The scope of this work was to evaluate the role of PKA in modulating PPAR activity

The scope of this work was to evaluate the role of PKA in modulating PPAR activity. Early work from Shalev (14) has shown that insulin treatment can phosphorylate PPAR. vectors. After lipofection, 10 M of H89 was added to the medium 1 h before ligands. Cells were grown for 36 h in the presence of 1 M WY 14,643 (WY), 50 M Bromopalmitate (Bro) and 5 M BRL 49,653 (BRL), with 1g/ml CT when indicated. Results are shown as the mean SD (n = 6) of CAT activity after normalization for -galactosidase activity. B. 30 g of whole cell extracts from transfected cells were loaded on SDS-PAGE and probed by western blot using RASGRP1 mPPAR antibody. The first lane corresponds to HEK-293 cells transfected with the empty pSG5 vector and the remaining lanes correspond to 293 cells transfected with pSG5-mPPAR vector and treated or not (?) with WY, CT or H89 under the same conditions as in Fig. 5A. C. 5 g of the same WCE were used in gel shift assays using the ACoA probe. RXR contributes to PPAR activation by PKA As RXR is an obligate heterodimerization partner of the PPARs for DNA binding and transactivation, we determined whether RXR could be involved in the PKA activation of PPAR (Fig 6A). In the absence of transfected RXR or PPAR, the activity of the 2CYPA6-TK-CAT construct was very low and not modulated by the WY 14,643 and CT. In the absence of transfected RXR, transfected PPAR was active and modulated by PKA in HEK-293 cells, as these cells express low levels of endogenous RXR. In contrast, transfection of RXR alone in these cells had almost no effect on the expression of the 2CYP4A6-TK-CAT reporter gene even in the presence of SR3335 9-cis-retinoic acid (9cRA, is a SR3335 ligand of RXR). However, we observed an enhancement of PPAR activity in the presence of 9cRA and CT both in the absence and in the presence of WY 14,643. Indeed, enhancement of the PPAR activity was even more potent with CT + 9cRA than with WY SR3335 14,643 + 9cRA. On the contrary, in the presence of WY 14,643, 9c-RA had only a minor effect. By overexpressing simultaneously RXR and PPAR, in the absence of 9cRA, we observed an increase by about 30% of PPAR activation by WY 14,643, and a 2 fold activity enhancement in the presence of CT and without ligand compared to PPAR without cotransfected RXR. RXR affected only moderately PPAR activation (about 20%) by WY 14, 643 + CT. In the presence of RXR and 9cRA and in the absence of WY 14,643 and CT, we observed a 3 fold enhancement of PPAR activity compared to cells without 9cRA. In the presence of WY 14,643 or WY 14,643 + CT, 9cRA only increased SR3335 by 30% the activity seen in the absence of 9cRA. Finally, 9cRA was unable to affect CT induction of PPAR in the absence of WY 14,643. These data suggest that RXR cooperates with PPAR in the absence of exogenous ligand to increase both the basal and CT-induced activity of PPAR on PPREs. We next checked whether RXR was itself the target of PKA when bound to its preferred binding site (DR1). To do so, we used the DR1-TK-CAT construct containing a strong RXR binding site (Fig. 6B). We observed a strong activation of the construct by SR3335 RXR in the presence of RA. CT treatment increased both ligand-independent and ligand-dependent activity of RXR. Thus, RXR by being itself the target of PKA can enhance PPAR activity on PPREs. Open in a separate window Fig 6 RXR modulates PPAR activity in the presence of PKA activatorsA. 100 ng of pSG5, pSG5-mPPAR and pSG5-mRXR2 expression vectors per well in combination or alone were cotransfected in HEK-293 cells with 200 ng of.