Suppression of immunoglobulin E-mediated allergic responses by regulator of G protein signaling 13.

Mast cells elicit allergic responses through degranulation and release of proinflammatory mediators after antigen crosslinking of the immunoglobulin E receptor FcepsilonRI. Proteins of the 'regulator of G protein signaling' (RGS) family negatively control signaling mediated by G protein-coupled receptors through GTPase-accelerating protein activity. Here we show that RGS13 inhibited allergic responses by physically interacting with the regulatory p85alpha subunit of phosphatidylinositol-3-OH kinase in mast cells and disrupting its association with an FcepsilonRI-activated scaffolding complex. Rgs13-/- mice had enhanced immunoglobulin E-mediated mast cell degranulation and anaphylaxis. Thus, RGS13 inhibits the assembly of immune receptor-induced signalosomes in mast cells. Abnormal RGS13 expression or function may contribute to disorders of amplified mast cell activity, such as idiopathic anaphylaxis.

RGS13 controls g protein-coupled receptor-evoked responses of human mast cells.

IgE-mediated mast cell degranulation and release of vasoactive mediators induced by allergens elicits allergic responses. Although G protein-coupled receptor (GPCR)-induced signals may amplify IgE-dependent degranulation, how GPCR signaling in mast cells is regulated remains incompletely defined. We investigated the role of regulator of G protein signaling (RGS) proteins in the modulation of these pathways in human mast cells. Several RGS proteins were expressed in mast cells including RGS13, which we previously showed inhibited IgE-mediated mast cell degranulation and anaphylaxis in mice. To characterize how RGS13 affects GPCR-mediated functions of human mast cells, we analyzed human mast cell lines (HMC-1 and LAD2) depleted of RGS13 by specific small interfering RNA or short hairpin RNA and HMC-1 cells overexpressing RGS13. Transient RGS13 knockdown in LAD2 cells lead to increased degranulation to sphingosine-1-phosphate but not to IgE-Ag or C3a. Relative to control cells, HMC-1 cells stably expressing RGS13-targeted short hairpin RNA had greater Ca(2+) mobilization in response to several natural GPCR ligands such as adenosine, C5a, sphingosine-1-phosphate, and CXCL12 than wild-type cells. Akt phosphorylation, chemotaxis, and cytokine (IL-8) secretion induced by CXCL12 were also greater in short hairpin RGS13-HMC-1 cells compared with control. RGS13 overexpression inhibited CXCL12-evoked Ca(2+) mobilization, Akt phosphorylation and chemotaxis. These results suggest that RGS13 restricts certain GPCR-mediated biological responses of human mast cells.

R4 RGS proteins: regulation of G-protein signaling and beyond.

The regulators of G-protein signaling (RGS) proteins were initially characterized as inhibitors of signal transduction cascades initiated by G-protein-coupled receptors (GPCR) because of their ability to increase the intrinsic GTPase activity of heterotrimeric G proteins. This GTPase accelerating protein (GAP) activity enhances G protein deactivation and promotes desensitization. However, in addition to this signature trait, emerging data have revealed an expanding network of proteins, lipids, and ions that interact with RGS proteins and confer additional regulatory functions. This review highlights recent advances in our understanding of the physiological functions of one subfamily of RGS proteins with a high degree of homology (B/R4) gleaned from recent studies of knockout mice or cells with reduced RGS expression. We also discuss some of the newly appreciated interactions of RGS proteins with cellular factors that suggest RGS control of several components of G-protein-mediated pathways, as well as a diverse array of non-GPCR-mediated biological responses.

Mechanism of the susceptibility of remodeled pulmonary vessels to drug-induced cell killing.

BACKGROUND: Pulmonary arterial hypertension remains a devastating disease without a cure. The major complication of this disease is the abnormal growth of vascular cells, resulting in pulmonary vascular remodeling. Thus, agents, which affect the remodeled vessels by killing unwanted cells, should improve treatment strategies. The present study reports that antitumor drugs selectively kill vascular cells in remodeled pulmonary vessels in rat models of pulmonary hypertension. METHODS AND RESULTS: After developing pulmonary vascular remodeling in chronic hypoxia or chronic hypoxia/SU-5416 models, rats were injected with antitumor drugs including proteasome inhibitors (bortezomib and MG-132) and daunorubicin. Within 1 to 3 days, these agents reduced the media and intima thickness of remodeled pulmonary vascular walls, but not the thickness of normal pulmonary vessels. These drugs also promoted apoptotic and autophagic death of vascular cells in the remodeled vessels, but not in normal vessels. We provide evidence that the upregulation of annexin A1, leading to GATA4-dependent downregulation of Bcl-xL, is a mechanism for specific apoptotic killing, and for the role of parkin in defining specificity of autophagic killing of remodeled vascular cells. The reversal of pulmonary vascular remodeling increased the capacity of vasodilators to reduce pulmonary arterial pressure. CONCLUSIONS: These results suggest that antitumor drugs can specifically kill cells in remodeled pulmonary vascular walls and may be useful for improving the efficacy of current therapeutic strategies to treat pulmonary arterial hypertension.

Reactive oxygen species and antioxidants in pulmonary hypertension.

SIGNIFICANCE: Pulmonary hypertension is a devastating disorder without any available treatment strategies that satisfactorily promote the survival of patients. The identification of new therapeutic strategies to treat patients with pulmonary hypertension is warranted. RECENT ADVANCES: Human studies have provided evidence that there is increased oxidative stress (lipid peroxidation, protein oxidation, DNA oxidation, and the depletion of small-molecule antioxidants) in patients with pulmonary hypertension. A variety of compounds with antioxidant properties have been shown to have beneficial therapeutic effects in animal models of pulmonary hypertension, possibly supporting the hypothesis that reactive oxygen species (ROS) are involved in the progression of pulmonary hypertension. Thus, understanding the molecular mechanisms of ROS actions could contribute to the development of optimal, antioxidant-based therapy for human pulmonary hypertension. One such mechanism includes action as a second messenger during cell-signaling events, leading to the growth of pulmonary vascular cells and right ventricular cells. CRITICAL ISSUES: The molecular mechanisms behind promotion of cell signaling for pulmonary vascular cell growth and right ventricular hypertrophy by ROS are not well understood. Evidence suggests that iron-catalyzed protein carbonylation may be involved. FUTURE DIRECTIONS: Understanding precise mechanisms of ROS actions should be useful for designing preclinical animal experiments and human clinical trials of the use of antioxidants and/or other redox compounds in the treatment of pulmonary hypertension.

IL-22 activates oxidant signaling in pulmonary vascular smooth muscle cells.

Reactive oxygen species (ROS) mediate cell-signaling processes in response to various ligands and play important roles in the pathogenesis of cardiovascular diseases. The present study reports that interleukin-22 (IL-22) elicits signal transduction in vascular smooth muscle cells (SMCs) through a ROS-dependent mechanism. We find that pulmonary artery SMCs express IL-22 receptor alpha 1 and that IL-22 activates STAT3 through this receptor. IL-22-induced signaling is found to be mediated by NADPH oxidase, as indicated by the observations that the inhibition and siRNA knock-down of this enzyme inhibit IL-22 signaling. IL-22 triggers the oxidative modifications of proteins through protein carbonylation and protein glutathionylation. Mass spectrometry identified some proteins that are carbonylated in response to IL-22 stimulation, including α-enolase, heat shock cognate 71kDa protein, mitochondrial 60kDa heat shock protein, and cytoplasmic 2 actin and determined that α-tubulin is glutathionylated. Protein glutathionylation and STAT3 phosphorylation are enhanced by the siRNA knock-down of glutaredoxin, while IL-22-mediated STAT3 phosphorylation is suppressed by knocking down thioredoxin interacting protein, an inhibitor of thioredoxin. IL-22 is also found to promote the growth of SMCs via NADPH oxidase. In rats, pulmonary hypertension is found to be associated with increased smooth muscle IL-22 expression. These results show that IL-22 promotes the growth of pulmonary vascular SMCs via a signaling mechanism that involves NADPH oxidase-dependent oxidation.

Proposed role of primary protein carbonylation in cell signaling.

Reactive oxygen species (ROS) mediate various cell signaling processes, but the mechanism for how ROS promote cell signaling is poorly understood. Protein carbonylation occurs because of the direct metal-catalyzed oxidation of amino acid side chains (primary protein carbonylation) or the addition of reactive aldehydes to amino acid side chains (secondary protein carbonylation). We hypothesize that primary protein carbonylation plays a role in the mechanism of ROS signaling. Specifically, we propose that (i) primary protein carbonylation mediates cell signaling and (ii) primary protein carbonylation is reversible.

Oxidant signaling for interleukin-13 gene expression in lung smooth muscle cells.

The growth of airway and vascular smooth muscle cells occurs in various lung diseases including asthma, chronic obstructive pulmonary disease, bronchopulmonary dysplasia, lymphangioleiomyomatosis, and pulmonary hypertension. Although inflammatory responses are critical in these diseases, the relationship between smooth muscle cell growth and inflammatory mediators is poorly understood. This study demonstrates that platelet-derived growth factor (PDGF) promotes the expression of interleukin-13 (IL-13) in lung smooth muscle cells through an oxidant signaling mechanism. Treatment of cultured human airway or pulmonary vascular smooth muscle cells with PDGF promotes IL-13 mRNA and protein expression. IL-13 expression is also detected in smooth muscle of airways and pulmonary vessels in allergen-stimulated mice. PDGF activates the proximal 980-bp region of the IL-13 promoter. PDGF-induced IL-13 expression is suppressed by the inhibition of reactive oxygen species signaling such as by NAD(P)H oxidase inhibition, reactive oxygen species scavenging, and metal chelation. Treatment of cells with hydrogen peroxide at as low as 1 µM also promotes IL-13 gene expression. PDGF-induced cell growth is suppressed by the neutralizing antibody against IL-13 as well as by reactive oxygen inhibitors, and recombinant IL-13 promotes the growth of airway smooth muscle cells. These results demonstrate that oxidant signaling activates IL-13 gene transcription in lung smooth muscle cells and that this signaling mechanism regulates cell growth.

RGS16 inhibits breast cancer cell growth by mitigating phosphatidylinositol 3-kinase signaling.

Aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway supports growth of many tumors including those of breast, lung, and prostate. Resistance of breast cancer cells to targeted chemotherapies including tyrosine kinase inhibitors (TKI) has been linked to persistent PI3K activity, which may in part be due to increased membrane expression of epidermal growth factor (EGF) receptors (HER2 and HER3). Recently we found that proteins of the RGS (regulator of G protein signaling) family suppress PI3K activity downstream of the receptor by sequestering its p85alpha subunit from signaling complexes. Because a substantial percentage of breast tumors have RGS16 mutations and reduced RGS16 protein expression, we investigated the link between regulation of PI3K activity by RGS16 and breast cancer cell growth. RGS16 overexpression in MCF7 breast cancer cells inhibited EGF-induced proliferation and Akt phosphorylation, whereas shRNA-mediated extinction of RGS16 augmented cell growth and resistance to TKI treatment. Exposure to TKI also reduced RGS16 expression in MCF7 and BT474 cell lines. RGS16 bound the amino-terminal SH2 and inter-SH2 domains of p85alpha and inhibited its interaction with the EGF receptor-associated adapter protein Gab1. These results suggest that the loss of RGS16 in some breast tumors enhances PI3K signaling elicited by growth factors and thereby promotes proliferation and TKI evasion downstream of HER activation.