita tässä väitöskirjatyössä tutkittiin eri koemalleissa. Sydämen täyttöpaineen nousun aiheuttama sydänlihassolujen mekaaninen venytys aktivoi natriureettisten peptidien (eteispeptidi, ANP ja B-tyypin natriureettinen peptidi, BNP) synteesiä ja vapautumista verenkiertoon. BNP geenin säätelyä mekaanisen venytyksen aikana tutkittiin rotan sydänlihassoluviljelmissä. Mitogeeni-aktivoituvat proteiinikinaasit (MAPK) sekä proteiinikinaasi A (PKA) säätelivät mekaanisen ärsykkeen aiheuttamaa BNP geenin ekspressiota. Venytys aktivoi myös transkriptiotekijöitä TEF-1 (transcriptional enhancer factor-1) ja ATF3 (activating transcription factor 3). TEF-1 sääteli venytyksen aiheuttamaa BNP:n aktivaatiota ERK:n (extracellular signal-regulated kinase) välityksellä BNP geenin säätelyalueella olevan sitoutumispaikkansa (M-CAT elementti) kautta. ATF3:n säätelyssä PKA:lla oli keskeinen merkitys. Tutkimus osoitti myös, että p38 MAPK:n alatyypeistä p38α lisäsi fibroosiin liittyvien geenien aktiivisuutta, kun taas p38β aiheutti solujen hypertrofioitumista lisäävien geenien ekspressiota. Molemmat alatyypit aktivoivat BNP geenin ekspressiota, mutta aktivaatio tapahtui eri transkriptiotekijöiden kautta. Tutkimuksessa havaittiin myös, että ATF3:n yliekspressio adenovirusvälitteisellä geeninsiirrolla lisäsi kahden sydäntä suojaavan transkriptiotekijän (nuclear factor-κВ ja Nkx-2.5) aktiivisuutta, sekä vähensi sydämen tulehdusvastetta ja fibroosia lisäävien tekijöiden (interleukiini-6 ja plasminogeeniaktivaattorin inhibiittori-1) ekspressiota. Väitöskirjatutkimus antaa uutta tietoa solunsisäisistä signaalinvälitys-järjestelmistä, jotka säätelevät sydänlihaksen kuormitusvastetta sydän- ja verenkiertoelimistön sairauksissa. Näiden solutason mekanismien tunteminen osaltaan edesauttaa jatkossa uusien menetelmien kehittämistä sydämen vajaatoiminnan ehkäisyyn ja hoitoon.