The ligand-activated transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), regulates metabolism, cell proliferation, and inflammation. Pulmonary hypertension (PH) is associated with reduced PPARγ expression, and hypoxia exposure regimens that cause PH reduce PPARγ expression. The current study examines mechanisms of hypoxia-induced PPARγ downregulation in vitro and in vivo. Hypoxia reduced PPARγ mRNA and protein levels, PPARγ activity, and the expression of PPARγ regulated genes in human pulmonary artery smooth muscle cells (HPASMC) exposed to 1% oxygen for 72 hours. Similarly, exposure of mice to hypoxia (10% O2) for 3 weeks reduced PPARγ mRNA and protein in mouse lung. Inhibiting ERK1/2 with PD98059 or treatment with siRNA directed against either NF-κB p65 or Nox4 attenuated hypoxic reductions in PPARγ expression and activity. Furthermore, degradation of H2O2 using PEG-catalase prevented hypoxia-induced ERK 1/2 phosphorylation and Nox4 expression suggesting sustained ERK 1/2-mediated signaling and Nox4 expression in this response. Mammalian two hybrid assays demonstrated that PPARγ and p65 bind directly to each other in a mutually repressive fashion. Taken together, we conclude that hypoxic regimens that promote PH pathogenesis and HPASMC proliferation reduce PPARγ expression and activity through ERK1/2-, p65-, and Nox4-dependent pathways. These findings provide novel insights into mechanisms by which pathophysiological stimuli such as hypoxia cause loss of PPARγ activity and pulmonary vascular cell proliferation, pulmonary vascular remodeling, and PH. These results also indicate that restoration of PPARγ activity with pharmacological ligands may provide a novel therapeutic approach in selected forms of PH.

Objective Pulmonary hypertension (PH) is characterized by increased pulmonary vascular remodeling, resistance, and pressures. Reactive oxygen species (ROS) contribute to PH-associated vascular dysfunction. NADPH oxidases (Nox) and mitochondria are major sources of superoxide (O2•−) and hydrogen peroxide (H2O2) in pulmonary vascular cells. Hypoxia, a common stimulus of PH, increases Nox expression and mitochondrial ROS (mtROS) production. The interactions between these two sources of ROS generation continue to be defined. We hypothesized that mitochondria-derived O2•− (mtO2•−) and H2O2 (mtH2O2) increases Nox expression to promote PH pathogenesis and that mitochondria-targeted antioxidants can reduce mtROS, Nox expression, and hypoxia-induced PH. Approach and Results Exposure of human pulmonary artery endothelial cells to hypoxia for 72 hours increased mtO2•− and mtH2O2. To assess the contribution of mtO2•− and mtH2O2 to hypoxia-induced PH, mice that overexpress superoxide dismutase 2 (TghSOD2) or mitochondria-targeted catalase (MCAT) were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 weeks. Compared to hypoxic control mice, MCAT mice developed less hypoxia-induced increases in RVSP, α-SMA staining, extracellular H2O2 (Amplex Red), Nox2 and Nox4 (qRT-PCR and western blot), or cyclinD1 and PCNA (western blot). In contrast, TghSOD2 mice experienced exacerbated responses to hypoxia. Conclusions These studies demonstrate that hypoxia increases mtO2•− and mtH2O2. Targeting mtH2O2 attenuates PH pathogenesis, whereas, targeting mtO2•− exacerbates PH. These differences in PH pathogenesis were mirrored by RVSP, vessel muscularization, levels of Nox2 and Nox4, proliferation, and H2O2 release. These studies suggest that targeted reductions in mtH2O2 generation may be particularly effective at preventing hypoxia-induced PH.

The nuclear factor B (NF-B)2 represents a ubiquitously expressed family of transcription factor participating in various biological effects ranging from immune, inflammatory, and stress-induced responses to cell fate decisions such as proliferation, differentiation, apoptosis, and tumorigenesis (1, 2). The mammalian NF-B family is comprised of five members: RelA (p65), RelB, c-Rel, NF-B1 (p50 and its precursor p105), and NF-B2 (p52 and its precursor p100). A characteristic feature of these proteins is the presence of a conserved N-terminal 300-amino acid Rel homology domain that contains nuclear localization signal and is involved in dimerization, sequencespecific DNA binding, and interaction with inhibitory IB proteins. A distinguishing feature of RelA, RelB, and c-Rel from p50 and p52 is the possession of a transactivation domain within the C-terminal region. Typically, NF-B exists as a heterodimer of p50 and RelA/p65 subunits associated with IB␣, the prototype of a family of inhibitory proteins IBs that keeps NF-B in the cytoplasm by virtue of masking the nuclear localization signal of RelA/p65 (3, 4). Activation of NF-B requires phosphorylation of IB␣ on two specific serine residues (Ser 32 and Ser 36 ) by a macromolecular cytoplasmic IB kinase (IKK) complex composed of the catalytic subunits IKK␣ and IKK␤ and the regulatory subunit NEMO/IKK␥ (5, 6). Phosphorylation triggers the ubiquitination of IB␣ by the E3-SCF␤-TrCP ubiquitin ligase, which in turn marks it for degradation by the 26 S proteasome (7,8). The unleashed NF-B migrates to the nucleus to activate transcription of target genes including intercellular adhesion molecule-1 (ICAM-1) (9 -14), an inducible endothelial adhesive protein that serves as a ligand for ␤ 2 -integrins (CD11/ CD18) present on the surface of leukocytes (15-17). Interaction of ICAM-1 with ␤ 2 -integrins enables polymorphonuclear leukocytes to adhere firmly and stably to the vascular endothelium and to migrate across the endothelial barrier (18 -20). We have shown that RelA/p65 is an essential regulator of endothelial ICAM-1 following stimulation of protease-activated receptor-1 by thrombin, a serine protease released during intravascular coagulation initiated by tissue injury or sepsis (21,22). A key signal mediating RelA/p65 activation by thrombin involves stimulation of the small GTPase RhoA and its effector Rhoassociated kinase (23,24). Activated RhoA/ROCK leads to activation of IKK␤, which in turn mediates the release of RelA/p65 for its nuclear uptake and binding to the ICAM-1 promoter, secondary to phosphorylation and degradation of IB␣ (24).

Activation of the transcription factor NF-B involves its release from the inhibitory protein IB␣ in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the events responsible for its release have been elucidated, mechanisms regulating the nuclear transport of NF-B remain elusive. We now provide evidence for actin cytoskeleton-dependent and -independent mechanisms of RelA/p65 nuclear transport using the proinflammatory mediators, thrombin and tumor necrosis factor ␣, respectively. We demonstrate that thrombin alters the actin cytoskeleton in endothelial cells and interfering with these alterations, whether by stabilizing or destabilizing the actin filaments, prevents thrombin-induced NF-B activation and consequently, expression of its target gene, ICAM-1. The blockade of NF-B activation occurs downstream of IB␣ degradation and is associated with impaired RelA/p65 nuclear translocation. Importantly, thrombin induces association of RelA/p65 with actin and this interaction is sensitive to stabilization/destabilization of the actin filaments. In parallel studies, stabilizing or destabilizing the actin filaments fails to inhibit RelA/p65 nuclear accumulation and ICAM-1 expression by tumor necrosis factor ␣, consistent with its inability to induce actin filament formation comparable with thrombin. Thus, these studies reveal the existence of actin cytoskeleton-dependent and -independent pathways that may be engaged in a stimulus-specific manner to facilitate RelA/p65 nuclear import and thereby ICAM-1 expression in endothelial cells. NF-B2 is an ubiquitously expressed family of transcription factors controlling varied biological effects ranging from inflammatory, immune, and stress-induced responses to cell fate decisions such as proliferation, differentiation, tumorigenesis, and apoptosis (1-3). NF-B dimers, typically a heterodimer of p50 and RelA/p65 subunits, are mostly sequestered in the cytoplasm by IB␣, the prototype of a family of inhibitory proteins IBs that mask the nuclear localization signal of RelA/ p65 (4, 5). Activation of NF-B requires serine phosphorylation (Ser 32 and Ser 36 ) of IB␣ by a macromolecular IB kinase (IKK) complex (6 -8). Phosphorylation marks IB␣ for polyubiquitination by the E3-SCF␤-TrCP ubiquitin ligase leading to its degradation by the 26 S proteasome (9 -11). The released NF-B undergoes rapid nuclear translocation and subsequent binding to NF-B responsive elements to activate transcription of target genes including intercellular adhesion molecule-1 (ICAM-1; CD54), an inducible endothelial adhesive protein that serves as a counter-receptor for ␤ 2 -integrins (CD11/CD18) present on the surface of leukocytes (12-14). Interaction of ICAM-1 with ␤ 2 -integrins enables polymorphonuclear leukocytes to adhere firmly and stably to the vascular endothelium, and to migrate across the endothelial barrier (15-18). have shown that activation of NF-B is essential for ICAM-1 expression in endothelial cells after stimulation with the proinflammatory cytokine tumor necrosis factor ...

We addressed the regulatory function of mammalian target of rapamycin (mTOR) in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. Pretreatment of HUVECs with rapamycin, an inhibitor of mTOR, augmented thrombin-induced ICAM-1 expression. Inhibition of mTOR by this approach promoted whereas over-expression of mTOR inhibited thrombin-induced transcriptional activity of NF-κB, an essential regulator of ICAM-1 transcription. Analysis of the NF-κB signaling pathway revealed that inhibition of mTOR potentiated IκB kinase activation resulting in a rapid and persistent phosphorylation of IκBα on Ser32 and Ser36, a requirement for IκBα degradation. Consistent with these data, we observed a more efficient and stable nuclear localization of RelA/p65 and, subsequently, the DNA binding activity of NF-κB by thrombin following mTOR inhibition. These data define a novel role of mTOR in down-regulating thrombin-induced ICAM-1 expression in endothelial cells by controlling a delayed and transient activation of NF-κB.