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. 2015 Jun 23;290(32):19544–19557. doi: 10.1074/jbc.M115.638924

FIGURE 5.

FIGURE 5.

PGC1α transduces RGZ/GPR40/p38 MAPK signaling to PPARγ in PAECs. A, RGZ induced PGC1α phosphorylation, an effect blocked by siRNA knockdown of p38α MAPK or GPR40 (p = 0.001 for RGZ versus CTRL within CTRL siRNA; p ≥ 0.45, RGZ versus CTRL within p38α or GPR40 siRNA conditions). PAECs were transfected with specific siRNA or scrambled CTRL siRNA, as indicated. At 48 h after transfection, cells were treated with RGZ (10 μm) or vehicle CTRL for 1 h prior to the extraction of nuclear protein for immunoprecipitation with phosphothreonine and phosphoserine antibodies. Phospho-PGC1α (pPGC1α) in immunoprecipitates was detected by Western blotting with anti-PGC1α. B, RGZ induced PGC1α protein expression in PAECs, an effect blocked by siRNA knockdown of p38α MAPK or GPR40 (p = 0.01 for RGZ versus CTRL within CTRL siRNA; p ≥ 0.62, RGZ versus CTRL within p38α or GPR40 siRNA). Cells were transfected as in A. At 48 h after transfection, cells were treated with RGZ (10 μm) or vehicle CTRL for 4 h prior to the extraction of nuclear protein for measurement of PGC1α by Western blotting. C, RGZ slightly decreased (p < 0.001 for RGZ versus CTRL within CTRL siRNA), but SIRT1 siRNA increased PGC1α acetylation in PAECs (p < 0.001 for SIRT1 siRNA versus CTRL siRNA in the absence and presence of RGZ). At 48 h after transfection, PAECs were treated with RGZ (10 μm) or vehicle CTRL for 4 h prior to the extraction of whole cell lysates for immunoprecipitation with anti-acetyl-lysine antibodies. Acetyl-PGC1α (Ac-PGC1α) in immunoprecipitates was detected by Western blotting with anti-PGC1α. D, siRNA knockdown of PGC1α or SIRT1 inhibited RGZ-induced PPARγ target genes in PAECs (p < 0.001 for RGZ versus RGZ plus PGC1α or SIRT1 siRNA for CD36, CYP1A1, and FABP4). Results are presented as geometric means ± S.E. (error bars) of five independent experiments using different PAEC donors. E and F, specific siRNA for PGC1α and SIRT1 reduced their protein expression in PAECs, respectively (p ≤ 0.001, main effects of PGC1α and SIRT1 siRNA). Cells were transfected with siRNA for PGC1α or SIRT1 or scrambled CTRL siRNA. At 48 h after transfection, PAECs were treated with RGZ (10 μm) or vehicle CTRL for 24 h prior to isolation of total RNA for measurement of PPARγ target genes by real-time PCR or for 4 h prior to the preparation of nuclear protein or whole cell lysates for measurement of PGC1α and SIRT, respectively, by Western blotting. Densitometry results of independent experiments using different PAEC donors (top panels) and a representative blot (bottom panels) are shown in A and B (n = 4), C (n = 5), E (n = 3), and F (n = 4).