Sildenafil Inhibits Hypoxia-Induced Transient Receptor Potential Canonical Protein Expression in Pulmonary Arterial Smooth Muscle via cGMP-PKG-PPARγ Axis

et al. 2014

J Mol Med

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In this study, we investigated the role of peroxisome proliferator-activated receptor γ (PPARγ) on store-operated calcium entry (SOCE) and expression of the main store-operated calcium channels (SOCCs) components, canonical transient receptor potential (TRPC) in chronic hypoxia (CH) and monocrotaline (MCT)-induced PH rat models. siRNA knockdown and adenoviral overexpression strategies were constructed for both loss-of-function and gain-of-function experiments. PPARγ agonist rosiglitazone attenuates the pathogenesis of both CHPH and MCT-PH, suppresses Hif-1α, TRPC1, TRPC6 expression in the distal pulmonary artery (PA) and SOCE in freshly isolated rat distal pulmonary arterial smooth muscle cells (PASMCs). By comprehensive use of knockdown and overexpression studies, bioinformatically analysis of the TRPC gene promoter and luciferase reporter assay, we demonstrated that PPARγ exerts roles of anti-proliferation, anti-migration, and pro-apoptosis in PASMCs, likely by inhibiting the elevated SOCE and TRPC expression. These effects were inhibited under the conditions of hypoxia or Hif-1α accumulation. We also found that under hypoxia, accumulated Hif-1α protein acts as upstream of suppressed PPARγ level, however, targeted PPARγ rescue acts negative feedback on suppressing Hif-1α level and Hif-1α mediated signaling pathway. PPARγ inhibits PH by targeting SOCE and TRPC via inhibiting Hif-1α expression and signaling transduction.

“…Similar to the previous finding [22], our results showed similar pattern of hypoxia increased TRPC1 and TRPC6, decreased PPARγ mRNA level (Fig. 4A and 4B), as well as protein level (Fig.…”

Section: Resultssupporting

confidence: 92%

Peroxisome proliferator-activated receptor γ inhibits pulmonary hypertension targeting store-operated calcium entry

Wang

et al. 2014

J Mol Med

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“…These beneficial roles are likely mediated through inhibiting SOCE and the expression of SOCCs channel protein TRPCs, which then lead to suppressed proliferation and migration of PASMCs (11,13). Previous studies have reported that, in cancer cell lines, caveolin-1 acts as a direct transcription target of the main hypoxic regulator, hypoxia-inducible factors 1 and 2, and facilitates the proproliferative consequences during the process of tumorigenesis (47,48).…”

Section: Discussionmentioning

confidence: 99%

“…Peroxisome proliferator-activated receptor (PPAR) g belongs to a kind of ligand-activated nuclear hormone receptor superfamily, which is ubiquitously expressed in pulmonary vascular endothelial and smooth muscle cells, and acts as a transcription factor to modulate the transcription of a number of genes (8). Previous studies reported that PPARg is down-regulated in the lungs (9,10) and distal PAs (11) of experimental PH models, whereas restoration of PPARg by specific agonists can markedly attenuate the PH pathogenesis by normalizing the elevated right ventricle systolic pressure and distal PA remodeling (12)(13)(14). Moreover, we further demonstrated the molecular mechanisms that PPARg inhibits PA remodeling and PASMC proliferation, mainly by targeting SOCE and TRPC proteins (11,13).…”

mentioning

confidence: 99%

Peroxisome Proliferator–Activated Receptor γ–Mediated Inhibition on Hypoxia-Triggered Store-Operated Calcium Entry. A Caveolin-1–Dependent Mechanism

Am J Respir Cell Mol Biol

Qian

et al. 2015

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Our previous publication demonstrated that peroxisome proliferator-activated receptor g (PPARg) inhibits the pathogenesis of chronic hypoxia (CH)-induced pulmonary hypertension by targeting store-operated calcium entry (SOCE) in rat distal pulmonary arterial smooth muscle cells (PASMCs). In this study, we aim to determine the role of a membrane scaffolding protein, caveolin-1, during the suppressive process of PPARg on SOCE. Adult (6-8 weeks) male Wistar rats (200-250 g) were exposed to CH (10% O 2 ) for 21 days to establish CH-induced pulmonary hypertension. Primary cultured rat distal PASMCs were applied for the molecular biological experiments. First, hypoxic exposure led to 2.5-fold and 1-fold increases of caveolin-1 protein expression in the distal pulmonary arteries and PASMCs, respectively. Second, effective knockdown of caveolin-1 significantly reduced hypoxia-induced SOCE for 58.2% and 41.5%, measured by Mn 21 quenching and extracellular Ca 21 restoration experiments, respectively. These results suggested that caveolin-1 acts as a crucial regulator of SOCE, and hypoxia-up-regulated caveolin-1 largely accounts for hypoxia-elevated SOCE in PASMCs. Then, by using a high-potency PPARg agonist, GW1929, we detected that PPARg activation inhibited SOCE and caveolin-1 protein for 62.5% and 59.8% under hypoxia, respectively, suggesting that caveolin-1 also acts as a key target during the suppressive process of PPARg on SOCE in PASMCs. Moreover, by using effective small interfering RNAs against PPARg and caveolin-1, and PPARg antagonist, T0070907, we observed that PPARg plays an inhibitory role on caveolin-1 protein by promoting its lysosomal degradation, without affecting the messenger RNA level. PPARg inhibits SOCE, at least partially, by suppressing cellular caveolin-1 protein in PASMCs.Keywords: pulmonary hypertension; peroxisome proliferator-activated receptor g; caveolin-1; store-operated calcium entry; pulmonary arterial smooth muscle cells Pulmonary hypertension (PH) is a severe pulmonary vascular disease characterized by sustained increase in the pulmonary arterial pressure and excessive thickening and remodeling of the distal small pulmonary arteries (PAs). These functional and structural changes then lead to right ventricular hypertrophy and, eventually, heart failure. Nowadays, it is well accepted that the dysregulation of the proliferation and migration of the PA smooth muscle cells (PASMCs) are the main reasons contributing to the abnormally excessive thickening and remodeling of the distal PAs during PH pathogenesis (1-3).Previous studies have described that the increase of the intracellular free calcium concentration ([Ca 21 ] i ) acts a major factor

“…Measurement of PASMC proliferation. The proliferation of PASMC was measured with the Cell Proliferation Biotrak ELISA Kit (GE Healthcare) based on a BrdU incorporation assay, as previously described (41). Briefly, PASMC were seeded in 96-well plates in SMBM at a density of 5 ϫ 10 3 cells/well.…”

Section: Methodsmentioning

confidence: 99%

Noggin inhibits hypoxia-induced proliferation by targeting store-operated calcium entry and transient receptor potential cation channels

American Journal of Physiology-Cell Physiology

Jia

et al. 2015

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Abnormally elevated bone morphogenetic protein 4 (BMP4) expression and mediated signaling play a critical role in the pathogenesis of chronic hypoxia-induced pulmonary hypertension (CHPH). In this study, we investigated the expression level and functional significance of four reported naturally occurring BMP4 antagonists, noggin, follistatin, gremlin1, and matrix gla protein (MGP), in the lung and distal pulmonary arterial smooth muscle cell (PASMC). A 21-day chronic hypoxic (10% O2) exposure rat model was utilized, which has been previously shown to successfully establish experimental CHPH. Among the four antagonists, noggin, but not the other three, was selectively downregulated by hypoxic exposure in both the lung tissue and PASMC, in correlation with markedly elevated BMP4 expression, suggesting that the loss of noggin might account for the hypoxia-triggered BMP4 signaling transduction. Then, by using treatment of extrogenous recombinant noggin protein, we further found that noggin significantly normalized 1) BMP4-induced phosphorylation of cellular p38 and ERK1/2; 2) BMP4-induced phosphorylation of cellular JAK2 and STAT3; 3) hypoxia-induced PASMC proliferation; 4) hypoxia-induced store-operated calcium entry (SOCE), and 5) hypoxia-increased expression of transient receptor potential cation channels (TRPC1 and TRPC6) in PASMC. In combination, these data strongly indicated that the hypoxia-suppressed noggin accounts, at least partially, for hypoxia-induced excessive PASMC proliferation, while restoration of noggin may be an effective way to inhibit cell proliferation by suppressing SOCE and TRPC expression.