IP receptor-dependent activation of PPARgamma by stable prostacyclin analogues

Abstract

Stable prostacyclin analogues can signal through cell surface IP receptors or by ligand binding to nuclear peroxisome proliferator-activated receptors (PPARs). So far these agents have been reported to activate PPARalpha and PPARdelta but not PPARgamma. Given PPARgamma agonists and prostacyclin analogues both inhibit cell proliferation, we postulated that the IP receptor might elicit PPARgamma activation. Using a dual luciferase reporter gene assay in HEK-293 cells stably expressing the IP receptor or empty vector, we found that prostacyclin analogues only activated PPARgamma in the presence of the IP receptor. Moreover, the novel IP receptor antagonist, RO1138452, but not inhibitors of the cyclic AMP pathway, prevented activation. Likewise, the anti-proliferative effects of treprostinil observed in IP receptor expressing cells, were partially inhibited by the PPARgamma antagonist, GW9662. We conclude that PPARgamma is activated through the IP receptor via a cyclic AMP-independent mechanism and contributes to the anti-growth effects of prostacyclin analogues.

Supplementary FigS1

Cicaprost activates PPARγ in the presence of the IP receptor. HEK-293-IP cells transiently transfected with PPARγ constructs, were stimulated with 10% FBS ± cicaprost (CICA; 1 μM), the prostacyclin antagonist (IPRA; 1 μM) or a combination as shown (cells were pre-treated with IPRA for 1 hr). Luciferase activity was determined 24 hr after stimulation and normalised to Renilla activity. Results are expressed as mean fold increase in luciferase activity relative to untreated control ± s.e.m. (n=12-16, 3-4 separate transfections). ∗=P<0.05 when compared to control or as shown.

Supplementary FigS1

Treprostinil fails to elevate cAMP or inhibit growth in HEK-29 cells not stably expressing the IP receptor Cyclic AMP measurements in HEK-293-Zeo cells stimulated for 30 mins with 10% FBS ± either treprostinil (TREP; 100 nM) or IP receptor antagonist (IPRA; 1 μM) or a combination (A). Cells were pretreated with IPRA for 1 hr prior to stimulation with TREP. Cyclic AMP was measured using an enzyme immunolinked assay. Results expressed as mean pmol of cAMP per mg of total protein ± S.E.M. (measurements in duplicate of 3-4 experiments). Effect TREP (1 μM) and carbacyclin (CARBA; 1 μM) on cell proliferation in control HEK-293 cells (B). Growth arrested cells were stimulated with 10% FBS with or without DMSO (0.01%) or PGI2 analogue. Cells were counted 48 hrs later. Data expressed as % cell proliferation relative to response mediated by 10% FBS alone and shown as mean ± S.E.M. (n=12-24).

Fig. 1

Activation of PPARγ by rosiglitazone and stable prostacyclin analogues. HEK-293 cells stably expressing the IP receptor (HEK-293-IP) were transiently transfected with pGAL5TKpGL3, pMLUC-2 and either GAL4-pcDNA3 (control—white bars) or GAL4-hPPARγ-pcDNA3 (reporter—black bars). After 48 h, cells were stimulated with 10% FBS in the absence (Control) or presence of DMSO (0.01%; A), rosiglitazone (ROSI; A), treprostinil (TREP; B and C) or carbacyclin (CARBA; C). In (A and C), cells were pre-treated with the PPARγ antagonist, GW9662 for 1 h prior to the addition of agonists. Unless stated, all drug concentrations were 1 μM. Luciferase activity was determined 24 h after FBS stimulation and normalised to renilla activity. Results are expressed as mean fold increase in luciferase activity above untreated control ± s.e.m. (n = 12, 3 separate transfections). ^∗∗∗P < 0.001 when compared to control or as shown. ^#P<0.05 when compared with TREP alone.

Fig. 2

IP receptor-dependent activation of PPARγ. (A) Cyclic AMP levels measured in HEK-293-IP cells stimulated for 30 min with 10% FBS ± either TREP (100 nM) or the IP receptor antagonist, R01138452 (IPRA; 1 μM) or a combination. Data are expressed as mean pmol of cyclic AMP per mg of total protein ± s.e.m. (in duplicate of 3–4 experiments). Luciferase activity was measured in HEK-293-IP (B and C) and HEK-293-Zeo (D) cells as described in Fig. 1. Cells were stimulated with 10% FBS alone (Control) or in the presence of either treprostinil (TREP), carbacyclin (CARBA), IPRA, rosiglitazone (ROSI) or a combination as shown. Unless otherwise stated, all drug concentrations were at 1 μM. Results are expressed as mean fold increase in luciferase activity ± s.e.m. (n = 12; 3 separate transfections). ^∗∗∗P < 0.001 when compared to control or as shown and ^∗P < 0.05 when compared to ROSI alone.

Fig. 3

IP receptor activation of PPARγ is independent of cyclic AMP and PKA. HEK-293-IP (A) and HEK-293-Zeo (B) cells were transfected with constructs as described in Fig. 1. After 48 h, cells were stimulated with 10% FBS alone (control) or in the presence of forskolin (10 μM), treprostinil (TREP; 1 μM), 2′5′-dideoxyadenosine (DDA; 100 μM), the PKA antagonist, Rp-cAMPS (100 μM) or in combination as shown. Results are expressed as mean fold increase in luciferase activity from untreated control ± s.e.m. (n = 12, 3 separate transfections). Cyclic AMP was measured in HEK-293-IP (C) or HEK-293-Zeo (D) cells stimulated with 10% FBS ± either forskolin (10 μM), DDA (100 μM) or TREP (100 nM) or a combination as shown. Results are expressed as mean pmol of cAMP per mg of total protein ± s.e.m. (measurements in duplicate of three experiments). ^∗∗∗P < 0.001 when compared to control or as shown.

Fig. 4

PPARγ mediates in part the antiproliferative effects of treprostinil. Effect of treprostinil on FBS-induced proliferation in HEK-293-IP cells. Growth-arrested cells were stimulated with MEM + 10% FBS in the absence and presence of GW9662 (1 μM) treprostinil (TREP) or a combination. Cells were counted 48 hr later and data expressed as % cell proliferation relative to response mediated by 10% FBS alone and shown as mean ± s.e.m. (n = 6). ^∗P < 0.05.