Hypoxia modulates the expression of leucine zipper-positive MYPT1 and its interaction with protein kinase G and Rho kinases in pulmonary arterial smooth muscle cells

Abstract

We have shown previously that acute hypoxia downregulates protein kinase G (PKG) expression and activity in ovine fetal pulmonary vessels and pulmonary arterial smooth muscle cells (SMC). Here, we report that acute hypoxia also reduces the expression of leucinezipper-positive MYPT1 (LZ(+)MYPT1), a subunit of myosin light chain (MLC) phosphatase, in ovine fetal pulmonary arterial SMC. We found that in hypoxia, there is greater interaction between LZ(+) MYPT1 and RhoA and Rho kinase 1 (ROCK1)/Rho kinase 2 (ROCK2) and decreased interaction between LZ(+) MYPT1 and PKG, resulting in increased MLC(20) phosphorylation, a higher pMLC(20)/MLC(20) ratio and SMC contraction. In normoxic SMC PKG overexpression, LZ(+) MYPT1 expression is upregulated while PKG knockdown had an opposite effect. LZ(+) MYPT1 overexpression enhanced the interaction between PKG and LZ(+) MYPT1. Overexpression of a mutant LZ(-) MYPT1 isoform in SMC mimicked the effects of acute hypoxia and decreased pMLC(20)/MLC(20) ratio. Collectively, our data suggest that hypoxia downregulates LZ(+) MYPT1 expression by suppressing PKG levels, reduces the interaction of LZ(+) MYPT1 with PKG and promotes LZ(+) MYPT1 interaction with RhoA or ROCK1/ROCK2, thereby promoting pulmonary arterial SMC contraction.

Keywords: cGMP; hypoxia-induced pulmonary hypertension; pulmonary vasoconstriction; signal transduction.

Figure 1

Effect of hypoxia on protein kinase G (PKG) and LZ⁺MYPT1 expression.Fetal pulmonary arterial smooth muscle cells were exposed to acute hypoxia (4 h) and cell lysates were prepared and probed with anti-PKG1á, anti-LZ⁺MYPT1 and â-actin antibodies. (a and c) Western blot analysis of PKG and LZ⁺MYPT1, respectively.(b and d) Quantification of PKG and LZ⁺MYPT1 expression. Bands were quantified and normalized to that of â-actin bands. The values are shown as percent of normoxia control after normalizing the data to respective â-actin values. Data represent means±SE, from at leastthree3 independent experiments. *P<0.05 compared with normoxia.

Figure 2

The effect of protein kinase G (PKG) knockdown on LZ+MYPT1 expression. Fetal pulmonary arterial smooth muscle cellsFPASMC were transfected with siRNA specific for PKG or non-silencings iRNA, as a negative control. Cell lysates were isolated and probed with antibodies specific for PKG, LZ+MYPT1, and ↓-actin. (a) Representative Western blot analysis of PKG expression. (b) Quantification of PKG expression. Bands were quantified and normalized to that of ↓-actin bands. (c and d) Analysis of LZ+MYPT1 expression in PKG-–siRNA siRNA-transfected cells. Samples as in panel A, were probed with antibodies specific for LZ+MYPT1 and ↓-actin. (d) Densitometry quantification of LZ+MYPT1 expression. Data represent means±SE from at least three to six3-6 independent experiments. *P<0.05 compared with the respective control (un-transfected cells).

Figure 3

Overexpression of protein kinase G (PKG) inhibits LZ+MYPT1 down-regulation by hypoxia. Fetal pulmonary arterial smooth muscle cells FPASMC were transfected with a plasmid encoding a full-length PKG1α tagged with green fluorescent protein (GFP). PKG-– GFP GFP-expressing cells were exposed to acute hypoxia (4 h) and then expression levels of PKG (a) and LZ+MYPT1 (c) were analyzed. Panels b and d are the quantified levels of PKG and LZ+MYPT1 expression analyzed by immunoblot analysis. Data represent means + SE (n=3). *P<0.05 compared with respective control (normoxia).

Figure 4

Hypoxia decreases LZ+MYPT1 expression via reactive oxygeb species-dependent protein kinase G (PKG) reduction. Cells were exposed to acute hypoxia (4 h) in the presence or absence of trolox (100 ìM) or 1 mM N-acetyl cysteine. (a, c and e) Representative immunoblots showing the levels of PKG, LZ+MYPT1 and pMYPT1 (Ser⁶⁹⁵), respectively. (b, d, f and g) Quantitative analysis of PKG (B) LZ+MYPT1 (D), pMYPT1 (Ser⁶⁹⁵, F) and MYPT1/LZ+MYPT1 (g). The values are represented as percent of control (normoxia) after normalizing the data to the respective â-actin. Data represent means+SE from at least three independent experiments. *P<0.05 compared with the respective control (normoxia) groups.

Figure 5

Hypoxia decreases LZ⁺MYPT1 expression via reactive oxygen species-dependent protein kinase G (PKG) reduction. Cells were transfected with siRNA specific for PKG or nonsilencings iRNA and then exposed to acute hypoxia (4 h) in the presence or absence of trolox (100 ìM) or 1 mM NAC. (a, c and e) Representative immunoblots showing the levels of PKG, LZ+MYPT1 and pMYPT1 (Ser⁶⁹⁵), respectively. (b, d, f and g) Quantitative analysis of PKG (b) LZ+MYPT1 (D), pMYPT1 (Ser⁶⁹⁵, F) and pMYPT1/LZ+MYPT1 (g). The values represented as percent of control (normoxia) after normalizing the data to respective â-actin. Data represent means + SE from at least three independent experiments. *P<0.05 compared with the respective control (normoxia) groups.

Figure 6

Deletion of the LZ motif in MYPT1 mimics hypoxia and decreases protein kinase G-mediated MYPT1 and MLC phosphatase phosphorylation. Fetal pulmonary arterial smooth muscle cells were transfected with MYPT1 expression vector without leucine zipper (^-LZ) or with Leucine zipper (⁺LZ). Empty vector was used for mock transfections and untransfected cells as control. Cell lysates were immunoprobed with anti-LZ⁺MYPT1, phospho (Ser⁶⁹⁵) specific-MYPT1, native MYPT1, MLC₂₀, pMLC₂₀ and â-actin antibodies. (a) Representative Western blot probed with antibodies as indicated is shown. (b) Quantification of Western blot data for LZ+MYPT1 relative to control (untransfected cells). (c) Quantitative analysis of pMYPT1 (Ser⁶⁹⁵)/MYPT1 levels. values are represented as percent of control ratios between pMYPT1 (Ser⁶⁹⁵) and total MYPT1 after normalizing to their respective â-actin. (d) Normalized ratios of pMLC₂₀/MLC₂₀ as percent of control (untransfected cells) after normalizing the data to respective â-actin. Data represent means + SE from at least three independent experiments, *P<0.05 compared with the respective control (untransfected cells).

Figure 7

Hypoxia promotes interaction between RhoA or Rho kinase (ROCK) and LZ⁺MYPT1. To determine the interactions between protein kinase G (PKG) and LZ⁺MYPT1, fetal pulmonary arterial smooth muscle cells were exposed to normoxia or hypoxia for 4 h and cell lysates (200 ìg) were immunoprecipitated and probed with the indicated antibodies. (a) Representative Western blot data showing reduced interaction between PKG and LZ⁺MYPT1 and increased interaction between LZ⁺MYPT1 with RhoA or ROCK1/ROCK2 following hypoxia exposure. (b) Quantitative analysis of Western blots from three independent experiments and values represented as percent of control (normoxia). Data represent means ± SE, *P<0.05 compared with control (normoxia).

Figure 8

Overexpression of MYPT1 (^-LZ) mimics hypoxia-induced effects on protein kinase G (PKG) and LZ⁺MYPT1 interactions. Cells were transfected with MYPT1 expression vector with (+LZ) or without (^-LZ) leucine zipper. Empty vector was used as a control. Cell lysates (200 ìg) were immunoprecipitated with PKG, Rho kinase 1 (ROCK1) or Rho kinase 2 (ROCK2) antibodies and then immunoblotted with LZ⁺MYPT1 antibody. Panel a: Western blot showing the interaction between PKG and LZ⁺MYPT1. Cell lysates were immunoprecipitated with PKG antibody and then immunoblotted with LZ⁺MYPT1 antibody. Panel b: Quantification of data in Panel a. Panel c: Western blot probed with LZ⁺MYPT1 antibody showing interactions between LZ⁺MYPT1 and ROCK1. Cell lysates were immunoprecipitated with ROCK1 antibody and then probed with LZ⁺MYPT1 antibody. Panel d: Quantitative analysis of immunoblots in Panel c. Panel e: Representative immunoblot probed with LZ⁺MYPT1 antibody to detect interaction between LZ⁺MYPT1 and ROCK2 after immunoprecipitation with anti-ROCK2 antibodies. Panel f: Quantification of the data from panel e. Data represent means + SE from three to five independent experiments, *P<0.05 compared with the respective control (untransfected cells).

Figure 9

Schematic representation of signaling in smooth muscle cell in normoxia and hypoxia.