Els estrògens poden regular la progressió del cicle cel·lular, tot i que la contribució concreta i els mecanismes d'acció del receptor d'estrògens beta (ER?) no s'ha descobert encara. En aquest treball s'ha analitzat els nivells d'ER?1 i d'ER?2 a través del cicle cel·lular, com també els mecanismes d'acció i les conseqüències de la sobrexpressió d'ER?1 en la línia cel·lular humana de càncer de pròstata LNCaP. Els resultats d'aquest treball mostren que el mRNA i la proteïna d'ER?1 augmenten des de la fase G1 a S del cicle cel·lular i disminueixen abans d'arribar a la fase G2/M, mentre que els nivells d'ER?2 disminueixen durant la fase S i augmenten de nou en G2/M. La proteïna d'ER?1 es va detectar tant en la fracció nuclear com en la no nuclear de totes les fases del cicle, i ER?2 únicament en la fracció nuclear. Respecte el mecanisme d'acció, la proteïna endògena d'ER?1 fou capaç d'activar la transcripció sobre les seqüències ERE únicament en la fase S del cicle cel·lular d'una manera lligand dependent. No es van detectar canvis en la transactivació d'AP1 i NF-kB després del tractament amb estradiol o l'inhibidor del receptor d'estrògens ICI 182,780. La sobrexpressió de la proteïna ER?1 nativa o amb dues mutacions en el domini d'unió al DNA provocaven un bloqueig en la fase G1a del cicle cel·lular. Aquest bloqueig va anar acompanyat per la interacció d'ER?1 sobrexpressat amb la proteïna cinasa c-Jun N-terminal (JNK1) i una disminució en la expressió del gen ciclina D1. El tractament de les cèl·lules amb ICI eliminava la interacció entre ER?1 i JNK1, un augment de la expressió de la ciclina D1 i una entrada en la fase G1b del cicle cel·lular, on continuaven estant bloquejades. En conclusió, els nostres resultats demostren que, en les cèl·lules LNCaP de càncer de pròstata, la expressió de totes dues isoformes del receptor està regulada diferencialment durant la progressió del cicle per un mecanisme ERE-dependent i ERE-independent. El bloqueig induït per la sobrexpressió de la proteïna reforça la idea que ER? es comportaria com un gen supressor de tumors. / It is well known that estrogens regulate cell cycle progression, but the specific contributions and mechanisms of action of the estrogen receptor beta (ER?) remain elusive. In this work has been analyzed the levels of ER?1 and ER?2 throughout the cell cycle, as well as the mechanisms of action and the consequences of the over-expression of ER?1 in the human prostate cancer LNCaP cell line. Results of this work shows that ER?1 mRNA and protein expression increased from the G1 to the S phase and decreased before entering the G2/M phase, whereas ER?2 levels decreased during the S phase and increased again in the G2/M phase. ER?1 protein was detected in both the nuclear and non-nuclear fractions, and ER?2 was found in only the nuclear fractions. Regarding the mechanisms of action, endogenous ER? was able to activate transcription via ERE during the S phase in a ligand-dependent manner, whereas no changes in AP1 and NFkB transactivation were observed after exposure to estradiol or the specific inhibitor ICI 182,780. Over-expression of either wild type ER?1 or ER?1 mutated in the DNA-binding domain caused an arrest in early G1. This arrest was accompanied by the interaction of over-expressed ER?1 with c-Jun N-terminal protein Kinase 1 (JNK1) and the decrease in cyclin D1 expression. The administration of ICI abolishes the JNK1-ER?1 interaction, increases c-jun phosphorylation and cyclin D1 expression and allowed the cells to progress to late G1, where they became arrested. In conclusion, our results demonstrate that, in LNCaP prostate cancer cells, the expression of both ER? isoforms is differentially regulated during the cell cycle and that ER? regulates cell cycle by ERE-dependent mechanisms during the S-phase and ERE-independent signaling pathways during the G1-phase. Moreover, the G1-cell cycle arrest caused by ER?1 over-expression reinforces the idea that ER? behaves as a tumor suppressor gene.

The infections agent of Lyme disease, Borrelia Burgdorferi is a complex microorganism with a highly diverse genome. One of the most remarkable aspects of the B. burgdorferi genome is the large number of sequences encoding predicted or known lipoproteins, including outer-surface proteins. The B. burgdorferi genome encodes no recognizable toxins. Instead, this extracellular pathogen causes pathology by migration through tissues, adhesion to host cells, and evasion of immune clearance. Inflammation elicited by infection with B. burgdorferi depends on the ability of the spirochete to survive in the mammalian host, as well as the immune response that arises upon the interaction of the bacterium with phagocytic, T and other cell types. Innate immune responses are critical in recognition and clearance of pathogens, and also play an important role in the outcome of adaptive immune responses. The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns (PAMPs) and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. In this study we demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK also controls tumor necrosis factor production and TLR-mediated macrophage responses to B. burgdorferi. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete. MCJ is a newly identified member of the DnaJ protein family of cochaperones that contains unique features different than the normally described DnaJ proteins. However, there is little known about its function and the role it plays in different cells and systems. It has been previously shown that MCJ is required for the repression of the ABCB1 drug transporter expression in breast cancer cells, and that this repression is mediated through the control of c-Jun protein stability. We were therefore interested in determining the role that MCJ plays in macrophages in response to B. burgdorferi antigens. We now provide evidence that MCJ controls inflammatory responses of macrophages through the regulation of c-Jun protein stability, and the expression and release of the inflammatory cytokine TNF through the regulation of the expression of TNF converting enzyme (TACE) inhibitor tissue inhibitor of metalloproteinase 3 (TIMP-3).

Approximately 1 in 5 Americans smoke despite the widely known negative health consequences of the habit. One factor that contributes to the high rates of nicotine addiction and its continued use is the ability of the drug to alter long-term memory. Learning in the presence of nicotine results in changes to the cellular and molecular processes that support the formation and storage of long-term memories. The consolidation of long-term memory requires a number of mechanisms, such as gene transcription. Previous work has found that learning a contextual fear conditioning task in the presence of nicotine results in the upregulation of the c-jun N-terminal kinase (JNK1) gene in the hippocampus and that JNK protein activation is necessary for the nicotine induced enhancement of contextual conditioning. The present study examines the transcription factors involved in the transcriptional regulation of jnk1 in the hippocampi of mice following learning in the presence of nicotine. The hypothesis that cAMP response element binding protein (CREB) regulates jnk1 transcription was examined. Further, a protein/DNA transcription factor array was used as an unbiased examination of changes in transcription factor activity following learning in the presence of nicotine. Using chromatin immunoprecipitation (ChIP), transcription factors identified from the array and CREB were examined for changes in their binding to the jnk1 promoter following fear conditioning in the presence of nicotine. An increase in the binding of phosphorylated CREB was found in the jnk1 promoter of mice trained in the presence of nicotine. This implicates CREB activation in the increase of jnk1 transcription following learning in the presence of nicotine. Additionally, data from the transcription factor array suggest other factors such as PARP, TR, USF-1 and E2F-1 as potentially playing a role in the cognitive effects of nicotine. These findings are discussed with respect to how they inform our understanding of the signaling cascades and genetics involved in the memory enhancing effects of this addictive drug. / Psychology

Receptor tyrosine kinase (RTK) inhibitors are emerging as an effective therapeutic option for treatment of breast cancer patients overexpressing particular RTKs. However, more patients may benefit from an inhibitor targeting a common effector protein downstream several RTKs. The presented studies herein identify c-Jun N-Terminal Kinase 2 (JNK2), a kinase downstream multiple RTKs, as a novel target to effectively inhibit Phosphatidylinositol 3-kinase/AKT activation and metastasis. Knockdown of JNK2 in the highly metastatic 4T1.2 mammary cancer cells significantly decreased growth factor induced invasion in Boyden chambers, orthotopic tumor growth, and metastatic lesions in lungs and bone. Intra-cardiac introduction of cancer cells is utilized to specifically study the later steps in the metastatic cascade including travel of disseminated cancer cells to a secondary location. Thus, earlier steps such as the process of acquiring a malignant phenotype leading to escape from the primary tumor are bypassed. Survival was prolonged in mice receiving intra-cardiac injection of cells deficient of JNK2 either in the host or in the tumor cells, suggesting a potential role for JNK2 as a therapeutic target for advanced stage breast cancer patients. Using siRNA and inhibitors against Src and PI3K, we determined that JNK2 activity is dependent on Src and PI3K, positioning JNK2 downstream of two critical factors involved in tumor progression. Microarray analysis of JNK2 deficient tumors revealed that JNK2 positively regulates the adaptor protein Grb2 associated binding protein 2 (Gab2). Knockdown of Gab2 in 4T1.2 cells resulted in decreased tumor growth and a trend for decreased lung metastasis. In vitro, stimulation of 4T1.2 shJNK2 or 4T1.2 shGab2 cells with HGF, heregulin, or insulin resulted in impaired AKT activation, suggesting involvement of Gab2 and JNK2 in multiple RTK signaling pathways. Understanding of the intricate mechanisms involved in RTK signal transduction can contribute to drug design geared towards more effective targets, namely JNK2. The secondary goal of this research was to decipher the individual roles of JNK2 and JNK1 in metastatic breast cancer. Survival was significantly shortened in mice injected intra-cardiac with 4T1.2 shJNK1 cells. In congruence, serum Cathepsin K levels were increased and bone lesions observed were higher in mice injected with shJNK1 expressing tumor cells compared to mice injected with control cells. In sharp contrast, jnk1-/- mice displayed dramatically increased survival and fewer bone lesions upon intra-cardiac injections of 4T1.2 cells. Collectively, these data suggest cell and isoform specific roles for JNKs. / text

JNK1 (c-Jun N-terminal kinase 1) has been studied in numerous biological phenomena, but its role in skin inflammation diseases has not been fully defined yet. We therefore evaluated the role of JNK1 in imiquimod-induced dermatitis, a classical model that shares many features with human psoriasis. We showed that JNK1 was necessary for the expression of inflammatory markers and for acanthosis induced by imiquimod. We demonstrated that the loss of JNK1 in dendritic cells or myeloid cells reduced inflammatory markers but did not affect acanthosis induced by imiquimod. In vitro experiments in bone marrow-derived macrophages (BMMs) supported the role of JNK1 in the activation of the inflammasome pathway by the Aldara® cream. Next, we observed that the loss of JNK1 in keratinocytes did not reduce imiquimod-induced expression of most inflammatory markers but acanthosis and proliferation of epidermal cells was decreased. To better understand the role of JNK1 in keratinocytes, we evaluated the transcriptome and the epigenomic landscape of JNK1-deficient epidermal cells from mice treated with imiquimod. These data highlighted the potential role of JNK1 downstream of the EGFR pathway. We further observed that the inhibition of the EGFR pathway decreased imiquimod-induced acanthosis. Our work shows the dual role of JNK1 in skin inflammation induced by imiquimod. On one hand, JNK1 influences the expression of inflammatory mediators by myeloid cells, probably through the inflammasome pathway. On the other hand, JNK1 modulates the response of keratinocytes to EGFR ligands. Taken together, these data suggest that JNK1 could represent a valuable therapeutic target for the management of psoriasis. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished