Guofei Zhou

Hypoxia-induced alveolar epithelial-mesenchymal transition requires mitochondrial ROS and hypoxia-inducible factor 1.

Patients with acute lung injury develop hypoxia, which may lead to lung dysfunction and aberrant tissue repair. Recent studies have suggested that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. We sought to determine whether hypoxia induces EMT in alveolar epithelial cells (AEC). We found that hypoxia induced the expression of alpha-smooth muscle actin (alpha-SMA) and vimentin and decreased the expression of E-cadherin in transformed and primary human, rat, and mouse AEC, suggesting that hypoxia induces EMT in AEC. Both severe hypoxia and moderate hypoxia induced EMT. The reactive oxygen species (ROS) scavenger Euk-134 prevented hypoxia-induced EMT. Moreover, hypoxia-induced expression of alpha-SMA and vimentin was prevented in mitochondria-deficient rho(0) cells, which are incapable of ROS production during hypoxia. CoCl(2) and dimethyloxaloylglycine, two compounds that stabilize hypoxia-inducible factor (HIF)-alpha under normoxia, failed to induce alpha-SMA expression in AEC. Furthermore, overexpression of constitutively active HIF-1alpha did not induce alpha-SMA. However, loss of HIF-1alpha or HIF-2alpha abolished induction of alpha-SMA mRNA during hypoxia. Hypoxia increased the levels of transforming growth factor (TGF)-beta1, and preincubation of AEC with SB431542, an inhibitor of the TGF-beta1 type I receptor kinase, prevented the hypoxia-induced EMT, suggesting that the process was TGF-beta1 dependent. Furthermore, both ROS and HIF-alpha were necessary for hypoxia-induced TGF-beta1 upregulation. Accordingly, we have provided evidence that hypoxia induces EMT of AEC through mitochondrial ROS, HIF, and endogenous TGF-beta1 signaling.

The sphingosine kinase 1/sphingosine-1-phosphate pathway in pulmonary arterial hypertension

RATIONALE Sphingosine kinases (SphKs) 1 and 2 regulate the synthesis of the bioactive sphingolipid sphingosine-1-phosphate (S1P), an important lipid mediator that promotes cell proliferation, migration, and angiogenesis. OBJECTIVES We aimed to examine whether SphKs and their product, S1P, play a role in the development of pulmonary arterial hypertension (PAH). METHODS SphK1(-/-), SphK2(-/-), and S1P lyase heterozygous (Sgpl1(+/-)) mice, a pharmacologic SphK inhibitor (SKI2), and a S1P receptor 2 (S1PR2) antagonist (JTE013) were used in rodent models of hypoxia-mediated pulmonary hypertension (HPH). S1P levels in lung tissues from patients with PAH and pulmonary arteries (PAs) from rodent models of HPH were measured. MEASUREMENTS AND MAIN RESULTS mRNA and protein levels of SphK1, but not SphK2, were significantly increased in the lungs and isolated PA smooth muscle cells (PASMCs) from patients with PAH, and in lungs of experimental rodent models of HPH. S1P levels were increased in lungs of patients with PAH and PAs from rodent models of HPH. Unlike SphK2(-/-) mice, SphK1(-/-) mice were protected against HPH, whereas Sgpl1(+/-) mice were more susceptible to HPH. Pharmacologic SphK1 and S1PR2 inhibition prevented the development of HPH in rodent models of HPH. Overexpression of SphK1 and stimulation with S1P potentially via ligation of S1PR2 promoted PASMC proliferation in vitro, whereas SphK1 deficiency inhibited PASMC proliferation. CONCLUSIONS The SphK1/S1P axis is a novel pathway in PAH that promotes PASMC proliferation, a major contributor to pulmonary vascular remodeling. Our results suggest that this pathway is a potential therapeutic target in PAH.

Na,K-ATPase α1-subunit dephosphorylation by protein phosphatase 2A is necessary for its recruitment to the plasma membrane

In alveolar epithelial cells, G‐protein coupled‐receptors agonists (GPCR) induce the recruitment of the Na,K‐ATPase to the plasma membrane. Here we report that for the recruitment of the Na,K‐ ATPase to occur, dephosphorylation of its α1‐subunit at serine 18 is necessary, as demonstrated by in vitro phosphorylation, mutation of the serine 18 to alanine, and use of a specific phospho‐antibody. Several approaches strongly suggest dephosphorylation to be mediated by protein phosphatase 2A (PP2A): 1) Na,K‐ ATPase dephosphorylation and recruitment were prevented by okadaic acid (OA); 2) the Na,K‐ATPase α1‐subunit is an in vitro substrate for PP2A; and 3) glutathione S‐transferase (GST)‐fusion proteins binding assays demonstrate a direct interaction between the catalytic subunit of PP2A and the first 90 amino acids of the Na,K‐ATPase α1‐subunit. Finally, GPCR agonists induced a rapid translocation of PP2A from the cytosol to the membrane fraction, which corresponded with increased coimmunoprecipitation and colocalization of PP2A and the Na,K‐ATPase. Accordingly, we provide evidence that GPCR agonists promote PP2A translocation to the membrane fraction, leading to the dephosphorylation of the Na,K‐ATPase α1‐subunit at the serine 18 residue and its recruitment to the cell plasma membrane, which is of biological and physiological importance.—Lecuona, E., Dada, L. A., Sun, H., Butti, M. L., Zhou, G., Chew, T.‐L., Sznajder, J. I. Na,K‐ ATPase α1‐subunit dephosphorylation by protein phosphatase 2A is necessary for its recruitment to the plasma membrane. FASEB J. 20, E2146–E2155 (2006)

MicroRNAs in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a devastating disease without effective treatment. Despite decades of research and the development of novel treatments, PAH remains a fatal disease, suggesting an urgent need for better understanding of the pathogenesis of PAH. Recent studies suggest that microRNAs (miRNAs) are dysregulated in patients with PAH and in experimental pulmonary hypertension. Furthermore, normalization of a few miRNAs is reported to inhibit experimental pulmonary hypertension. We have reviewed the current knowledge about miRNA biogenesis, miRNA expression pattern, and their roles in regulation of pulmonary artery smooth muscle cells, endothelial cells, and fibroblasts. We have also identified emerging trends in our understanding of the role of miRNAs in the pathogenesis of PAH and propose future studies that might lead to novel therapeutic strategies for the treatment of PAH.

Adenosine monophosphate-activated protein kinase is required for pulmonary artery smooth muscle cell survival and the development of hypoxic pulmonary hypertension.

Human pulmonary artery smooth muscle cells (HPASMCs) express both adenosine monophosphate-activated protein kinase (AMPK) α1 and α2. We investigated the distinct roles of AMPK α1 and α2 in the survival of HPASMCs during hypoxia and hypoxia-induced pulmonary hypertension (PH). The exposure of HPASMCs to hypoxia (3% O2) increased AMPK activation and phosphorylation, and the inhibition of AMPK with Compound C during hypoxia decreased their viability and increased lactate dehydrogenase activity and apoptosis. Although the suppression of either AMPK α1 or α2 expression led to increased cell death, the suppression of AMPK α2 alone increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. It also resulted in the decreased expression of myeloid cell leukemia sequence 1 (MCL-1). The knockdown of MCL-1 or MCL-1 inhibitors increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. On the other hand, the suppression of AMPK α1 expression alone prevented hypoxia-mediated autophagy. The inhibition of autophagy induced cell death in HPASMCs. Our results suggest that AMPK α1 and AMPK α2 play differential roles in the survival of HPASMCs during hypoxia. The activation of AMPK α2 maintains the expression of MCL-1 and prevents apoptosis, whereas the activation of AMPK α1 stimulates autophagy, promoting HPASMC survival. Moreover, treatment with Compound C, which inhibits both isoforms of AMPK, prevented and partly reversed hypoxia-induced PH in mice. Taking these results together, our study suggests that AMPK plays a key role in the pathogenesis of pulmonary arterial hypertension, and AMPK may represent a novel therapeutic target for the treatment of pulmonary arterial hypertension.

cAMP-dependent protein kinase is essential for hypoxia-mediated epithelial-mesenchymal transition, migration, and invasion in lung cancer cells.

Lung cancer is the leading cause of cancer-related death worldwide. Hypoxia is known to increase cancer cell migration and invasion. We have previously reported that hypoxia induces epithelial-mesenchymal transition (EMT) in lung cancer cells. However, it is unknown whether hypoxia promotes lung cancer cell migration and invasion via EMT and whether cyclic AMP (cAMP) dependent protein kinase (PKA) plays a role in this process. We found that hypoxia increased PKA activity and induced mRNA and protein expression of PKA catalytic subunit α (PKACA), and regulatory subunits R1A and R1B. Knockdown of HIF-1/2α prevented hypoxia-mediated induction of PKACA mRNA expression and PKA activity. Inhibition of PKA activity with chemical inhibitors prevented EMT induced by hypoxia and tumor growth factor β1. However, activation of PKA by forskolin and 8-Br-cAMP did not induce EMT. Furthermore, treatment with H89 and knockdown of PKACA prevented hypoxia-mediated, EMT, cell migration, and invasion, whereas overexpression of mouse PKACA rescued hypoxia-mediated migration and invasion in PKACA deficient cancer cells. Our results suggest that hypoxia enhances PKA activity by upregulating PKA gene expression in a HIF dependent mechanism and that PKA plays a key role in hypoxia-mediated EMT, migration, and invasion in lung cancer cells.

Regulation of alveolar epithelial function by hypoxia

Patients with acute respiratory distress syndrome and high-altitude pulmonary oedema build up excess lung fluid, which leads to alveolar hypoxia. In patients with acute respiratory distress syndrome and hypoxia, there is a decrease in oedema fluid clearance, due in part to the downregulation of plasma membrane sodium–potassium adenosine triphosphatase (Na,K-ATPase). In alveolar epithelial cells, acute hypoxia promotes Na,K-ATPase endocytosis from the plasma membrane to intracellular compartments, resulting in inhibition of Na,K-ATPase activity. Exposure to prolonged hypoxia leads to degradation of plasma membrane Na,K-ATPase. The downregulation of plasma membrane Na,K-ATPase reduces adenosine triphosphate demand, as part of a survival mechanism of cellular adaptation to hypoxia. Hypoxia has also been shown to disassemble and degrade the keratin intermediate filament network, a fundamental component of the cell cytoskeleton, affecting epithelial barrier function. Accordingly, better understanding of the mechanisms regulating cellular adaptation to hypoxia may lead to the development of novel therapeutic strategies for acute respiratory distress syndrome and high-altitude pulmonary oedema patients.

Hypoxia-mediated Na-K-ATPase degradation requires von Hippel Lindau protein

Hypoxia inhibits Na‐K‐ATPase activity and leads to its degradation in mammalian cells. Von Hippel Lindau protein (pVHL) and hypoxia inducible factor (HIF) are key mediators in cellular adaptation to hypoxia; thus, we set out to investigate whether pVHL and HIF participate in the hypoxia‐mediated degradation of plasma membrane Na‐K‐ATPase. We found that in the presence of pVHL hypoxia decreased Na‐K‐ ATPase activity and promoted the degradation of plasma membrane Na‐K‐ATPase. In pVHL‐deficient cells, hypoxia did not decrease the Na‐K‐ATPase activ ity and the degradation of plasma membrane Na‐K‐ ATPase was prevented. pVHL‐mediated degradation of Na‐K‐ATPase required the functional pVHL E3 ligase and Ubc5 since pVHL mutants and dominant negative Ubc5 prevented Na‐K‐ATPase from degradation. The generation of reactive oxygen species was necessary for pVHL‐mediated Na‐K‐ATPase degradation during hypoxia. Desferrioxamine, which stabilizes HIF1/2α, did not affect the half‐life of plasma mem brane Na‐K‐ATPase. In addition, stabilizing HIF1/2α by infecting mammalian cells with adenoviruses con taining the oxygen‐dependent degradation domain of HIFlα did not affect the plasma membrane Na‐K‐ ATPase degradation. In cells with suppression of pVHL by short hairpin RNA, the Na‐K‐ATPase was not de graded during hypoxia, whereas cells with knockdown of HIF1/2α retained the ability to degrade plasma membrane Na‐K‐ATPase. These findings suggest that pVHL participates in the hypoxia‐mediated degradation of plasma membrane Na‐K‐ATPase in a HIF‐ independent manner.—Zhou, G., Dada, L. A., Chandel, N. S., Iwai, K., Lecuona, E., Ciechanover, A., Sznajder, J. I. Hypoxia‐mediated Na‐K‐ATPase degradation re quires von Hippel Lindau protein. FASEB J. 22, 1335–1342 (2008)

Loss of MicroRNA-17∼92 in Smooth Muscle Cells Attenuates Experimental Pulmonary Hypertension via Induction of PDZ and LIM Domain 5

RATIONALE Recent studies suggest that microRNAs (miRNAs) play important roles in regulation of pulmonary artery smooth muscle cell (PASMC) phenotype and are implicated in pulmonary arterial hypertension (PAH). However, the underlying molecular mechanisms remain elusive. OBJECTIVES This study aims to understand the mechanisms regulating PASMC proliferation and differentiation by microRNA-17∼92 (miR-17∼92) and to elucidate its implication in PAH. METHODS We generated smooth muscle cell (SMC)-specific miR-17∼92 and PDZ and LIM domain 5 (PDLIM5) knockout mice and overexpressed miR-17∼92 and PDLIM5 by injection of miR-17∼92 mimics or PDLIM5-V5-His plasmids and measured their responses to hypoxia. We used miR-17∼92 mimics, inhibitors, overexpression vectors, small interfering RNAs against PDLIM5, Smad, and transforming growth factor (TGF)-β to determine the role of miR-17∼92 and its downstream targets in PASMC proliferation and differentiation. MEASUREMENTS AND MAIN RESULTS We found that human PASMC (HPASMC) from patients with PAH expressed decreased levels of the miR-17∼92 cluster, TGF-β, and SMC markers. Overexpression of miR-17∼92 increased and restored the expression of TGF-β3, Smad3, and SMC markers in HPASMC of normal subjects and patients with idiopathic PAH, respectively. Knockdown of Smad3 but not Smad2 prevented miR-17∼92-induced expression of SMC markers. SMC-specific knockout of miR-17∼92 attenuated hypoxia-induced pulmonary hypertension (PH) in mice, whereas reconstitution of miR-17∼92 restored hypoxia-induced PH in these mice. We also found that PDLIM5 is a direct target of miR-17/20a, and hypertensive HPASMC and mouse PASMC expressed elevated PDLIM5 levels. Suppression of PDLIM5 increased expression of SMC markers and enhanced TGF-β/Smad2/3 activity in vitro and enhanced hypoxia-induced PH in vivo, whereas overexpression of PDLIM5 attenuated hypoxia-induced PH. CONCLUSIONS We provided the first evidence that miR-17∼92 inhibits PDLIM5 to induce the TGF-β3/SMAD3 pathway, contributing to the pathogenesis of PAH.

Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cell Proliferation Is Controlled by Forkhead Box M1

Pulmonary arterial hypertension (PAH) is a devastating disease, and no effective treatments are available. Hypoxia-induced pulmonary artery remodeling, including smooth muscle cell proliferation, contributes to PAH, but the exact mechanisms underlying this abnormal process are largely undefined. The forkhead box M1 (FoxM1) transcription factor regulates cancer cell growth by modulating gene expression critical for cell cycle progression. Here, we report for the first time, to the best of our knowledge, a novel function of FoxM1 in the hypoxia-stimulated proliferation of human pulmonary artery smooth muscle cells (HPASMCs). Exposure to hypoxia caused a marked up-regulation of FoxM1 gene expression, mainly at the transcription level, and this induction correlated with HPASMC cell proliferation. The knockdown of FoxM1 inhibited the hypoxia-stimulated proliferation of HPASMCs. We found that the knockdown of HIF-2α, but not HIF-1α, diminished FoxM1 induction in response to hypoxia. However, the knockdown of FoxM1 did not alter expression levels of HIF-2α or HIF-1α, suggesting that HIF-2α is an upstream regulator of FoxM1. Furthermore, the knockdown of FoxM1 prevented the hypoxia-induced expression of aurora A kinase and cyclin D1. Collectively, our results suggest that hypoxia induces FoxM1 gene expression in an HIF-2α-dependent pathway, thereby promoting HPASMC proliferation.