Faculty Bibliography

INrf2:Nrf2 are sensors of chemical/radiation stress. Nrf2 dissociates from INrf2 in response to a stress and translocates in the nucleus. This leads to induction of a battery of antioxidant genes that protect cells. Nrf2 is then exported out and degraded. INrf2 functions as an adaptor of ubiquitin ligase for ubiquitination and degradation of Nrf2. Here we demonstrate the presence of a novel feedback autoregulatory loop between INrf2 and Nrf2 that controls cellular abundance of INrf2 and Nrf2. Nrf2 controls its own degradation by regulating expression and induction of the INrf2 gene. The antioxidant treatment of cells led to nuclear localization and stabilization of Nrf2 and induction of INrf2 gene expression. Mutagenesis, transfection, and chromatin immunoprecipitation assays identified an antioxidant-response element in the reverse strand of the proximal INrf2 promoter that binds to Nrf2 and regulates expression and antioxidant induction of the INrf2 gene. In addition, short interfering RNA inhibition or overexpression of Nrf2 led to a respective decrease and increase in INrf2 gene expression. These results implicated Nrf2 in the regulation of expression and induction of INrf2. The induction of INrf2 followed ubiquitination and degradation of Nrf2 and suppression of INrf2 gene expression. In conclusion, Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by degrading it.

NAD(P)H:quinone oxidoreductase 1(-/-) (NQO1(-/-)), NQO1(+/-) along with NRH:quinone oxidoreductase 2(-/-) (NQO2(-/-)), and wild-type (WT) mice were exposed to five once weekly doses of mitomycin C. The mice were euthanized 15 weeks after the first dose. Blood cell counts and histologic analyses were done. WT and NQO2(-/-) mice showed hypocellularity and a significant increase in adipocytes in bone marrow. They also showed anemia because of the loss of RBC and hemoglobin. The neutrophils and platelets were reduced, whereas other blood cell types and tissues were normal. Interestingly, NQO1(-/-) mice showed a complete resistance to mitomycin C-induced bone marrow cytotoxicity and reduction in RBC, hemoglobin, and neutrophils. NQO1(+/-) mice also showed limited resistance to mitomycin C-induced bone marrow cytotoxicity. These data show a major in vivo role of NQO1 in metabolic activation of mitomycin C with implications in mitomycin C chemotherapy.

Aiming at more precise detection of melanoma cells in sentinel lymph nodes and better understanding of the mechanisms underlying metastatic spread, expression of L1, CEACAM1, and binding of the lectins HPA, ML-I and PNA, was assessed in benign nevi (n=12), primary melanomas (PTs: n=67), their corresponding sentinel lymph nodes (SLNs: n=40), and distant metastases (DMs: n=35). Sensitivity and specificity of CEACAM1 (95-97%; 66%) and L1 (90-93%; 100%) exceeded that of the standard markers MelanA, S100, and HMB45 in single marker use. Lectin binding was found in PTs and DMs (HPA: 69% and 77%; ML-I: 82% and 77%, respectively), but rarely in SLNMs (HPA: 20%, ML-I: 20%, PNA: 5%, respectively). The highly specific and sensitive L1-11A against L1 and 4D1/C2 against CEACAM1 antibodies are a worthy completion to standard antibody panels for diagnosis of melanoma cells. Both CAMs seem to be functionally involved in lymphatic and haematogenous spread, and are thus promising target molecules for immunotoxins.

Transcriptional dysregulation and aberrant chromatin remodeling are central features in the pathology of Huntington's disease (HD). In order to more fully characterize these pathogenic events, an assessment of histone profiles and associated gene changes were performed in transgenic N171-82Q (82Q) and R6/2 HD mice. Analyses revealed significant chromatin modification, resulting in reduced histone acetylation with concomitant increased histone methylation, consistent with findings observed in HD patients. While there are no known interventions that ameliorate or arrest HD progression, DNA/RNA-binding anthracyclines may provide significant therapeutic potential by correcting pathological nucleosome changes and realigning transcription. Two such anthracyclines, chromomycin and mithramycin, improved altered nucleosome homeostasis in HD mice, normalizing the chromatin pattern. There was a significant shift in the balance between methylation and acetylation in treated HD mice to that found in wild-type mice, resulting in greater acetylation of histone H3 at lysine 9 and promoting gene transcription. Gene expression profiling in anthracycline-treated HD mice showed molecular changes that correlate with disease correction, such that a subset of downregulated genes were upregulated with anthracycline treatment. Improved nucleosomal dynamics were concurrent with a significant improvement in the behavioral and neuropathological phenotype observed in HD mice. These data show the ability of anthracycline compounds to rebalance epigenetic histone modification and, as such, may provide the rationale for the design of human clinical trials in HD patients.

Rab11 and Rab6 guanosine triphosphatases are associated with membranes of the recycling endosomes (REs) and Golgi complex, respectively. Evidence indicates that they sequentially regulate a retrograde transport pathway between these two compartments, suggesting the existence of proteins that must co-ordinate their functions. Here, we report the characterization of two isoforms of a protein, Rab6-interacting protein 1 (R6IP1), originally identified as a Rab6-binding protein. R6IP1 also binds to Rab11A in its GTP-bound conformation. In interphase cells, R6IP1 is targeted to the Golgi in a Rab6-dependent manner but can associate with Rab11-positive compartments when the level of Rab11A is increased within the cells. Fluorescence resonance energy transfer analysis using fluorescence lifetime imaging shows that the overexpression of R6IP1 promotes an interaction between Rab11A and Rab6 in living cells. Accordingly, the REs marked by Rab11 and transferrin receptor are depleted from the cell periphery and accumulate in the pericentriolar area. However, endosomal and Golgi membranes do not appear to fuse with each other. We also show that R6IP1 function is required during metaphase and cytokinesis, two mitotic steps in which a role of Rab6 and Rab11 has been previously documented. We propose that R6IP1 may couple Rab6 and Rab11 function throughout the cell cycle.

The extent to which Rab GTPases, Rab-interacting proteins, and cargo molecules cooperate in the dynamic organization of membrane architecture remains to be clarified. Langerin, a recycling protein accumulating in the Rab11-positive compartments of Langerhans cells, induces the formation of Birbeck granules (BGs), which are membrane subdomains of the endosomal recycling network. We investigated the role of Rab11A and two members of the Rab11 family of interacting proteins, Rip11 and RCP, in Langerin traffic and the biogenesis of BGs. The overexpression of a dominant-negative Rab11A mutant or Rab11A depletion strongly influenced Langerin traffic and stability and the formation of BGs, whereas modulation of other Rab proteins involved in dynamic regulation of the endocytic-recycling pathway had no effect. Impairment of Rab11A function led to a missorting of Langerin to lysosomal compartments, but inhibition of Langerin degradation by chloroquine did not restore the formation of BGs. Loss of RCP, but not of Rip11, also had a modest, but reproducible effect on Langerin stability and BG biogenesis, pointing to a role for Rab11A-RCP complexes in these events. Our results show that Rab11A and Langerin are required for BG biogenesis, and they illustrate the role played by a Rab GTPase in the formation of a specialized subcompartment within the endocytic-recycling system.

Lymphocyte transformation induced by Theileria parasites involves constitutive activation of c-Jun N-terminal kinase (JNK) and the AP-1 transcription factor. We found that JNK/AP-1 activation is associated with elevated levels of Rab11 protein in Theileria-transformed B cells. We show that AP-1 regulates rab11a promoter activity in B cells and that the induction of c-Jun activity in mouse fibroblasts also leads to increased transcription of the endogenous rab11a gene, consistent with it being an AP-1 target. Pharmacological inhibition of the JNK pathway reduced Rab11 protein levels and endosome recycling of transferrin receptor (TfR) and siRNA knockdown of JNK1 and Rab11A levels also reduced TfR surface expression. We propose a model, where activation of the JNK/AP-1 pathway during cell transformation might assure that the regulation of recycling endosomes is co-ordinated with cell-cycle progression. This might be achieved via the simultaneous upregulation of the cell cycle machinery (e.g. cyclin D1) and the recycling endosome regulators (e.g. Rab11A).

The biogenesis and accumulation of the beta amyloid protein (Abeta) is a key event in the cascade of oxidative and inflammatory processes that characterises Alzheimer's disease. The presenilins and its interacting proteins play a pivotal role in the generation of Abeta from the amyloid precursor protein (APP). In particular, three proteins (nicastrin, aph-1 and pen-2) interact with presenilins to form a large multi-subunit enzymatic complex (gamma-secretase) that cleaves APP to generate Abeta. Reconstitution studies in yeast and insect cells have provided strong evidence that these four proteins are the major components of the gamma-secretase enzyme. Current research is directed at elucidating the roles that each of these protein play in the function of this enzyme. In addition, a number of presenilin interacting proteins that are not components of gamma-secretase play important roles in modulating Abeta production. This review will discuss the components of the gamma-secretase complex and the role of presenilin interacting proteins on gamma-secretase activity.

The Gli code hypothesis postulates that the three vertebrate Gli transcription factors act together in responding cells to integrate intercellular Hedgehog (Hh) and other signaling inputs, resulting in the regulation of tissue pattern, size and shape. Hh and other inputs are then just ways to modify the Gli code. Recent data confirm this idea and suggest that the Gli code regulates stemness and also tumor progression and metastatic growth, opening exciting possibilities for both regenerative medicine and novel anticancer therapies.

Melanoma is one of the most aggressive cancers, and its incidence is increasing. These tumors derive from the melanocyte lineage and remain incurable after metastasis. Here we report that SONIC HEDGEHOG (SHH)-GLI signaling is active in the matrix of human hair follicles, and that it is required for the normal proliferation of human melanocytes in culture. SHH-GLI signaling also regulates the proliferation and survival of human melanomas: the growth, recurrence, and metastasis of melanoma xenografts in mice are prevented by local or systemic interference of HH-GLI function. Moreover, we show that oncogenic RAS-induced melanomas in transgenic mice express Gli1 and require Hh-Gli signaling in vitro and in vivo. Finally, we provide evidence that endogenous RAS-MEK and AKT signaling regulate the nuclear localization and transcriptional activity of GLI1 in melanoma and other cancer cells. Our data uncover an unsuspected role of HH-GLI signaling in melanocytes and melanomas, demonstrate a role for this pathway in RAS-induced tumors, suggest a general integration of the RAS/AKT and HH-GLI pathways, and open a therapeutic approach for human melanomas.