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. 2015 Jan 9;290(2):950-9.
doi: 10.1074/jbc.M114.589325. Epub 2014 Nov 11.

Relaxin activates peroxisome proliferator-activated receptor γ (PPARγ) through a pathway involving PPARγ coactivator 1α (PGC1α)

Sudhir Singh  1 , Ronda L Simpson  1 , Robert G Bennett  2
Affiliations

Relaxin activates peroxisome proliferator-activated receptor γ (PPARγ) through a pathway involving PPARγ coactivator 1α (PGC1α)

Sudhir Singh et al. J Biol Chem. .

Abstract

Relaxin activation of its receptor RXFP1 triggers multiple signaling pathways. Previously, we have shown that relaxin activates PPARγ transcriptional activity in a ligand-independent manner, but the mechanism for this effect was unknown. In this study, we examined the signaling pathways of downstream of RXFP1 leading to PPARγ activation. Using cells stably expressing RXFP1, we found that relaxin regulation of PPARγ activity requires accumulation of cAMP and subsequent activation of cAMP-dependent protein kinase (PKA). The activated PKA subsequently phosphorylated cAMP response element-binding protein (CREB) at Ser-133 to activate it directly, as well as indirectly through mitogen activated protein kinase p38 MAPK. Activated CREB was required for relaxin stimulation of PPARγ activity, while there was no evidence for a role of the nitric oxide or ERK MAPK pathways. Relaxin increased the mRNA and protein levels of the coactivator protein PGC1α, and this effect was dependent on PKA, and was completely abrogated by a dominant-negative form of CREB. This mechanism was confirmed in a hepatic stellate cell line stably that endogenously expresses RXFP1. Reduction of PGC1α levels using siRNA diminished the regulation of PPARγ by relaxin. These results suggest that relaxin activates the cAMP/PKA and p38 MAPK pathways to phosphorylate CREB, resulting in increased PGC1α levels. This provides a mechanism for the ligand-independent activation of PPARγ in response to relaxin.

Keywords: Peroxisome Proliferator-activated Receptor γ Coactivator 1-α (PGC-1a)(PPARGC1A); Peroxisome Proliferator-activated receptor (PPAR); Protein Kinase A (PKA); Relaxin; Relaxin Family Peptide Receptor 1 (RXFP1); cAMP Response Element-binding Protein (CREB); p38 MAPK.

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Figures

FIGURE 1.
FIGURE 1.
The cAMP/PKA pathway activates transcription through a PPRE reporter. A, HEK-RXFP1 cells were pretreated for 30 min in the presence of indicated concentration of the PKA inhibitor H89, then treated 30 min with 1 nm relaxin, and PKA activity was measured by phosphorylation of a PKA substrate. B, HEK-RXFP1 cells transfected with PPRE and Renilla luciferase reporters were pretreated 30 min in the absence and presence of H89 (20 μm), then treated with 10 μm forskolin or 1 nm relaxin for 24 h prior to dual luciferase assay. Data are presented as PPRE luciferase assay relative to control, mean ± S.E. *, p < .05; **, p < .01; ***, p < .001, n = 3. C, white bars: HEK-RXFP1 cells transfected with PPRE and Renilla luciferase reporters were treated for 24 h with 10 μm forskolin, 100 μm Sp-6-Bnz-cAMPS, 10 μm Sp-8CPT-cAMPS, 100 μm SNAP, or 500 μm GSNO, then subject to dual luciferase assay. Black bars: cells were pretreated for 30 min with 20 μm H89, 1 μg/ml PTX, 2 μm LY294002, 20 μm PD169316, 10 μm PD98059, or 100 μm L-NAME, then stimulated with 1 nm relaxin for 24 h. Data are presented as luciferase activity relative to control, mean ± S.E., n = 3. *, p < 0.05 compared with untreated control; †, p < 0.05 compared with relaxin alone. D, HEK-RXFP1 cells were cotransfected with plasmids encoding PKI or an inactive PKI mutant (PKImut) and PPRE and Renilla luciferase reporters, then treated with relaxin as described above, then subject to dual luciferase assay. ***, p < 0.001; n = 3. E, Western blot showing knockdown of PKA or p38 MAPK after treatment with siRNA. F, HEK-RXFP1 cells were cotransfected with PKA siRNA or nontargeting control siRNA and PPRE and Renilla luciferase reporters, then treated with relaxin as described above, then subject to dual luciferase assay. ***, p < 0.001, n = 3.
FIGURE 2.
FIGURE 2.
p38 MAPK is a downstream mediator of PKA-induced PPAR activity. A, HEK-RXFP1 cells transfected with PPRE and Renilla luciferase reporters were pretreated 30 min with the p38 MAPK inhibitor PD169316 (20 μm) or vehicle, then treated for 24 h with or without 1 nm relaxin then subject to PPRE luciferase assay. The data are expressed as the PPRE luciferase activity relative to untreated cells, mean ± S.E., n = 3. *, p < 0.05; **, p < 0.01. B, HEK-RXFP1 cells were treated with 1 nm relaxin for the indicated times, and lysates were analyzed by Western blot for total and phosphorylated p38 MAPK. C, cells were pretreated 30 min with 20 μm H89 or vehicle, then treated with 1 nm relaxin for 15 min, and lysates were analyzed by Western blot for total and phosphorylated p38 MAPK. D, HEK-RXFP1 cells were cotransfected with p38 MAPK siRNA or nontargeting control siRNA and PPRE and Renilla luciferase reporters, then treated with relaxin as described above, then subject to dual luciferase assay. *, p < .05; ***, p < .001, n = 3. E, cells were cotransfected with PKA siRNA or nontargeting control siRNA, then treated with relaxin for 15 min before Western blot analysis for phosphorylated and total p38 MAPK.
FIGURE 3.
FIGURE 3.
CREB mediates relaxin-induced PPAR activity. A, HEK-RXFP1 cells were transfected with CRE and Renilla luciferase reporters. The cells were treated for 24 h with or without 1 nm relaxin and subjected to dual luciferase assay. B, HEK-RXFP1 cells were transfected with the dominant- negative CREB construct (pCMV-KCREB), or empty vector (pCMV), and the PPRE and Renilla luciferase reporters. The cells were treated for 24 h with or without 1 nm relaxin and subjected to dual luciferase assay. The data are expressed as the luciferase activity relative to untreated cells, mean ± S.E., n = 3. ***, p < 0.001 compared with untreated control. C, HEK-RXFP1 cells were pretreated for 30 min with 20 μm H89, 20 μm PD169316 (PD16), or vehicle, then treated 30 min in the presence or absence of 1 nm relaxin. Lysates were analyzed by Western blotting for total and phosphorylated CREB. C, HEK-RXFP1 cells were transfected with PKA, p38 MAPK, or nontargeting siRNA, then were treated 30 min in the presence or absence of 1 nm relaxin. Lysates were analyzed by Western blotting for total and phosphorylated CREB.
FIGURE 4.
FIGURE 4.
PGC1α is the mechanism for activation of PPARγ activity by relaxin. A, HEK-RXFP1 cells were treated at the indicated times with 1 nm relaxin, and the mRNA for PGC1α was quantified by real time RT-PCR relative to that of TBP. Data are expressed as fold expression of PGC1α compared with untreated cells. B, HEK-RXFP1 cells were transfected with PGC1α-promoter and Renilla luciferase reporters, treated 24 h with 1 nm relaxin, then subject to dual-luciferase assay. C, HEK-RXFP1 cells were treated with 1 nm relaxin for the indicated times, and lysates were analyzed by Western blotting PGC1α and GAPDH as indicated. D, HEK-RXFP1 cells were pretreated 20 μm H89 or vehicle, then incubated for 2 h in the presence or absence of 1 nm relaxin. The mRNA for PGC1α was quantified by real time RT-PCR relative to that of TBP. Data are expressed as fold expression of PGC1α compared with untreated cells. E, HEK-RXFP1 cells were transfected with KCREB or control plasmid, then cells were treated with 1 nm relaxin for 4.5 h. Lysates were analyzed by Western blotting for PGC1α and GAPDH as indicated. F and G, HEK-RXFP1 cells were cotransfected with control or PGC1α siRNA, PPRE and Renilla reporter vectors. After 24 h, the levels of PGC1α were reduced as determined by Western blot (F). Cells were then treated with or without 1 nm relaxin for 24 h and subjected to dual luciferase assay (G). The data are expressed as the luciferase activity relative to untreated cells, mean ± S.E., n = 3. *, p < 0.05; **, p < 0.01.
FIGURE 5.
FIGURE 5.
Relaxin regulates PGC1α in LX2 cells endogenously expressing RXFP1. A, LX2 cells were treated with or without 1 nm relaxin in the presence of the phosphodiesterase inhibitors IBMX (0.5 mm) and imidazolidone (0.1 mm) for 30 min, then cAMP levels were determined. B, cells were pretreated with 20 μm H89 for 30 min, then treated 30 min with 1 nm relaxin. PKA activity was measured by phosphorylation of a PKA substrate. C, total RNA was extracted from THP1, HEK-RXFP1, HepG2, and LX2 cells, and subject to RT-PCR for detection of RXFP1 or GAPDH transcripts. D, LX2 cells were transfected with the PPRE and Renilla luciferase reporters, then treated with or without 1 nm relaxin for 24 h and subjected to dual luciferase assay. The data are expressed as the luciferase activity relative to untreated cells. E, cells were treated with 1 nm relaxin for the indicated times, then lysates were analyzed by Western blotting for total and phosphorylated p38 MAPK. F, LX2 cells were pretreated 30 min with 20 μm H89, 20 μm PD169316 (PD16), or vehicle, then treated with or without 1 nm relaxin for 30 min. Lysates were analyzed by Western blotting for total and phosphorylated CREB. G, LX2 cells were treated with 1 nm relaxin for 2 h, and the mRNA for PGC1α was quantified by real time RT-PCR relative to that of TBP. Data are expressed as fold expression of PGC1α compared with untreated cells. Mean ± S.E., n = 3. **, p < .01; ***, p < .001.
FIGURE 6.
FIGURE 6.
Schematic representation of relaxin signaling pathways leading to activation of PPARγ. Relaxin binding to RXFP1 results in a rapid activation of adenylyl cyclase (AC), followed by a delayed phase that involves PI3K. This results in elevated cAMP levels and activation of PKA. The activated PKA phosphorylates CREB, through p38 MAPK-dependent and -independent mechanisms. The phosphorylated CREB then increases PGC1α gene expression. The elevated level of PGC1α then increases the ligand-independent transcriptional activity of PPARγ.
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