The incubation with primary antibodies was performed for 2 h at RT, followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibody. this process. GLS2 and TAp63 expression increases during the in vitro differentiation of primary R788 (Fostamatinib) human keratinocytes, and depletion of GLS2 inhibits skin differentiation both at molecular and cellular levels. We found that GLS2 and TAp63 expression are concomitantly induced in cancer cells exposed to oxidative stresses. siRNA-mediated depletion of GLS2 sensitizes cells to ROS-induced apoptosis, suggesting that the TAp63/GLS2 axis can be functionally important as a cellular antioxidant pathway in the absence of p53. Accordingly, we found that GLS2 is upregulated in colon adenocarcinoma. Altogether, our findings demonstrate that GLS2 is a bona fide TAp63 target gene, and that the TAp63-dependent regulation of GLS2 is important for both physiological and pathological processes. strong class=”kwd-title” Keywords: glutaminolysis, p63, skin differentiation, colon carcinoma Introduction p63 is a transcription factor belonging to the p53 family.1 Like other members of the p53 family,2 p63 is expressed as multiple isoforms arising by alternative promoter usage or differential splicing events at the C terminus.3,4 In particular, TAp63 isoforms contain a complete N-terminal transcriptional domain and are capable of efficiently transactivating different p53-responsive genes, thus largely mimicking p53 tumor-suppressive activities.5 Accordingly, ectopic expression of TAp63 can induce cell cycle arrest and apoptosis in different tumor cell lines, and different cellular stresses induce TAp63 expression by both transcriptional and post-translational mechanisms.6-9 Np63, which lacks the N-terminal transcriptional domain, mainly acts as a dominant negative of p53 and TAp63 isoforms, even though it posses two transactivation domains,10-12 which endows it with a specific transcriptional activity, mainly towards epithelial specific genes.13-15 Accordingly, genetic studies in mice have indicated that p63 is crucially involved in the maintenance of epithelial stem cells,16-18 and mice genetically depleted of p63 display a complete lack of skin as well as defects in limb development and other epithelial-derived tissues.16,19,20 The role of p63 in skin formation has been further supported by ex vivo and in vitro experiments indicating a prevalent expression of Np63 in the basal layer of the epidermis.21,22 Although the p63 gene is rarely mutated in human tumors, numerous studies have highlighted the importance of p63 in tumor development, and recent reports have demonstrated that TAp63 acts as a putative tumor suppressor gene.23-25 Loss or impaired expression of TAp63 has been associated with tumor progression and poor prognosis in some invasive human cancers,26 and TAp63-knockout mice develop metastatic tumors, suggesting that the deregulated expression of TAp63 isoforms could be important in regulating tumor progression and metastasis. 27 Although TAp63 activity has been recently linked to the regulation of lipid and glucose metabolism,28 its role in the regulation of energy metabolism and cellular antioxidant defenses is not R788 (Fostamatinib) completely characterized. Enhanced mitochondrial glutamine catabolism and increased rate of glycolysis are the two major metabolic alterations exhibited by many cancer cells in order to support their rapid proliferation.29-36 Glutaminolysis involves the initial deamination of glutamine by glutaminase, yielding glutamate and ammonia. In mammals, two different genes located on distinct chromosomes encode for glutaminase enzymes, EMR2 the kidney-type isozymes (GLS1) and the liver-type isozymes GLS2.37,38 Although GLS1 and GLS2 share a considerable degree of sequence similarity, the two enzymes have different kinetic, immunological and molecular features. GLS1 is activated by high phosphate levels and is inhibited by the end-product glutamate, while GLS2 is activated by low phosphate levels and is not inhibited by glutamate.39 Although GLS2 has been initially reported to be expressed specifically in postnatal liver, recent evidence indicate that this enzyme is also R788 (Fostamatinib) expressed in other tissues and in some cancer cells.40,41 On R788 (Fostamatinib) the basis of their ability to convert glutamine into glutamate, an important intermediate for energy production and antioxidant cellular defense, it is not surprising that glutaminase expression is often deregulated in cancer cells. Loss of GLS2 expression has been reported in hepatocellular carcinomas, in highly malignant glioblastoma and anaplastic astrocytomas.40,41 Moreover, the.