[PubMed] [CrossRef] [Google Scholar] 37

[PubMed] [CrossRef] [Google Scholar] 37. this effect, suggesting that STAT4 phosphorylation is not essential for but enhances transcription. Additionally, we demonstrate that CLL B lymphocytes have a STAT4 expression defect which partly accounts for Phenolphthalein their p66Shc deficiency, as supported by reconstitution experiments. Finally, we show that p66Shc participates in a positive feedback loop to promote Phenolphthalein STAT4 expression. These results provide new insights into the mechanism of p66Shc expression in B cells and its defect in CLL, identifying the STAT4/IL-12 pathway as a potential therapeutic target in this neoplasia. locus regulating the transcripts encoding p52Shc/p46Shc and p66Shc, respectively [8]. The regulatory region of is characterized by the presence of a CpG-rich region that can be hyper-methylated, leading to promoter silencing [8, 9]. Although DNA modifications are responsible for silencing in epithelial as well as in T cells, the mechanism of p66Shc regulation in other cell types has yet not been elucidated. The absence of transcription factors specifically able to bind and activate the promoter may provide an alternative or additional mechanism, as exemplified by nuclear erythroid 2-related factor 2 (Nrf2), which binds to an antioxidant response element on the promoter [10, 11]. We have recently shown that neoplastic B cells from Chronic Lymphocytic Leukemia (CLL) patients exhibit a defect in expression, with the lowest levels displayed by patients with unfavorable prognosis [6]. Interestingly, although the presence of methylated CpG sites in the promoter may account in part for the relatively low expression levels of p66Shc in healthy B cells, neither the overall methylation status of the CpG-rich region nor the methylation of individual CpG sites differ between healthy and CLL B cells [6], indicating that a transcriptional rather than epigenetic mechanism may account for the p66Shc expression defect in neoplastic cells. Here we show that STAT4 Phenolphthalein regulates p66Shc expression in B cells by interacting with several specific binding sites in the promoter. Of note, the p66Shc defect in CLL B cells correlates with impaired STAT4 expression. Interestingly, we found that p66Shc is in turn able to promote the expression of several Phenolphthalein genes participating in the IL-12 pathway and regulated by STAT4, including STAT4 itself, and reconstitution of p66Shc in CLL B cells normalizes the levels of STAT4. The data highlight a new mechanism of transcriptional regulation of p66Shc in B cells mediated by STAT4 binding to the promoter and provide evidence of a feedback regulatory loop whereby p66Shc modulates STAT4. They identify moreover STAT4 deficiency as a potential player in the response of CLL B cells with the tumor microenvironment. RESULTS AND DISCUSSION Gene expression profile analysis associates p66Shc to expression of IL-12 responsive genes in B cells We have shown that p66Shc is able to modulate the expression of several genes critical to B-cell survival Phenolphthalein and homing through both its adaptor and pro-oxidant activities [6, 12]. To achieve insights into the processes regulated by p66Shc we used an unbiased approach involving a gene expression profile analysis on B cells stably transfected with a plasmid encoding p66Shc (MEC-p66) or the respective empty vector (MEC-Ctr). The MEC-1 cell line was used for these experiments as endogenous is completely silenced by promoter methylation, as supported by the fact that treatment with the demethylating agent 5-Aza-2-deoxycytidine (AZA, decitabine) restored its expression (Supplementary Figure S1A) [13]. Two independent mRNA extractions were profiled for each sample using the Affymetrix HuGene 2.0-st-v1 array. An ANOVA model to identify genes differentially expressed between the two groups was created and the transcripts with a fold-change higher than 2 and a statistically significant and (Figure ?(Figure1A),1A), as well as of and (Figure ?(Figure1B),1B), mRNA, were confirmed to be up-regulated in p66Shc-overexpressing cells compared to the empty vector transfectant. Consistent with the qRT-PCR data, IFN-, IL-1 and IL-10, whose mRNA levels showed the largest fold-changes, were up-regulated in MEC-p66 cells compared to control cells, as assessed by flow cytometry (Figure ?(Figure1C1C). Table 1 List of IL-12 regulated genes found to be differentially up-regulated in Proc p66Shc-overexpressing MEC-1 cells versus control and mRNA in MEC-1 cells stably transfected with a construct encoding p66Shc (MEC-p66) or empty vector (MEC-Ctr). The relative abundance of gene transcripts was determined on triplicate samples from 3 independent mRNA extractions using.