Anuli C. Anyanwu

Glycogen synthase kinase-3β/β-catenin signaling regulates neonatal lung mesenchymal stromal cell myofibroblastic differentiation

In bronchopulmonary dysplasia (BPD), alveolar septa are thickened with collagen and α-smooth muscle actin-, transforming growth factor (TGF)-β-positive myofibroblasts. We examined the biochemical mechanisms underlying myofibroblastic differentiation, focusing on the role of glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. In the cytoplasm, β-catenin is phosphorylated on the NH(2) terminus by constitutively active GSK-3β, favoring its degradation. Upon TGF-β stimulation, GSK-3β is phosphorylated and inactivated, allowing β-catenin to translocate to the nucleus, where it activates transcription of genes involved in myofibroblastic differentiation. We examined the role of β-catenin in TGF-β1-induced myofibroblastic differentiation of neonatal lung mesenchymal stromal cells (MSCs) isolated from tracheal aspirates of premature infants with respiratory distress. TGF-β1 increased β-catenin expression and nuclear translocation. Transduction of cells with GSK-3β S9A, a nonphosphorylatable, constitutively active mutant that favors β-catenin degradation, blocked TGF-β1-induced myofibroblastic differentiation. Furthermore, transduction of MSCs with ΔN-catenin, a truncation mutant that cannot be phosphorylated on the NH(2) terminus by GSK-3β and is not degraded, was sufficient for myofibroblastic differentiation. In vivo, hyperoxic exposure of neonatal mice increases expression of β-catenin in α-smooth muscle actin-positive myofibroblasts. Similar changes were found in lungs of infants with BPD. Finally, low-passage unstimulated MSCs from infants developing BPD showed higher phospho-GSK-3β, β-catenin, and α-actin content compared with MSCs from infants not developing this disease, and phospho-GSK-3β and β-catenin each correlated with α-actin content. We conclude that phospho-GSK-3β/β-catenin signaling regulates α-smooth muscle actin expression, a marker of myofibroblast differentiation, in vitro and in vivo. This pathway appears to be activated in lung mesenchymal cells from patients with BPD.

Suppression of inflammatory cell trafficking and alveolar simplification by the heme oxygenase-1 product carbon monoxide

Bronchopulmonary dysplasia (BPD), a lung disease of prematurely born infants, is characterized in part by arrested development of pulmonary alveolae. We hypothesized that heme oxygenase (HO-1) and its byproduct carbon monoxide (CO), which are thought to be cytoprotective against redox stress, mitigate lung injury and alveolar simplification in hyperoxia-exposed neonatal mice, a model of BPD. Three-day-old C57BL/6J mice were exposed to air or hyperoxia (FiO2, 75%) in the presence or absence of inhaled CO (250 ppm for 1 h twice daily) for 21 days. Hyperoxic exposure increased mean linear intercept, a measure of alveolar simplification, whereas CO treatment attenuated hypoalveolarization, yielding a normal-appearing lung. Conversely, HO-1-null mice showed exaggerated hyperoxia-induced hypoalveolarization. CO also inhibited hyperoxia-induced pulmonary accumulation of F4/80+, CD11c+, and CD11b+ monocytes and Gr-1+ neutrophils. Furthermore, CO attenuated lung mRNA and protein expression of proinflammatory cytokines, including the monocyte chemoattractant CCL2 in vivo, and decreased hyperoxia-induced type I alveolar epithelial cell CCL2 production in vitro. Hyperoxia-exposed CCL2-null mice, like CO-treated mice, showed attenuated alveolar simplification and lung infiltration of CD11b+ monocytes, consistent with the notion that CO blocks lung epithelial cell cytokine production. We conclude that, in hyperoxia-exposed neonatal mice, inhalation of CO suppresses inflammation and alveolar simplification.

Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

Increased medial arterial thickness is a structural change in pulmonary arterial hypertension (PAH). The role of smooth muscle hypertrophy in this process has not been well studied. Bone morphogenetic proteins (BMPs), transforming growth factor (TGF)-beta1, serotonin (or 5-hydroxytryptamine; 5-HT), and endothelin (ET)-1 have been implicated in PAH pathogenesis. We examined the effect of these mediators on human pulmonary artery smooth muscle cell size, contractile protein expression, and contractile function, as well on the roles of glycogen synthase kinase (GSK)-3beta and p70 ribosomal S6 kinase (p70S6K), two proteins involved in translational control, in this process. Unlike epidermal growth factor, BMP-4, TGF-beta1, 5-HT, and ET-1 each increased smooth muscle cell size, contractile protein expression, fractional cell shortening, and GSK-3beta phosphorylation. GSK-3beta inhibition by lithium or SB-216763 increased cell size, protein synthesis, and contractile protein expression. Expression of a non-phosphorylatable GSK-3beta mutant blocked BMP-4-, TGF-beta1-, 5-HT-, and ET-1-induced cell size enlargement, suggesting that GSK-3beta phosphorylation is required and sufficient for cellular hypertrophy. However, BMP-4, TGF-beta1, 5-HT, and ET-1 stimulation was accompanied by an increase in serum response factor transcriptional activation but not eIF2 phosphorylation, suggesting that GSK-3beta-mediated hypertrophy occurs via transcriptional, not translational, control. Finally, BMP-4, TGF-beta1, 5-HT, and ET-1 treatment induced phosphorylation of p70S6K and ribosomal protein S6, and siRNAs against p70S6K and S6 blocked the hypertrophic response. We conclude that mediators implicated in the pathogenesis of PAH induce pulmonary arterial smooth muscle hypertrophy. Identification of the signaling pathways regulating vascular smooth muscle hypertrophy may define new therapeutic targets for PAH.

Purinergic dysregulation in pulmonary hypertension.

Despite the fact that nucleotides and adenosine help regulate vascular tone through purinergic signaling pathways, little is known regarding their contributions to the pathobiology of pulmonary arterial hypertension, a condition characterized by elevated pulmonary vascular resistance and remodeling. Even less is known about the potential role that alterations in CD39 (ENTPD1), the ectonucleotidase responsible for the conversion of the nucleotides ATP and ADP to AMP, may play in pulmonary arterial hypertension. In this study we identified decreased CD39 expression on the pulmonary endothelium of patients with idiopathic pulmonary arterial hypertension. We next determined the effects of CD39 gene deletion in mice exposed to normoxia or normobaric hypoxia (10% oxygen). Compared with controls, hypoxic CD39(-/-) mice were found to have a markedly elevated ATP-to-adenosine ratio, higher pulmonary arterial pressures, more right ventricular hypertrophy, more arterial medial hypertrophy, and a pro-thrombotic phenotype. In addition, hypoxic CD39(-/-) mice exhibited a marked increase in lung P2X1 receptors. Systemic reconstitution of ATPase and ADPase enzymatic activities through continuous administration of apyrase decreased pulmonary arterial pressures in hypoxic CD39(-/-) mice to levels found in hypoxic CD39(+/+) controls. Treatment with NF279, a potent and selective P2X1 receptor antagonist, lowered pulmonary arterial pressures even further. Our study is the first to implicate decreased CD39 and resultant alterations in circulating purinergic signaling ligands and cognate receptors in the pathobiology of pulmonary arterial hypertension. Reconstitution and receptor blocking experiments suggest that phosphohydrolysis of purinergic nucleotide tri- and diphosphates, or blocking of the P2X1 receptor could serve as treatment for pulmonary arterial hypertension.

Correction: Neonatal periostin knockout mice are protected from hyperoxia-induced alveolar simplication

The word “Simplification” is misspelled in the title of the article. The correct title is: Neonatal Periostin Knockout Mice Are Protected from Hyperoxia-Induced Alveolar Simplification. The correct citation is: Bozyk PD, Bentley JK, Popova AP, Anyanwu AC, Linn MD, Goldsmith AM, et al. (2012) Neonatal Periostin Knockout Mice Are Protected from Hyperoxia-Induced Alveolar Simplification. PLoS ONE 7(2): e31336. doi:10.1371/journal.pone.0031336