Nr4a1 and Nr4a2 are transcription factors and immediate early genes belonging to the nuclear receptor Nr4a family. In this study, we examine their role in long-term memory formation for object location and object recognition. Using siRNA to block expression of either Nr4a1 or Nr4a2, we found that Nr4a2 is necessary for both long-term memory for object location and object recognition. In contrast, Nr4a1 appears to be necessary only for object location. Indeed, their roles in these different types of long-term memory may be dependent on their expression in the brain, as NR4A2 was found to be expressed in hippocampal neurons (associated with object location memory) as well as in the insular and perirhinal cortex (associated with object recognition memory), whereas NR4A1 showed minimal neuronal expression in these cortical areas. These results begin to elucidate how NR4A1 and NR4A2 differentially contribute to object location versus object recognition memory.[Supplemental material is available for this article.]It is well established that long-term memory (LTM) formation requires transcription (for review, see Alberini 2009). Transcription regulated specifically by cAMP responsive element binding protein (CREB) has been shown to be essential for long-term memory (Bourtchuladze et al. 1994;Yin et al. 1994;Guzowski and McGaugh 1997;Pittenger et al. 2002;Sekeres et al. 2010; but see Balschun et al. 2003). Two CREB-dependent immediate early genes that have been implicated in LTM are Nr4a1 and Nr4a2 (Pena de Ortiz et al. 2000; von Hertzen and Giese 2005a,b;Colon-Cesario et al. 2006). Nr4a1 (Nur77) and Nr4a2 (Nurr1) are members of the nuclear steroid/thyroid hormone receptor superfamily that bind in an apparently ligand-independent manner to Nerve Growth Factor1-B (NGFI-B) response elements (Baker et al. 2003;Wang et al. 2003).Expression of both Nr4a1 and Nr4a2 has been shown to increase in the hippocampus following learning. Nr4a1 expression increased in the CA1 region of the hippocampus during context shock memory consolidation, and Nr4a2 increased in CA1 and CA3 pyramidal cell layers of the rat hippocampus following a spatial discrimination task (Pena de Ortiz et al. 2000; von Hertzen and Giese 2005a,b;Keeley et al. 2006;Hawk and Abel 2011). An exception to these findings was a study from Malkani and Rosen (2000) who found increased Nr4a1 expression in the cortex and amygdala following context shock memory consolidation, but failed to see a change in area CA1 of the hippocampus. These patterns of expression suggest a role for the Nr4a family in learning and memory.Transcription of both Nr4a1 and Nr4a2 appear to be regulated by chromatin modification via histone acetylation and deacetylation. Histone deacetylase (HDAC) activity was shown to interfere with formation of the pre-initiation complex at the Nr4a1 promoter, suggesting that acetylation is necessary for its transcription (Fass et al. 2003). In addition, during memory consolidation, the HDAC inhibitor Trichostatin A (TSA) maintained the expression of both Nr4a1 ...

Abstract-Melanocytes and melanoma cells contain melanin, a complex polymer that modulates redox changes in these cells. Relative intracellular hydrogen peroxide levels measured by dichlorodihydrofluorescein are similar in the two cell types, but the levels of superoxide anion measured by dihydroethidium were markedly increased in melanoma cells. Chelator-induced oxidative stress is efficiently suppressed by melanocytes without substantial recruitment of the transcription factors NF-B and AP-1 as measured by electrophoretic mobility shift assay and quantitated by densitometry or by a change in frequency of apoptosis as determined by annexin V binding. In contrast, NF-B in melanoma cells is strongly recruited by changes in redox status and exhibits a correlative relationship to intracellular hydrogen peroxide (but not superoxide anion). However, the response of the NF-B pathway to intracellular hydrogen peroxide is anomalous, including downregulation of p65 and IB␣ RNA expression (Northern blot). Additionally, recruitment of AP-1 binding in melanoma cells was directly correlated with intracellular levels of superoxide anion (but not hydrogen peroxide). Neither the degree of NF-B nor AP-1 binding in melanoma cells was related to the frequency of apoptosis. The responsiveness of NF-B and AP-1 recruitment to intracellular levels of hydrogen peroxide and superoxide anion without concomitant control of apoptosis provides a general mechanism by which these cells can escape noxious injury (e.g., chemotherapy). The marked enhancement of apoptosis in melanoma cells by chelators indicates, however, that this alteration can be circumvented and offers a unique therapeutic window to explore.

Nuclear factor kappa B (NFκB) is an essential regulator of gene transcription for hundreds of genes, including many critically involved in apoptosis. NFκB complexes containing cRel generally activate pro‐apoptotic genes, while those with RelA activate anti‐apoptotic genes. We have previously shown that NFκB binding by RelA is constitutively elevated in human metastatic melanoma cultures relative to normal melanocytes. Here we extended our investigation to immunohistochemical analysis of human tissue biopsies. We found that RelA expression is significantly elevated in melanocytes of human naevi and melanomas relative to normal skin, but expression of its inhibitor IκB‐α is significantly lower in metastatic melanomas than in intradermal naevi. Antibodies specific for the nuclear localization signal of RelA also showed significantly increased staining in metastatic melanoma biopsies. Notably, in melanomas and in naevi, we also found that RelA is phosphorylated at serine 529, and this activated form accumulates in the nuclei of melanomas. This suggests that increased expression and phosphorylation of RelA occurs at the stage of the benign naevus, but IκB‐α is able to sequester RelA in the cytoplasm and regulate RelA transcriptional transactivation. We also found that antibodies against cRel show a progressive increase in staining from naevi to melanoma. However, staining for IκB‐ɛ, which primarily inhibits the nuclear localization of cRel was also progressively increased, and cRel expression was predominantly cytoplasmic in melanomas. These results confirm that the altered expression of RelA found in metastatic melanoma cells in tissue culture is relevant to human tumors and offer new insights into the deregulation of NFκB signaling.

melanocytes. We also found that melanoma cells expressed Metastatic melanomas are typically resistant to radiation and chemotherapy. The underlying basis for this phenomenon may higher cytoplasmic levels of RelA, p105/p50 and the inhibitory protein, inhibitor of kappa B alpha (IkBa) than melanocytes. result in part from defects in apoptotic pathways. Nuclear factor kappa B (NFkB) has been shown to control apoptosis in To directly test whether RelA expression has an impact on melanoma cell survival, we used antisense RelA phosphorothmany cell types and normally functions as an immediate stress ioate oligonucleotides and found that melanoma cell viability response mechanism that is rigorously controlled by multiple inhibitory complexes. We have previously shown that NFkB was significantly decreased compared with untreated or conbinding is elevated in metastatic melanoma cells relative to trol cultures. The constitutive activation of NFkB in metastatic melanoma cell cultures may, therefore, support an normal melanocytes. In the current study, Western blot analysis showed that, compared with normal melanocytes, inappropriate cell survival pathway that can be therapeutically manipulated. melanoma cell lines have higher nuclear levels of the NFkB subunits p50 (7-fold) and RelA (5-10-fold). In response to Key words: Melanocyte, Melanoma, NFkB, Antisense, TNF, tumor necrosis factor-alpha (TNFa), both melanocytes and UVB melanoma cells showed increased nuclear p50 and RelA levels, but levels in melanoma cells remained higher than in Nuclear factor kappa B (NFkB) activation has been shown to have both pro-and anti-apoptotic functions in various cell types (5, 6). There are five mammalian NFkB/ Rel family members, p50, p52, RelA, RelB, and cRel, that share a highly conserved 300-amino acid Rel homology domain containing dimerization, nuclear localization, and DNA binding regions (5 -8). These proteins can form homoand heterodimers, which bind DNA at NFkB sequences found in the promoters of a variety of genes and specific dimer pairs elicit differential induction of these genes (5,8,9). Several studies have suggested that NFkB transcription

We demonstrated previously that c‐Jun, JunB and c‐Fos RNA were dysregulated in metastatic melanoma cells compared with normal human melanocytes. The purpose of this study was to evaluate the distribution in composition of AP‐1 dimers in human melanoma pathogenesis. We investigated AP‐1 dimer pairing in radial growth phase‐like (RGP) (w3211) and vertical growth phase‐like (VGP) (w1205) human melanoma cells and metastatic cell lines (cloned from patients, c83‐2c, c81‐46A, A375, respectively) compared with melanocytes using electrophoretic mobility shift assay (EMSA), Western blot and transfection analyses. There are progressive variations in AP‐1 composition in different melanoma cell lines compared with normal melanocytes, in which c‐Jun, JunD and FosB were involved in AP‐1 complexes. In w3211, c‐Jun, JunD and Fra‐1 were involved in AP‐1 binding, while in w1205, overall AP‐1 binding activity was decreased significantly and supershift binding was detected only with JunD antibodies. In metastatic c81‐46A and A375 cells, only JunD was involved in AP‐1 binding activity, but in a third (c83‐2c) c‐Jun, JunD and Fra‐1 were present. Western blot evaluation detected c‐Jun in melanocytes and w3211, but this component was decreased significantly or was not detectable in w1205, c81‐46A and A375 cells. In contrast, JunD protein was elevated in c81‐46A and c83‐2c cells compared with melanocytes and RGP and VGP cell lines. Normal melanocytes and c83‐2c cells (which have c‐Jun involved in AP‐1 binding), transfected with c‐Jun antisense and treated with cisplatin, showed higher viability compared with untransfected cells, while in c81‐46A cells (in which only JunD is detectable) no change in cell viability was observed following treatment with cisplatin and c‐jun antisense transfection. A dominant‐negative c‐Jun mutant (TAM67) significantly increased the soft agar colony formation of w3211 and c83‐2c cells. These results suggest that components of AP‐1, especially c‐Jun, may offer a new target for the prevention or treatment of human melanoma progression.