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. 2018 Apr 1;314(4):C428-C438.
doi: 10.1152/ajpcell.00143.2017. Epub 2018 Jan 3.

Pharmacological activation of PPARγ inhibits hypoxia-induced proliferation through a caveolin-1-targeted and -dependent mechanism in PASMCs

Kai Yang  1 , Mingming Zhao  2 , Junyi Huang  1 , Chenting Zhang  1 , Qiuyu Zheng  1 , Yuqin Chen  1 , Haiyang Jiang  3 , Wenju Lu  1 , Jian Wang  1   4
Affiliations

Pharmacological activation of PPARγ inhibits hypoxia-induced proliferation through a caveolin-1-targeted and -dependent mechanism in PASMCs

Kai Yang et al. Am J Physiol Cell Physiol. .

Abstract

Previously, we and others have demonstrated that activation of peroxisome proliferator-activated receptor γ (PPARγ) by specific pharmacological agonists inhibits the pathogenesis of chronic hypoxia-induced pulmonary hypertension (CHPH) by suppressing the proliferation and migration in distal pulmonary arterial smooth muscle cells (PASMCs). Moreover, these beneficial effects of PPARγ are mediated by targeting the intracellular calcium homeostasis and store-operated calcium channel (SOCC) proteins, including the main caveolae component caveolin-1. However, other than the caveolin-1 targeted mechanism, in this study, we further uncovered a caveolin-1 dependent mechanism within the activation of PPARγ by the specific agonist GW1929. First, effective knockdown of caveolin-1 by small-interfering RNA (siRNA) markedly abolished the upregulation of GW1929 on PPARγ expression at both mRNA and protein levels; Then, in HEK293T, which has previously been reported with low endogenous caveolin-1 expression, exogenous expression of caveolin-1 significantly enhanced the upregulation of GW1929 on PPARγ expression compared with nontransfection control. In addition, inhibition of PPARγ by either siRNA or pharmacological inhibitor T0070907 led to increased phosphorylation of cellular mitogen-activated protein kinases ERK1/2 and p38. In parallel, GW1929 dramatically decreased the expression of the proliferative regulators (cyclin D1 and PCNA), whereas it increased the apoptotic factors (p21, p53, and mdm2) in hypoxic PASMCs. Furthermore, these effects of GW1929 could be partially reversed by recovery of the drug treatment. In combination, PPARγ activation by GW1929 reversibly drove the cell toward an antiproliferative and proapoptotic phenotype in a caveolin-1-dependent and -targeted mechanism.

Keywords: caveolin-1; extracellular signal-regulated kinase 1/2; p38; peroxisome proliferator-activated receptor-γ; pulmonary arterial smooth muscle cells.

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Figures

Fig. 1.
Fig. 1.
PPARγ agonist GW1929 inhibits prolonged hypoxia (4% O2)-induced upregulation of caveolin-1 expression in a reversible manner in cultured rat distal pulmonary arterial smooth muscle cells (PASMCs). A and B: Western blot (A) and bar graph (B) showing the protein expression level of caveolin-1 upon GW1929 (GW; 10 μM) treatment in rat distal PASMCs under normoxic and hypoxic conditions. C: treatment strategy of straight GW1929 and of GW1929 plus drug withdrawal. D and E: Western blot (D) and bar graph (E) showing the protein expression level of caveolin-1 under GW1929 treatment (72 h) and GW1929 treatment (48 h) plus drug withdrawal (24 h) in hypoxic PASMCs. GAPDH serves as housekeeping protein. Bar graph represents means ± SE; n = 4 in each group. *P < 0.05 vs. control (B, 48 or 72 h) or control (E); #P < 0.05 vs. hypoxia (48 or 72 h) or GW 72 h (E).
Fig. 2.
Fig. 2.
GW1929 activates PPARγ via a caveolin-1-dependent mechanism. A–D: knockdown of caveolin-1 (siCv) eliminated GW1929-upregulated PPARγ expression in PASMCs. Western blot (A) shows the siRNA knockdown efficiency of caveolin-1 as relevant to β-tubulin. Bar graph (B and D) and Western blot (C) show the mRNA (normalized to 18S) and protein expression (normalized to GAPDH) levels of PPARγ under treatment of caveolin-1 knockdown and GW1929 in rat distal PASMCs. E–G: overexpression of caveolin-1 in HEK293T enhanced the GW1929-mediated upregulation of PPARγ and downregulation of caveolin-1. Western blot (E) and bar graph (F and G) represent the protein expression levels of PPARγ and caveolin-1 under treatment of caveolin-1 overexpression and GW1929 in HEK293T. Bar graph represents means ± SE; n = 6 in each group. *P < 0.05 vs. nontargeted siRNA (siNT; B and D) or Ad-GFP control (F and G); #P < 0.05 vs. siNT + GW (B and D) or Ad-Cav-1 control (F and G).
Fig. 3.
Fig. 3.
Inhibition of PPARγ and caveolin-1 altered the basal phosphorylation of p38 and ERK1/2 in PASMCs. A–F: Western blots (A and D) showing the protein expression levels of p-p38, t-p38, p-ERK1/2, and t-ERK1/2 upon treatment of either PPARγ knockdown (siPP) or PPARγ antagonist T0070907 (T007; 10 μM) for 48 h and 72 h in rat distal PASMCs. Bar graphs (B, C, E, and F) represent the phosphorylation rates of p-p38 (B and E) and p-ERK1/2 (C and F) as normalized to t-p38 and t-ERK1/2. G–I: Western blots (G) showing the protein expression levels of p-p38, t-p38, p-ERK1/2, and t-ERK1/2 upon treatment of either nontargeted siRNA (siNT) or caveolin-1 (siCv) knockdown for 48 h in rat distal PASMCs. Bar graphs represent the phosphorylation rates of p-p38 (H) and p-ERK1/2 (I) as normalized to t-p38 and t-ERK1/2, respectively. Bar graph represents means ± SE; n = 4–6 in each group. *P < 0.05 vs. siNT or Cont (48 h); #P < 0.05 vs. siNT or Cont (72 h); ns, no significant difference.
Fig. 4.
Fig. 4.
Neither PDGF-BB- nor BMP4-induced phosphorylation of p38 and ERK1/2 was affected by GW1929 treatment in hypoxic PASMCs. Western blots (A and C) show the protein expression levels of p-p38, t-p38, p-ERK1/2, and t-ERK1/2 in cultured rat distal PASMCs upon treatment of either PDGF-BB (50 ng/ml; A) or BMP4 (50 ng/ml; C) for different time courses. Bar graphs (B and D) represent the phosphorylation rates of p-ERK1/2 as normalized to t-p38 and t-ERK1/2, respectively. Bar graph represents means ± SE; n = 3 in each group.
Fig. 5.
Fig. 5.
Knockdown of caveolin-1 failed to mediate the functional consequences of pharmacological PPARγ activation on cell proliferation and apoptosis in hypoxic PASMCs. A: cell apoptosis assay showing the effects of hypoxia (4% O2, 72 h), GW1929 (10 μM, 48 h), and/or siRNA knockdown of caveolin-1 (25 nM, 48 h) on cell apoptosis of PASMCs. B: cell proliferation assay showing the effects of hypoxia (4% O2, 72 h), GW1929 (10 μM, 48 h), and/or siRNA knockdown of caveolin-1 (25 nM, 48 h) on cell proliferation property of PASMCs. Bar graph represents means ± SE; n = 6 in each group. *P < 0.05 vs. siNT; #P < 0.05 vs. hypoxia + siNT.
Fig. 6.
Fig. 6.
GW1929 activation of PPARγ reversibly inhibited cellular proliferative markers and increased apoptotic markers in hypoxic PASMCs. A: Western blots showing the protein expression levels of cyclin D1, PCNA, p53, mdm2, p21, and β-tubulin under treatment of GW1929 (10 μM, 72-h) and GW1929 treatment (48 h) plus drug withdrawal (24 h) in hypoxic PASMCs. B–F: bar graphs representing the expression levels of cyclin D1 (B), PCNA (C), p21 (D), p53 (E), and mdm2 (F) as normalized to β-tubulin. Bar graph represents means ± SE; n = 4 in each group. *P < 0.05 vs. control; #P < 0.05 vs. GW(72) -treated groups (72 h).
Fig. 7.
Fig. 7.
Schematic working model. Pharmacological activation of PPARγ leads to an antiproliferative and proapoptotic phenotype, which relies on a caveolin-1-targeted and -dependent mechanism. Activation of PPARγ contributes to a dominant beneficial consequence in PASMCs and leads to a gateway for the treatment of pulmonary hypertension.
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