Experience drives changes in gene expression that mold and reorganize neuronal circuits. In response to neuronal activity, the transcription factor CREB binds to a regulatory site on Bdnf promoter IV to modulate BDNF protein levels. CREB and BDNF are extensively implicated in animal behaviour, but the role of the interaction between these proteins has not been studied. I used transgenic mice carrying mutations at the CREB binding site of Bdnf promoter IV (CREmKI mutation) to specifically disrupt this interaction. F1 (N = 52) and F2 (N = 69) mice underwent a battery of behavioural tests. All mice showed normal motor learning and spatial memory. Critically, F1 mutants showed impaired auditory fear memory, while F2 mutants showed heightened anxiety. I suspect that differences in Bdnf expression and compensatory effects contributed to discrepancies between the two generations. My findings highlight the relevance of BDNF expression levels for memory- and anxiety-related behaviours.

Η cAMP αποτελεί ένα σημαντικό δεύτερο μήνυμα που ρυθμίζει την ανοσολογική απόκριση. Η αύξηση της ενδοκυττάριας cAMP αυξάνει την παραγωγή της IL-10 από μονοκύτταρα. Σκοπός της μελέτης είναι η αποσαφήνιση της συμμετοχής της cAMP στην παραγωγή της IL-10 από Τ-λεμφοκύτταρα όπου τα δεδομένα είναι ακόμα ασαφή. Ανθρώπινα Τ-λεμφοκύτταρα περιφερικού αίματος διεγέρθηκαν με anti-CD3/anti-CD28, anti-CD3 ή Ionomycin/PMA παρουσία ή απουσία παραγόντων που αυξάνουν την ενδοκυττάρια cAMP (10-6 Μ Forskolin, 10-6 Μ PGE2, 5x10-6 Μ Rolipram και 10-6 Μ 8-Br-cAMP). Το πρωτεϊνικό προϊόν της IL-10 μετρήθηκε με ELISA ενώ η παραγωγή mRNA της IL-10 με Real Time PCR. Η ενεργότητα του υποκινητή της IL-10 ελέγχθηκε με διαμόλυνση των κυττάρων με πλασμίδια που φέρουν τον υποκινητή του γονιδίου (1327 bp) ή τμήματα αυτού (-1010, -500, -310, -235, -135 bp). Η δέσμευση των μεταγραφικών παραγόντων MEF-2 και CREB ελέγχτηκε σε πυρηνικά πρωτεϊνικά εκχυλίσματα με πειράματα EMSA, ενώ η ενεργότητα τους ελέγχθηκε με πειράματα διαμολύνσεως με πλασμίδια που ελέγχουν την ενεργότητα της λουσιφεράσης υπό τον έλεγχο των MEF-2 και CREB. Αύξηση της cAMP ελαττώνει την παραγωγή της IL-10 σε πρωτεϊνικό επίπεδο κατά 50-60% μετά από διέγερση με Ion/PMA, και κατά 80-90% με anti-CD3 ή με anti-CD3/anti-CD28. Η IL-10 παράγεται ακόμα και μετά από διέγερση μόνο με anti-CD3, εύρημα ειδικό για την IL-10, καθώς δεν παρατηρήθηκε αύξηση της παραγωγής άλλων κυτταροκινων (IL-2 & IL-4). Η ελάττωση της παραγωγής της IL-10 αντανακλάται και σε επίπεδο mRNA όπου οι αντίστοιχες μειώσεις είναι κατά 50% με όλους τους τρόπους διέγερσης. Η ενεργότητα του υποκινητή της IL-10 δεν επηρεάζεται από αλλαγές στα επίπεδα της cAMP όταν η διεγερση παρακάμπτει τον Τ κυτταρικό υποδοχέα. Ωστοσο, μειώνεται παρουσία αυξημένων συγκεντρώσεων cAMP όταν τα κύτταρα διεγείρονται μέσω του Τ κυτταρικού υποδοχέα. Το τμήμα του υποκινητή της IL-10 που επηρεάζεται από την ανασταλτική δραση της cAMP (50 % αναστολή) βρίσκεται στις πρώτες 500 bp πριν το TATA box, και περιέχει σημεία πρόσδεσης των μεταγραφικών παραγόντων MEF-2 και CREB, όπως ελέγχθηκε με το πρόγραμμα Consite. Η δέσμευση του MEF-2 σε πυρηνικά εκχυλίσματα διεγερμένων Τ-λεμφοκυττάρων μειώνεται κατά 70% παρουσία αυξημένης cAMP ενώ η ενεργότητα του δεν επηρεάζεται σημαντικά. Αντίθετα, η αύξηση της cAMP αυξάνει τόσο τη δέσμευση (x 2,5) όσο και την ενεργότητα του CREB (x 2). Η δράση της cAMP στην παραγωγή της IL-10 είναι ειδική για τα Τ-λεμφοκύτταρα και εξαρτάται από τον τρόπο διέγερσής τους. Η ρύθμισή της γίνεται τόσο σε μεταγραφικό όσο και σε μετά-μεταγραφικό επίπεδο. Η αύξηση της cAMP μπορεί να επηρεάσει την παραγωγή IL-10 από τα Τ-λεμφοκύτταρα παρεμβαίνοντας στην δέσμευση και την ενεργότητα των παραγόντων μεταγραφής MEF-2 και CREB. Ο τρόπος της αλληλεπίδρασης/συνεργασίας των MEF-2 και CREB παραμένει υπό διερεύνηση. / cAMP is a second messenger playing a crucial role in the signal transduction which controls the immune response, while IL-10 is considered to be an important regulator of this response. Elevation of intracellular concentration of cAMP has been shown to increase IL-10 production by monocytes. The aim of this study was the elucidation of the role of cAMP in IL-10 production by normal T lymphocytes, a mechanism that remains unclear. Fresh Human Τ-lymphocytes derived from PBMC of healthy donors where stimulated with anti-CD3/anti-CD28 or Ionomycin/PMA, in the presence or absence of cAMP elevating agents (10-6 Μ Forskolin, 10-6 Μ PGE2, 5x10-6 Μ Rolipram and 10-6 Μ 8-Br-cAMP). The protein product of IL-10 was measured by ELISA, the production of IL-10 mRNA by Real Time PCR and IL-10 mRNA stability was determined by the use of Actinomycin D (10 μM). The activity of IL-10 promoter was measured by luciferase reporter assay, after transfection of cells with plasmids carrying the wild type promoter (1037bp) or promoter fragments (constructs of -1010, -500, -310, -235, -135bp). PKA role was examined either by cotransfection experiments with a plasmid carrying a constitutively active mutant of the catalytic subunit of PKA-α isoform, or by the use of a specific PKA inhibitor Rp-8- Br-cAMP (10-50 μM). The presence of binding sites of transcription factors in the first 500bp of the IL-10 promoter, was validated using the web-based program CONSITE. Binding of the transcription factors MEF2 and CREB was investigated in nuclear extracts of stimulated human T cells with EMSA experiments. The activity of MEF2 and CREB was investigated independently with transfection experiments using plasmids containing the lusiferase reporter under the control of the transcription factors. Intracellular cAMP elevation, inhibits IL-10 protein production by 50-60%, when T cells are stimulated with Ionomycin/PMA, and by 80-90% after stimulation with anti-CD3 or anti-CD3/anti-CD28, while PKA blocking by Rp-8- Br-cAMP reversed cAMP mediated inhibition.. IL-10 steady state mRNA levels follow the same pattern of inhibition only after anti-CD3/anti-CD28 stimulation. cAMP elevation decreases IL-10 mRNA stability after I/PMA stimulation, whereas in the anti-CD3/anti-CD28 stimulated cells, the mechanism of inhibition is mainly transcriptional. IL-10 promoter activity is reduced up to 60% when cells are stimulated with anti CD3/anti CD28 in the presence of cAMP elevating agents, but is not affected after stimulation with Ionomycin/PMA or cotransfection of the cells with constitutively active PKA mutant. Transfection assays with the different IL-10 promoter fragments revealed that the most responsible part of IL-10 promoter to cAMP mediated inhibition, is the first 500 bp after the TATA box. This part contains binding sites for the transcription factors MEF-2 and CREB, as validated by the web-based program Consite. Increased intracellular cAMP reduces the binding of MEF2 to nuclear extracts of stimulated T cells by 70 %, however its activity is not affected significantly. On the contrary, both the binding and the activity of CREB are increased in the presence of elevated cAMP. cAMP mediated inhibition of IL-10 production is PKA mediated and specific for T lymphocytes, depending on the nature/strength of stimulation. cAMP-dependent regulation of IL-10 production is controlled by transcriptional and/or post-transcriptional mechanisms depending on the nature of stimulus. Transcriptional mechanisms involve the transcription factors MEF2 and CREB, however the exact mechanisms of action of these factors deserves further elucidation. Cell and stimulus specific mechanism of regulation of IL-10 production is necessary for its immunoregulatory function.

Colorectal cancer, among other tumors, is characterized by elevated levels of prostaglandins due to the up-regulation of cyclooxygenase -2 (COX-2), a key enzyme in the eicosanoid biosynthesis pathway. Prostaglandin E2 (PGE2) is an important prostaglandin that exerts its biological function via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is the most important. The relevance of EP4 receptor to the carcinogenic process and the consequences of its interaction with PGE2 were explored in this dissertation.Despite the importance of the EP4 receptor in colon carcinogenesis, studies looking at the receptor expression during cancer progression have not been extensive. One study showed that the protein levels of EP4 receptor were elevated in colon cancer whereas another study indicated that mRNA levels were decreased in tumor compared to normal. We expanded these observations and now report that the elevated protein levels of EP4 receptor in cancer are due to increased translation of proteins.In addition, we identified S100P as a novel downstream target of the PGE2/EP4 receptor signaling pathway. S100P has been previously implicated in a number of gastro-intestinal cancers such as pancreatic, gastric and colon cancers. However, its regulation via the PGE2/EP4 receptor signaling pathway has never been investigated. Here, we show that PGE2 via the EP4 receptor signaling leads to the transcriptional activation of S100P and that this activation happens exclusively in the presence of CREB. In summary, this dissertation brings to light novel therapeutic targets which could be used as potential markers to stratify colon cancer patients as well as avenues for clinical intervention for the management of colon carcinogenesis.

Huntington’s disease (HD) is caused by expression of the huntingtin gene containing an expanded CAG repeat. N-terminal mutant huntingtin protein (N-mHtt) accumulates in the nucleus and impairs transcription of a subset of genes through incorporation into transcriptional complexes or sequestration of proteins away from the promoter. CREB-binding protein (CBP) is a transcriptional co-activator and acetyltransferase (AT) that binds to N-mHtt. We hypothesized that overexpressing CBP fragments that lack a promoter association domain would block N-mHtt-mediated transcriptional dysregulation and toxicity. We found that overexpressing full-length CBP or CBP fragments did not reverse transcriptional dysregulation, but did decrease toxicity in a cell model of HD. Overexpressing fragments of CBP containing the AT domain increased toxicity in wild-type cells, while overexpressing a fragment lacking this domain had no effect. We conclude that excess AT activity was detrimental in wild-type cells, while overexpressing CBP or CBP fragments was protective in HD cells.

Memory is subdivided into short- and long-term memory. The interaction between transcription factors (TF) and expressed genes are essential steps in memory formation. Some TFs that might be involved in memory formation include CREB, NF-kB and Egr-2. We hypothesized that there would be a difference in the expression levels of these TFs following learning in the Morris Water Maze (MWM). In study one, CD1 mice were categorized into two groups. Group 1 assigned as non-trained control group. Group 2 (experimental group) underwent 9 consecutive days of MWM training. In the second study, male C57BL/6 mice were categorized into four groups. Group 1 was a non-trained control group (allowed to swim randomly). Groups 2, 3, and 4 (experimental groups) had variations in their MWM training. Search strategies, escape latency, time spending in the target quadrant and number of attempts passing the missing platform, were measured. To evaluate the expression levels of TFs pre- versus post-learning, mice were sacrificed at the end of MWM. Hippocampi were separated and Western blot and immunohistochemical procedures were done. In study one, the escape latency decreased progressively toward the end of the acquisition phase in the trained group. The search pattern showed that the mice used primarily spatial strategies. Mice spent more time in the target quadrant during the retention phase. The number of passes over the missing platform peaked on the first day of the retention phase. NF-kB and CREB were expressed significantly higher in the control group versus the MWM trained mice (p = 0.0031 and p < 0.0001 respectively). There was no statistically significant difference in expression of Egr-2 between the two groups (p = 0.3092). In study two, Group 4 showed the highest and Group 1 the lowest levels of CREB expression. CREB and NF-kB were decreased following MWM training in study one. In study two CREB levels were highest in the Group 4 which had interval between the acquisition and retention phases. These differences could be due to multiphasic expression patterns and/or other experimental design issues. Further studies are warranted to examine time dependent differential expression of TFs in memory.

Memory is subdivided into short- and long-term memory. The interaction between transcription factors (TF) and expressed genes are essential steps in memory formation. Some TFs that might be involved in memory formation include CREB, NF-kB and Egr-2. We hypothesized that there would be a difference in the expression levels of these TFs following learning in the Morris Water Maze (MWM). In study one, CD1 mice were categorized into two groups. Group 1 assigned as non-trained control group. Group 2 (experimental group) underwent 9 consecutive days of MWM training. In the second study, male C57BL/6 mice were categorized into four groups. Group 1 was a non-trained control group (allowed to swim randomly). Groups 2, 3, and 4 (experimental groups) had variations in their MWM training. Search strategies, escape latency, time spending in the target quadrant and number of attempts passing the missing platform, were measured. To evaluate the expression levels of TFs pre- versus post-learning, mice were sacrificed at the end of MWM. Hippocampi were separated and Western blot and immunohistochemical procedures were done. In study one, the escape latency decreased progressively toward the end of the acquisition phase in the trained group. The search pattern showed that the mice used primarily spatial strategies. Mice spent more time in the target quadrant during the retention phase. The number of passes over the missing platform peaked on the first day of the retention phase. NF-kB and CREB were expressed significantly higher in the control group versus the MWM trained mice (p = 0.0031 and p < 0.0001 respectively). There was no statistically significant difference in expression of Egr-2 between the two groups (p = 0.3092). In study two, Group 4 showed the highest and Group 1 the lowest levels of CREB expression. CREB and NF-kB were decreased following MWM training in study one. In study two CREB levels were highest in the Group 4 which had interval between the acquisition and retention phases. These differences could be due to multiphasic expression patterns and/or other experimental design issues. Further studies are warranted to examine time dependent differential expression of TFs in memory.

Τα γλοιώματα είναι η πιο συχνή κακοήθης μορφή καρκίνου του κεντρικού νευρικού συστήματος. Δυστυχώς, είναι επίσης μεταξύ των πιο δύσκολων μορφών όσο αναφορά τη θεραπεία, με αποτέλεσμα την κακή πρόγνωση των ασθενών. Γλοιώματα παρουσιάζουν πολύπλοκη κυτταρική και γενετική ετερογένεια, περιορίζοντας σημαντικά τις στοχευμένες θεραπευτικές προσεγγίσεις. Η ανακάλυψη γονιδίων και σηματοδοτικών μονοπατιών που ρυθμίζουν την επιβίωση και την ανάπτυξη των καρκινικών κυττάρων θα βοηθήσουν στην ανάπτυξη νέων και πιο αποτελεσματικών θεραπειών. Η πρωτεΐνη απόκρισης στο κυκλικό-AMP (CREB) είναι ένα πυρηνικό μόριο που ενεργοποιείται μέσω φωσφορυλίωσης από κινάσες σερίνης/θρεονίνης και ελέγχει τη μεταγραφή πολλών γονιδίων στα νευρικά κύτταρα. Ο ρόλος του CREB στη νευρωνική λειτουργία κυμαίνεται από την επιβίωση και τον πολλαπλασιασμό σε πιο πολύπλοκες εγκεφαλικές λειτουργίες, όπως η μνήμη και οι συμπεριφορές εθισμού. Δεδομένα από πρόσφατες έρευνες δείχνουν πως ανώμαλη έκφραση ή ενεργοποίηση του CREB μπορεί να προσδώσει καρκινικές ιδιότητες σε διάφορους τύπους ιστών και κυττάρων. Επίσης το CREB συμμετέχει σε διαδικασίες επιβίωσης και μετάστασης σε μεγάλο αριθμό καρκινικών κυτταρικών σειρών. Με βάση αυτά μελετήθηκε ο ρόλος του CREB σε κύτταρα καρκίνου του εγκεφάλου, με έμφαση στις περιπτώσεις γλοιοβλαστώματος. Δείξαμε ότι τόσο τα επίπεδα ενεργοποίησης όσο και ο αριθμός των κυττάρων που στα οποία το CREB είναι φωσφορυλιωμένο/ ενεργοποιημένο είναι σημαντικά αυξημένος σε σχέση με δείγματα παρακείμενου μη καρκινικού ιστού. Επιπλέων βρέθηκε πως η αποσιώπηση του CREB σε καρκινικές σειρές γλοιοβλαστώματος, με χρήση μορίων siRNA, επέφερε σημαντική μείωση στην βιωσιμότητα τους. Τα παραπάνω ευρήματα συσχετίζουν για πρώτη φορά το μεταγραφικό παράγοντα CREB με το γλοιοβλάστωμα και του προσδίδουν ένα αρκετά σημαντικό ρόλο όσο αναφορά την επιβίωση των συγκεκριμένων καρκινικών κυττάρων. / Gliomas are the most common malignant cancers of the nervous system. Unfortunately, they are also amongst the most difficult cancers to treat, resulting in poor patient prognosis. Gliomas exhibit complex cellular and genetic heterogeneity, limiting effective targeted therapy approaches. Discovering the genes and pathways that regulate cancer cells survival and growth will aid in the development of novel and more effective treatments. The Cyclic-AMP Response Element Binding protein (CREB) is a serine/threonine kinase-regulated nuclear factor modulating the transcription of numerous genes in nerve cells and has various roles in neuronal function, ranging from survival and proliferation to more complex brain functions, such as memory and drug addiction behaviours. Given recent findings that aberrant CREB expression can impart oncogenic properties on myeloid cells, liver cells and ovarian epithelial cells, we explored the potential role of CREB in brain cancer biology by examining its expression in a panel of human patient brain tumour specimens. We show that both the level of expression and the number of cells expressing activated/phosphorylated CREB is markedly elevated in tumours compared with adjacent non-tumour control brain tissue. Moreover using siRNA molecules we knocked-down the expression of CREB in glioblastoma cell lines and then we studied the viability of these cells throughout 96 hours with the use of MTT assay. A significant reduction in viability was observed at CREB siRNA transfected cells against the control. These observations are the first to highlight a link between CREB and brain tumours. Our hypothesis is that CREB has a role in brain tumour development/growth and that at least some of CREB's neuro-oncogenic properties are due to its role in neural cells survival and proliferation.

Made available in DSpace on 2015-12-06T23:46:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2012 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Associação Fundo de Incentivo à Psicofarmacologia (AFIP) / O condicionamento de medo e um dos modelos animais de memoria emocional mais utilizados atualmente para se estudar mecanismos subjacentes aos processos mnemonicos. Neste modelo um pareamento de um estimulo neutro com um estimulo aversivo torna o primeiro preditor do segundo e, por sua vez, adquire caracteristicas aversivas tambem. Muitas evidencias tem mostrado uma participacao critica da amigdala nesse modelo, mais especificamente, seus nucleos lateral, basal e central. Na versao contextual do condicionamento de medo, no qual o ambiente e o estimulo neutro inicial, tem-se mostrado tambem um envolvimento do hipocampo, que geralmente esta envolvimento com memoria espacial. Recentes evidencias sugerem que, nesta tarefa, hipocampo e amigdala interagem, influenciando processos de plasticidade sinaptica um do outro, influenciando a expressao de genes de expressao imediata. Neste estudo, nos injetamos o antagonista do receptor NMDA, AP5, no hipocampo dorsal de ratos wistar logo antes do condicionamento de medo ao contexto. Apos o condicionamento, metade dos animais foi testada 48 horas depois e metade foi submetida a um processo de perfusao e processamento imunohistoquimico para CREB e pCREB. Nossos resultados mostraram que o condicionamento de medo o contexto aumenta a taxa de pCREB de maneira especifica ao aprendizado associativo, e que o bloqueio de receptores NMDA do hipocampo por AP5 nao so diminui o comportamento de medo dos animais como tambem diminui a taxa de pCREB nos nucleos lateral, basal e central da amigdala. Nossos resultados sugerem que a atividade dos receptores NMDA hipocampais influencia a inducao de plasticidade sinaptica na amigdala apos o condicionamento de medo ao contexto / BV UNIFESP: Teses e dissertações

Tesis para optar al grado de Doctor en Bioquímica / Las enfermedades cardiovasculares son la primera causa de muerte en todos los países desarrollados, incluyendo Chile. En los últimos años se ha sugerido que la pérdida de cardiomiocitos debido a muerte celular es un factor importante en el desarrollo de la insuficiencia cardiaca. Sin embargo, no existe claridad acerca del mecanismo por el cual se produce la muerte de los cardiomiocitos y de su desaparición del tejido cardiaco. En el corazón se produce estrés hiperosmótico durante eventos de isquemia/reperfusión cardiaca. Existe un pobre conocimiento acerca de las vías de señalización cardiaca que conllevan a la muerte celular como una consecuencia del estrés osmótico. Nosotros hemos demostrado que el estrés hiperosmótico (sorbitol 600 mOsm) induce una rápida apoptosis en cardiomiocitos en cultivo. El futuro desarrollo de esta área requiere la identificación de moléculas que protejan a los cardiomiocitos de la muerte celular. En este aspecto, el agente más promisorio corresponde al factor de crecimiento análogo a insulina tipo 1 (IGF-1). Algunas células poseen eficientes mecanismos reguladores involucrados en la mantención de la homeostasis, sin embargo el cardiomiocito es especialmente vulnerable a la apoptosis inducida por estrés hiperosmótico debido a que regula su volumen muy lentamente. Aunque existe cierta caracterización molecular de los eventos desencadenados en la apoptosis inducida por sorbitol, se desconoce la participación del Ca2+ y de factores transcripcionales en este proceso. El propósito de esta tesis fue estudiar los mecanismos de señalización que regulan la apoptosis del cardiomiocito inducida por estrés hiperosmótico. Con este objetivo, se estudió la actividad y regulación transduccional del factor transcripcional CREB en respuesta a IGF-1 y/o estrés hiperosmótico. CREB es un importante mediador de la sobrevida celular promovida por IGF-1 en otros tipos celulares. Por lo tanto se investigó la participación de CREB en la señalización antiapoptótica gatillada por IGF-1 en este modelo de estrés hiperosmótico. Además, se estudiaron los efectos del estrés hiperosmótico en cultivos primarios de cardiomiocitos, en términos de cambios en los niveles intracelulares de Ca2+ y en las actividades enzimáticas de la proteína kinasa dependiente de Ca2+/CaM (Calmodulina quinasa II, CaMKII) y la proteína fosfatasa Calcineurina (Cn). Los resultados presentados en este estudio demostraron que tanto el estrés hiperosmótico como IGF-1 conllevan a la activación de CREB, a través de su fosforilación, aunque probablemente por vías distintas. La activación de CREB por estrés hiperosmótico dependió de Ca2+ i y las vías MAPK, ya que BAPTA-AM e inhibidores de las vías p38-MAPK y ERK impidieron el aumento de su fosforilación. Por otro lado, la preincubación de cardiomiocitos con BAPTA-AM o inhibidores de las vías p38-MAPK, ERK, PI3-K, Cn and CaMKII disminuyeron significativamente la fosforilación de CREB inducida por IGF-1. El estrés hiperosmótico bloqueó la activación de CREB inducida por IGF-1, con un patrón de activación similar al de el estímulo apoptótico. Finalmente, los resultados indicaron que CREB participa en la señalización antiapoptótica del IGF-1 en cardiomiocitos expuestos a estrés hiperosmótico, pero no en la respuesta de sobrevida celular frente al estímulo osmótico. El efecto protector de IGF-1 frente a estrés hiperosmótico fue bloqueado por la expresión de una forma inactiva de CREB, como se demuestra con la mayoría de los parámetros apoptóticos evaluados en este estudio; sin embargo, la apoptosis inducida con este modelo experimental no presentó cambios significativos tras la sobreexpresión de CREB inactivo. Los resultados, además, demostraron que el estrés hiperosmótico llevó a aumentos rápidos y transitorios de los niveles de Ca2+ i, como resultado tanto del influjo de este ión desde el medio extracelular como de su liberación desde depósitos intracelulares. Este aumento del Ca2+ i estuvo mediado al menos, por entrada de Ca2+ por canales de Ca2+ de tipo L y la liberación desde reservorios de Ca2+ activada por IP3. Los resultados también mostraron que la liberación de Ca2+ desde depósitos intracelulares inducida por el estrés hiperosmótico depende de la activación de Fosfolipasa C (PLC). El estrés hiperosmótico no produjo un aumento de las actividades enzimáticas CaMKII y Cn. El pretratamiento de los cardiomiocitos con KN62 (inhibidor de CaMKII) y CsA (inhibidor de Cn) no modificó significativamente la apoptosis inducida por estrés hiperosmótico, evaluada en términos de viabilidad celular, fragmentación del DNA, activación de caspasa-3 y -9 y externalización de fosfatidilserina. Estos resultados sugieren que dichas vías de señalización probablemente no estarían involucradas en la regulación de la apoptosis desencadenada por el estrés hiperosmótico inducido por sorbitol. De este trabajo de tesis se puede concluir que: 1) CREB participaría en la señalización antiapoptótica del IGF-1 en cardiomiocitos expuestos a estrés hiperosmótico, pero no en la muerte inducida por este tipo de estrés; 2) el estrés hiperosmótico produce un aumento de los niveles Ca2+ i como consecuencia del influjo de este ión desde el medio extracelular y de su liberación desde depósitos intracelulares; y finalmente, 3) CaMKII y Cn no participarían en la apoptosis inducida por el estímulo osmótico / Cardiovascular diseases are the leading cause of death in all developed countries, including Chile. In recent years, it has been suggested that loss of cardiomyocytes due to cell death is an important causative factor in the development of heart failure. However, the mechanism by which cardiomyocytes die and then disappear from the tissue is not clear. In the heart, osmotic stress occurs during myocardial ischemia/reperfusion. Cardiac signaling pathways leading to cell death as a consequence of osmotic stress remain poorly understood. We have shown that hyperosmotic stress (sorbitol 600 mOsm) rapidly induces apoptosis in cultured cardiomyocytes. Future development in this area will require the identification of molecules that protect cardiac cells from cell death. The most promising agent is insulin like growth factor-1 (IGF-1). Several cells display efficient regulatory mechanisms involved in maintaining homeostasis, however cardiomyocyte is especially vulnerable to hyperosmotic stress-induced apoptosis since its volume is regulated very slowly. Although there is some molecular characterization of the events engaged in estrés hiperosmótico-induced apoptosis, the involvement of Ca2+ and transcription factors remains unknown. The aim of this thesis was to study of the signaling mechanisms regulating hyperosmotic stress-induced apoptosis of cardiomyocyte. With that purpose, the activity and transductional regulation of transcriptional factor CREB in the response to IGF-1 and/or hyperosmotic stress were studied. Since CREB is an important mediator of IGF-1 promotion of cell survival in other cell types, the participation of CREB in the IGF-1-induced antiapoptotic signaling pathway during hyperosmotic stress was also investigated. In addition effects of sorbitolinduced hyperosmotic stress on primary cardiomyocyte cultures were studied, in terms of changes in Ca2+ intracellular levels, Ca2+/CaM-dependent protein kinase (Calmodulin kinase II, CaMKII) and the protein phosphatase Calcineurin (Cn) enzymatic activities. Data presented in this study showed that CREB activation (phosphorilation) was induced by both hyperosmotic stress and IGF-1, although most likely by different pathways. CREB activation by hyperosmotic stress depended on Ca2+ i and MAPK pathways since BAPTA-AM and p38-MAPK and ERK pathway inhibitors prevented its phosphorylation. In the other hand, pretreatment of cardiomyocytes with BAPTA-AM or p38-MAPK, ERK, PI3-K, Cn and CaMKII pathway inhibitors significantly decreased CREB phosphorylation induced by IGF-1. Hyperosmotic stress prevented CREB activation IGF-1-induced, maintaining the activation pattern of DNA affinity displayed after the apoptotic stimulus. Finally, the results indicated that CREB participates in the IGF-1 antiapoptotic signaling in cardiomyocytes treated with hyperosmotic stress, but not in the cell survival response. The protective effect of IGF-1 towards sorbitol was overridden by the overexpression of an inactive form of CREB, as shown by most of the apoptotic parameters evaluated in this study; however, stress-induced apoptosis with this experimental model remained unchanged by overexpression of inactive CREB. The results also showed that hyperosmotic stress led to fast and transient increases in intracellular Ca2+ levels, which were the result of both Ca2+ influx from the extracellular milieu and the release from intracellular stores. This Ca2+ i increase was mediated in part by Ca2+ inward by L-type Ca2+ channels and release from stores by IP3 receptors. Results also showed that hyperosmotic stress-induced Ca2+ release from intracellular stores was also dependent on PLC activation. Hyperosmotic stress did not induce CaMKII and Cn enzymatic activities. Pretreatment of cardiomyocytes with KN62 (CaMKII inhibitor) and CsA (Cn inhibitor) did not modified significantly hyperosmotic stress-induced apoptosis, evaluated in terms of cell viability, DNA fragmentation, caspases–3 and –9 activation and phosphatidylserine externalization. These results suggested that these two signaling pathways were not likely implicated in the regulation of apoptosis triggered by sorbitol-induced hyperosmotic stress. Conclusions drawn from this thesis work are: 1) CREB most likely is involved in IGF-1 antiapoptotic signal in cardiomyocytes subjected to hyperosmotic stress but not in this type of stress-induced cell death; 2) hyperosmotic stress leads to an increase in intracellular Ca2+ levels due to both influx from the extracellular milieu and release from intracellular stores; and finally, 3) CaMKII and Cn are not likely to be involved in this osmotic stimulus-induced apoptosis / FONDAP 15010006; FONDECYT 1010246; CONICYT

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13113号 / 論医博第2131号 / 新制||医||1022(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 安達 泰治, 教授 開 祐司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM