Transcriptional Regulation of LKB1 Figure 7. NF-Y, Sp1 and FOXO transcription factors activate transcription from the LKB1 promoter. Luciferase reporter assays in C33a cells after over-expression of Sp1 (A), NF-Y (B) and FOXO transcription factors (C). Reporter activity of LKB1 wild-type promoter is indicated in dark grey, while reporter activity of constructs lacking the corresponding transcription factor binding site is indicated in light grey. Luciferase activity (relative light units normalized to renilla luciferase activity) is expressed as the percentage of the signal obtained from co-transfection of 100 ng of the plasmid containing the LKB1 wild-type promoter (LKB1 Pro II, 2549 to +727) together with 150 ng of the empty expression vector (CTR). Instead of the empty vector 150 ng of the corresponding transcription factor expression plasmid have been co-transfected. In the case of NF-Y 50 ng of plasmids encoding each subunit NF-Ya, NFY-b and NF-Yc were transfected together. Each bar represents the means 6 standard deviation of three independent experiments. doi:10.1371/journal.pone.0032590.g007 family of transcription factors exhibit nearly identical binding motifs [37,38]. It therefore remains possible that beside FOXO3 and FOXO4, also other members of this transcription factors family may regulate LKB1 gene expression in a cell type specific manner. Since the expression pattern of each family member ranges from restricted to a single tissue to nearly ubiquitous, it also seems possible that these factors are either redundant or indispensable in certain tissues. Further experiments will be needed to dissect the role of other forkhead box transcription factors in the regulation of the LKB1 promoter in various cell types and in correlation with their metabolic state. Notably, the activity of FOXO transcription factors is not only controlled by tissue specific expression, but also by a variety of posttranslational modification, including phosphorylation, acety- lation and ubiquitination [23]. For instance, phosphorylation of FOXO3 by AMPK leads to the activation of its transcriptional activity [39]. Having shown that LKB1 transcription is induced by FOXO3 and since AMPK is a direct target of LKB1 [12,13], our analysis suggests that the expression of LKB1 might be stimulated by AMPK via a positive regulatory feedback loop, allowing a cross-talk between these enzymes to counterbalance each other in the coordination of anabolic and catabolic activities. How could LKB1 react to changes of the external milieu? Here, phosphorylation of three conserved serine/threonine residues within FOXO proteins by the proto-oncogene PKB may play an important role [24,25]. This specific phosphorylation is mainly triggered by growth factors like insulin or the insulin-like growth factors and results in the inhibition of FOXO factors due to their export from the nucleus [32]. Notably, in our experiments we could show that PKB phosphorylation site deficient mutants of FOXO3 and FOXO4 were even more potent in inducing LKB1 promoter activity than the wild type forms (Figure 7C). It therefore seems possible that the insulin-phosphatidylinositol 3-kinase – PKB signalling pathway negatively regulates LKB1 expression through inactivation of FOXO transcription factors. Although further experiments will be needed to prove this assumption, there are other reports supporting the existence of this functional link. Recently it was shown that LKB1 transcription was down- regulated upon induction of the serum and glucocorticoid- inducible kinase 1 (SGK-1) [40]. SGK-1, like PKB, belongs to the same family of protein kinases [41], is a downstream effector of the phosphatidylinositol 3-kinase pathway and is also able to phosphorylate FOXO transcription factors at the same residues like PKB [42]. Since there is now considerable evidence that insulin and the insulin-like growth factors play important roles in neoplasia [43], it will be one of the future goals to elucidate whether these hormones are involved in the down regulation of the LKB1 tumour suppressor in certain tumour entities. Materials and Methods Antibodies All antibodies were obtained from commercial suppliers and used without further purification. Mouse monoclonal anti-LKB1 (ab15095) was purchased from Abcam, rabbit monoclonal anti- phospho-AMPKa Thr172 (2535/40H9) was purchased from Cell Signaling Technology, goat polyclonal anti-NF-YA (sc-7712 X), rabbit polyclonal anti-FOXO3 (sc-11351 X), goat polyclonal anti- FOXO4 (sc-5221 X), rabbit polyclonal anti-Sp1 (sc-59 X), rabbit polyclonal anti-Sp1 (sc-14027 X), rabbit polyclonal anti-Sp3 (sc- 644 X), rabbit polyclonal anti glutathione-S-transferase (GST) (sc- 459) and normal goat IgG (sc-2028) were from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated secondary antibodies, polyclonal goat anti-rabbit IgG (W4011) and anti- mouse IgG (W4012) were from Promega. Cell Culture The HPV-negative cervical carcinoma cell line C33a and hepatocellular carcinoma cells HepG2 were obtained from American Type Culture Collection. The normal fibroblast line IMR-90 and the non-tumourigenic somatic cell hybrids made between HeLa cells and IMR-90 (referred to as ‘‘444’’) were kindly provided by E. Stanbridge [44,45]. All cells were maintained in Dulbecco modified Eagle medium (DMEM) (Sigma) supplemented with 10% (v/v) of foetal bovine serum (Gibco Life Technologies, Paisley, United Kingdom), penicillin (final concentration: 100 U/ml, Gibco) and streptomycin (final concentration: 0.1 mg/ml, Gibco). Plasmids The plasmids pJET1.2 (Fermentas), pGL3-Basic (Promega), pRL-null (Promega), pRL-TK (Promega) and pcDNA3 (Invitro- gen) have been purchased from commercial suppliers. PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e32590