Expression profiling elucidates a molecular gene signature for pulmonary hypertension in sarcoidosis.

Pulmonary hypertension (PH), when it complicates sarcoidosis, carries a poor prognosis, in part because it is difficult to detect early in patients with worsening respiratory symptoms. Pathogenesis of sarcoidosis occurs via incompletely characterized mechanisms that are distinct from the mechanisms of pulmonary vascular remodeling well known to occur in conjunction with other chronic lung diseases. To address the need for a biomarker to aid in early detection as well as the gap in knowledge regarding the mechanisms of PH in sarcoidosis, we used genome-wide peripheral blood gene expression analysis and identified an 18-gene signature capable of distinguishing sarcoidosis patients with PH (n = 8), sarcoidosis patients without PH (n = 17), and healthy controls (n = 45). The discriminative accuracy of this 18-gene signature was 100% in separating sarcoidosis patients with PH from those without it. If validated in a large replicate cohort, this signature could potentially be used as a diagnostic molecular biomarker for sarcoidosis-associated PH.

Src phosphorylation of endothelial cell surface intercellular adhesion molecule-1 mediates neutrophil adhesion and contributes to the mechanism of lung inflammation.

OBJECTIVE: The goal of this study was to determine whether tumor necrosis factor α (TNFα)-induced Src activation and intercellular adhesion molecule-1 (ICAM-1) phosphorylation rapidly increase endothelial cell adhesivity and polymorphonuclear leukocyte (PMN) sequestration independently of de novo ICAM-1 synthesis. METHODS AND RESULTS: TNFα exposure of mouse lungs for 5 minutes produced a 3-fold increase in (125)I-anti-ICAM-1 monoclonal antibody (mAb) binding and (111)In oxine-labeled PMN sequestration, as well as Src activation, ICAM-1 Tyr518 phosphorylation, and phospho- Tyr518-ICAM-1 coimmunoprecipitation with actin. The response was absent in Nox2(-/-) lungs or following Src inhibition. In COS-7 cells transfected with wild-type (WT), phospho-defective (Tyr518Phe), or phospho-mimicking (Tyr518Asp) mouse ICAM-1 cDNA constructs, TNFα increased the B(max) of YN1/1.7.4 anti-ICAM-1 mAb binding to WT-ICAM-1 but not to Tyr518Phe-ICAM-1, indicating increased binding avidity secondary to ICAM-1 phosphorylation. This effect was mimicked by expression of the Tyr518Asp-ICAM-1 mutant. TNFα also increased the staining intensity and cell surface clustering of YN1/1.7.4 mAb-labeled WT-ICAM-1 that colocalized with F-actin, which was not observed with Tyr518Phe-ICAM-1 but was recapitulated with Tyr518Asp-ICAM-1. Finally, overexpression of ICAM-1 in mouse lungs significantly increased lipopolysaccharide-induced transvascular albumin leakage and bronchoalveolar lavage PMN counts at 2 and 24 hours after lipopolysaccharide inhalation compared with lungs expressing the Tyr518Phe ICAM-1 mutant. CONCLUSION: Src-dependent phosphorylation of endothelial cell ICAM-1 Tyr518 induces PMN adhesion by promoting ICAM-1 clustering, which we propose mediates rapid-phase lung vascular accumulation of PMNs during inflammation.

Phosphatidylinositol 3-kinase gamma signaling through protein kinase Czeta induces NADPH oxidase-mediated oxidant generation and NF-kappaB activation in endothelial cells.

We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kgamma in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kgamma-mediated oxidant signaling in the mechanism of NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110gamma catalytic subunit of PI3Kgamma. Tumor necrosis factor (TNF) alpha challenge of wild type ECs caused p110gamma translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110gamma-/- ECs. ROS production was the result of TNFalpha activation of Ser phosphorylation of NADPH oxidase subunit p47(phox) and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110gamma-/- ECs. Additionally, the TNFalpha-activated NF-kappaB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kgamma. TNFalpha challenge of p110gamma-/- ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCzeta. Thus, PI3Kgamma lies upstream of PKCzeta in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-kappaB activation and ICAM-1 expression.

SHIP2 is recruited to the cell membrane upon macrophage colony-stimulating factor (M-CSF) stimulation and regulates M-CSF-induced signaling.

The Src homology 2-containing inositol phosphatase SHIP1 functions in hemopoietic cells to limit activation events mediated by PI3K products, including Akt activation and cell survival. In contrast to the limited cellular expression of SHIP1, the related isoform SHIP2, is widely expressed in both parenchymal and hemopoietic cells. The goal of this study was to determine how SHIP2 functions to regulate M-CSF signaling. We report that 1) SHIP2 was tyrosine-phosphorylated in M-CSF-stimulated human alveolar macrophages, human THP-1 cells, murine macrophages, and the murine macrophage cell line RAW264; 2) SHIP2 associated with the M-CSF receptor after M-CSF stimulation; and 3) SHIP2 associated with the actin-binding protein filamin and localization to the cell membrane, requiring the proline-rich domain, but not on the Src homology 2 domain of SHIP2. Analyzing the function of SHIP2 in M-CSF-stimulated cells by expressing either wild-type SHIP2 or an Src homology 2 domain mutant of SHIP2 reduced Akt activation in response to M-CSF stimulation. In contrast, the expression of a catalytically deficient mutant of SHIP2 or the proline-rich domain of SHIP2 enhanced Akt activation. Similarly, the expression of wild-type SHIP2 inhibited NF-kappaB-mediated gene transcription. Finally, fetal liver-derived macrophages from SHIP2 gene knockout mice enhanced activation of Akt in response to M-CSF treatment. These data suggest a novel regulatory role for SHIP2 in M-CSF-stimulated myeloid cells.

cAMP targeting of p38 MAP kinase inhibits thrombin-induced NF-kappaB activation and ICAM-1 expression in endothelial cells.

We investigated the mechanisms by which elevated intracellular cAMP concentration inhibits the thrombin-induced ICAM-1 expression in endothelial cells. Exposure of human umbilical vein endothelial cells to forskolin or dibutyryl cAMP, which increase intracellular cAMP by separate mechanisms, inhibited the thrombin-induced ICAM-1 expression. This effect of cAMP was secondary to inhibition of NF-kappaB activity, the key regulator of thrombin-induced ICAM-1 expression in endothelial cells. The action of cAMP occurred downstream of IkappaBalpha degradation and was independent of NF-kappaB binding to the ICAM-1 promoter. We observed that cAMP interfered with thrombin-induced phosphorylation of NF-kappaB p65 (RelA) subunit, a crucial event promoting the activation of the DNA-bound NF-kappaB. Because p38 MAPK can induce transcriptional activity of RelA/p65 without altering the DNA binding function of NF-kappaB, we addressed the possibility that cAMP antagonizes thrombin-induced NF-kappaB activity and ICAM-1 expression by preventing the activation of p38 MAPK. We observed that treating cells with forskolin blocked the activation of p38 MAPK, and inhibition of p38 MAPK interfered with phosphorylation of RelA/p65 induced by thrombin. Our data demonstrate that increased intracellular cAMP concentration in endothelial cells prevents thrombin-induced ICAM-1 expression by inhibiting p38 MAPK activation, which in turn prevents phosphorylation of RelA/p65 and transcriptional activity of the bound NF-kappaB.

Tumor necrosis factor-alpha induces early-onset endothelial adhesivity by protein kinase Czeta-dependent activation of intercellular adhesion molecule-1.

We tested the hypothesis that TNF-alpha induces early-onset endothelial adhesivity toward PMN by activating the constitutive endothelial cell surface ICAM-1, the beta2-integrin (CD11/CD18) counter-receptor. Stimulation of human pulmonary artery endothelial cells with TNF-alpha resulted in phosphorylation of ICAM-1 within 1 minute, a response that was sustained up to 15 minutes after TNF-alpha challenge. We observed that TNF-alpha induced 10-fold increase in PMN adhesion to endothelial cells in an ICAM-1-dependent manner and that this response paralleled the rapid time course of ICAM-1 phosphorylation. We also observed that the early-onset TNF-alpha-induced endothelial adhesivity was protein synthesis-independent and associated with cell surface ICAM-1 clustering. Pretreatment of cells with the pan-PKC inhibitor, chelerythrine, prevented the activation of endothelial adhesivity. As PKCzeta, an atypical PKC isoform abundantly expressed in endothelial cells, is implicated in signaling TNF-alpha-induced ICAM-1 gene transcription, we determined the possibility that PKCzeta was involved in mediating endothelial adhesivity through ICAM-1 expression. We observed that TNF-alpha stimulation of endothelial cells induced PKCzeta activation and its association with ICAM-1. Inhibition of PKCzeta by pharmacological and genetic approaches prevented the TNF-alpha-induced phosphorylation and the clustering of the cell surface ICAM-1 as well as activation of endothelial adhesivity. Thus, TNF-alpha induces early-onset, protein synthesis-independent expression of endothelial adhesivity by PKCzeta-dependent phosphorylation of cell surface ICAM-1 that precedes the de novo ICAM-1 synthesis. The rapid ICAM-1 expression represents a novel mechanism for promoting the stable adhesion of PMN to endothelial cells that is needed to facilitate the early-onset transendothelial migration of PMN.