Role of phosphatase and tensin homolog in hypoxic pulmonary vasoconstriction

Abstract

Aims: Hypoxic pulmonary vasoconstriction (HPV) redistributes blood flow from poorly ventilated to better aerated areas in the lung, thereby optimizing ventilation-perfusion ratio (V/Q). Pulmonary artery smooth muscle cell (PASMC) contraction in response to hypoxia is triggered by Ca2+ influx via transient receptor potential canonical 6 (TRPC6) cation channels that have translocated to caveolae in the plasma membrane. Since phosphatase and tensin homolog (PTEN) was suggested to regulate TRPC6 in endothelial cells, we aimed to define its role in the hypoxic response of PASMCs and as a putative mediator of HPV.

Methods and results: In isolated perfused mouse lungs, smooth muscle specific PTEN deficiency attenuated pulmonary vasoconstriction in response to hypoxia but not to angiotensin II (Ang II). Analogously, siRNA-mediated knock down of PTEN in human PASMC inhibited the hypoxia-induced increase in cytosolic Ca2+ concentration ([Ca2+]i). Co-immunoprecipitation and proximity ligation assays revealed increased interaction of PTEN with TRPC6 in human PASMC and murine lungs in response to hypoxia. In hypoxic PASMC, both PTEN and TRPC6 translocated to caveolae, and this response was blocked by pharmacological inhibition of Rho-associated protein kinase (ROCK) which in parallel prevented PTEN-TRPC6 interaction, hypoxia-induced [Ca2+]i increase, and HPV in PASMC and murine lungs, respectively.

Conclusion: Our data indicate a novel interplay between ROCK and [Ca2+]i signalling in HPV via PTEN, in that ROCK mediates interaction of PTEN and TRPC6 which then conjointly translocate to caveolae allowing for Ca2+ influx into and subsequent contraction of PASMC.

Keywords: Hypoxia; Phosphatase and tensin homolog (PTEN); Pulmonary artery smooth muscle cells (PASMC); Rho kinase (ROCK); Transient receptor potential canonical 6 (TRPC6).

Figure 1

HPV requires SMC PTEN. (A) Representative immunoblots and quantitative data (n = 4–8) show that SMC PTEN KO reduced PTEN expression in SMC-rich intestines, but not in the heart. (B) Quantitative data shows that baseline pressures are similar in IPL of KO animals compared with WT controls (n = 10 each). (C) Representative tracings of PAP in IPL experiments show attenuated vasoconstriction in response to hypoxia (1% O₂) in lungs of SMC PTEN KO mice compared with WT. (D) Group data show attenuated PAP increase (ΔPAP) 5 min after start of hypoxia but not in response to Ang II (1 µg bolus for 5 min) in SMC PTEN KO mice; (n = 5–6, respectively) compared with WT mice (n = 7 both). (E) VO-OHpic (10 µMol/L) did not affect the PAP response to hypoxia in isolated lungs of C57Bl/6 mice (n = 6 each). (F) Lungs were collected and snap-frozen after 30 min of perfusion in the presence or absence of VO-OHpic (10 µMol/L). Representative western blot of lung lysates and quantitative data show levels of p-Akt normalized to total Akt in the presence or absence of VO-OHpic (10 µMol/L) (n = 5). Group data are means ± SEM, *P < 0.05 vs. WT (A, D) or control (Ctrl; F).

Figure 2

PASMC [Ca²⁺]_i increase in response to hypoxia requires PTEN. (A) Representative immunoblot and quantitative data from four independent isolations show effective knockdown of PTEN by PTEN-specific siRNA (siPTEN) as compared with scrambled siRNA (siCtrl). Each experiment has been normalized to its corresponding control group from the same gel. (B) Representative tracings of the 340/380 nm fura-2 fluorescence ratio (normalized to baseline) in PASMC show a reduced [Ca²⁺]_i response to hypoxia (1% O₂) in PASMC transfected with siPTEN as compared with siCtrl. Group data show hypoxia-induced [Ca²⁺]_i increase in siPTEN and siCtrl PASMC (C) or murine PASMC isolated from SMC PTEN KO or WT mice (D) (data from three to five independent experiments, respectively).Group data are means ± SEM, *P < 0.05 vs. siCtrl (A,C) or WT (D).

Figure 3

Hypoxia increases the interaction of PTEN with TRPC6 in a ROCK-dependent manner. (A) PASMCs were exposed to either normoxia (n), hypoxia (h; 1% O₂), or hypoxia in the presence of Y27632 (Y; 5µMol/L), or to S1P (S; 10 µMol/L) for 5 min. Representative immunoblots show TRPC6 and PTEN expression in PASMC for whole cell lysate (input) and after immunoprecipitation for PTEN (Co-IP). Group data from n = 5–7 independent replicates showing TRPC6-over-PTEN ratio normalized to the normoxic control group from the same gel, demonstrate increased interaction of PTEN with TRPC6 in response to hypoxia and S1P, respectively. (B) Isolated lung were ventilated with normoxic or hypoxic (1% O₂) gas for 3 min, and tissue was snap-frozen and lysed. Representative immunoblots show TRPC6 and PTEN expression after immunoprecipitation for PTEN; group data from n = 6 replicates show quantification of TRPC6-over-PTEN ratio normalized to the normoxic control group from the same gel. (C) Representative images show PLA for the interaction between PTEN and TRPC6 in PASMC following exposure to either normoxia, hypoxia, or S1P(10 µMol/L) for 5 min, in the presence or absence of Y27632 (5 µMol/L), or LPA (3 µMol/L) for 15 min. Red puncta indicate sites of interaction between PTEN and TRPC6, nuclei are counterstained in blue with DAPI. (D) Group data show quantification of the PLA from eight cells from three independent experiments each. Group data are means ± SEM, *P < 0.05 vs. normoxia, #P < 0.05 vs. hypoxia and $P < 0.05 vs. S1P.

Figure 4

Hypoxia causes ROCK-dependent translocation of TRPC6 and PTEN to caveolae. Caveolar fractions were isolated from PASMC (input: whole cell lysate) by sucrose gradient centrifugation, and identified by the presence of the marker protein caveolin-1. Representative immunoblots show (A) absence of PTEN and TRPC6 from caveolae of normoxic PASMC, but (B) recruitment to caveolar fractions within 15 min of hypoxia (1% O₂), that was (C) blocked by Y27632 (5 µMol/L). (D) Group data from n = 3–5 independent experiments each show caveolar recruitment of TRPC6 and PTEN, expressed as fraction of protein detected in caveolar fractions. Group data are means ± SEM, *P < 0.05 vs. normoxia.

Figure 5

ROCK inhibition prevents [Ca²⁺]_i signalling and HPV response. (A) Representative tracings of the 340/380 nm fura-2 fluorescence ratio (normalized to baseline) in PASMC show reduced cytosolic [Ca²⁺]_i response to hypoxia (1% O₂) in PASMC in the presence of Y27632 (5 µMol/L). (B) Group data show effect of Y27632 on the hypoxia-induced [Ca²⁺]_i increase (data from 3 independent experiments each). (C) Group data show inhibition of the PAP increase (ΔPAP) by Y27632 in IPL 3 min after start of hypoxia (data from four independent experiments each). Group data are means ± SEM, *P < 0.05 vs. hypoxia (B) or control (Ctrl; C).

Figure 6

Schematic outline of the proposed role of PTEN in HPV: Hypoxia, either directly and/or via formation of S1P, activates ROCKin PASMC, which mediates the interaction between PTEN and TRPC6 and their translocation to caveolae, where TRPC6 facilitates Ca²⁺ entry and subsequent PASMC contraction.