3.5  The phosphoinositide-3-kinase (PI3K) pathway is also involved in the regulation of LPL gene transcription through Sp1/Sp3
Our previous studies showed that the PI3K inhibitor wortmannin prevented, at least in part, the IFN-γ-mediated inhibition of LPL enzymatic activity and mRNA expression in J774.2 macrophages [14]. More recently, we have shown that PI3K, along with CK2, is involved in the classical JAK-STAT pathway of IFN-γ signalling through the regulation of STAT1 phosphorylation at serine 727, which is necessary for maximal transcriptional activity [11]. PKB (also called Akt) is a key downstream mediator of PI3K activation [23]. The action of IFN-γ on PKB activity in J774.2 macrophages was therefore investigated using an in vitro kinase assay in which its ability to phosphorylate glycogen synthase kinase (GSK)-3α/β is measured. Consistent with previous studies in other cellular systems [24–26], IFN-γ activated PKB in J774.2 macrophages (Fig. 5). In order to ascertain whether PKB affects LPL gene transcription, the effect of DN PKB on LPL promoter activity in transfected cells was analysed. Fig. 6 shows that the IFN-γ-mediated decrease in LPL promoter activity could be prevented by expression of DN PKB.
To determine whether the PI3K pathway was also involved in the cytokine-mediated reduction of Sp1/Sp3 binding to its recognition sequence in the LPL gene, the effect of pre-treatment of the cells with three different concentrations of the PI3K inhibitor LY294002 on the IFN-γ-mediated decrease in Sp1/Sp3 binding was analysed by EMSA. Representative, comparative experiments were also carried out with SB415286, an inhibitor of GSK, as we found that IFN-γ had no effect on phosphorylation-mediated activation of GSK-3β or serine 9 (data not shown). Fig. 7A shows that the presence of LY294002 attenuated the IFN-γ-mediated reduction of Sp1/Sp3 binding to the LPL gene. Such an effect was not observed with the GSK inhibitor SB415286 (Fig. 7B). Our previous studies showed that the IFN-γ-mediated activation of PKB could be attenuated by inhibition of the PI3K pathway and JAK2 but not by CK2 [11]. Indeed, co-immunoprecipitation assays demonstrated a constitutive interaction between JAK2 and the p85 subunit of PI3K (data not shown). Consistent with such an interaction, inclusion of the JAK2 inhibitor, AG490, at three different concentrations, prevented the IFN-γ-mediated suppression of Sp1/Sp3 binding (Fig. 7C).
PKB-mediated phosphorylation of rhSp1 had no effect on DNA binding (data not shown), thereby suggesting that a downstream kinase(s) was potentially mediating the actions of PKB on Sp1/Sp3 binding. The mammalian targets of rapamycin (mTOR) proteins represent an important class of downstream targets of PKB, which have been implicated in the control of several cellular functions [27]. The action of the mTOR inhibitor rapamycin was therefore analysed. As shown in Fig. 7D, rapamycin attenuated the decrease in Sp1/Sp3 binding.