Jung Seok Hwang

Deacetylation-mediated interaction of SIRT1-HMGB1 improves survival in a mouse model of endotoxemia

Inflammatory signal-mediated release of high-mobility group box 1 (HMGB1) is a damage-associated molecular pattern or alarmin. The inflammatory functions of HMGB1 have been extensively investigated; however, less is known about the mechanisms controlling HMGB1 release. We show that SIRT1, the human homolog of the Saccharomyces cerevisiae protein silent information regulator 2, which is involved in cellular senescence and possibly the response to inflammation, forms a stable complex with HMGB1 in murine macrophage RAW264.7 cells. SIRT1 directly interacted with HMGB1 via its N-terminal lysine residues (28–30), and thereby inhibited HMGB1 release to improve survival in an experimental model of sepsis. By contrast, inflammatory stimuli such as lipopolysaccharide (LPS) and tumor necrosis factor-α promoted HMGB1 release by provoking its dissociation from SIRT1 dependent on acetylation, thereby increasing the association between HMGB1 and chromosome region maintenance 1, leading to HMGB1 translocation. In vivo infection with wild-type SIRT1 and HMGB1K282930R, a hypo-acetylation mutant, improved survival (85.7%) during endotoxemia more than infection with wild-type SIRT1 and HMGB1-expressing adenovirus, indicating that the acetylation-dependent interaction between HMGB1 and SIRT1 is critical for LPS-induced lethality. Taken together, we propose that SIRT1 forms an anti-inflammatory complex with HMGB1, allowing cells to bypass the response to inflammation.

The PPARδ-mediated inhibition of angiotensin II-induced premature senescence in human endothelial cells is SIRT1-dependent.

Cellular senescence has been implicated in endothelial dysfunctions affecting vascular tone and regeneration. The molecular mechanisms of vascular senescence are poorly understood. The present study demonstrates that upregulation of SIRT1 by peroxisome proliferator-activated receptor (PPAR) δ attenuates premature senescence in angiotensin (Ang) II-treated human coronary artery endothelial cells (HCAECs). Activation of PPARδ by the specific ligand GW501516 significantly inhibited Ang II-induced premature senescence and generation of reactive oxygen species (ROS) in HCAECs. A marked concentration- and time-dependent increase in the mRNA levels of SIRT1 was observed in GW501516-treated HCAECs. The effects of GW501516 were almost completely abolished in the presence of small interfering (si) RNA against PPARδ, indicating that PPARδ mediates the effects of GW501516. In addition, activation of PPARδ, but not PPARα or PPARγ, significantly enhanced SIRT1 promoter activity and protein expression. Down-regulation or inhibition of SIRT1 by siRNA or sirtinol abrogated the effects of PPARδ on Ang II-induced premature senescence and ROS generation, respectively. Furthermore, resveratrol, a well-known activator of SIRT1, mimicked the action of PPARδ on Ang II-induced premature senescence and ROS generation. Taken together, these results indicate that the anti-senescent activities of PPARδ may be achieved at least in part by fine tuning the expression of SIRT1 in the vascular endothelium.

Activation of Peroxisome Proliferator-Activated Receptor γ by Rosiglitazone Inhibits Lipopolysaccharide-Induced Release of High Mobility Group Box 1

Peroxisome proliferator-activated receptors (PPARs) are shown to modulate the pathological status of sepsis by regulating the release of high mobility group box 1 (HMGB1), a well-known late proinflammatory mediator of sepsis. Ligand-activated PPARs markedly inhibited lipopolysaccharide- (LPS) induced release of HMGB1 in RAW 264.7 cells. Among the ligands of PPAR, the effect of rosiglitazone, a specific ligand for PPARγ, was superior in the inhibition of HMGB1 release induced by LPS. This effect was observed in cells that received rosiglitazone before LPS or after LPS treatment, indicating that rosiglitazone is effective in both treatment and prevention. Ablation of PPARγ with small interfering RNA or GW9662-mediated inhibition of PPARγ abolished the effect of rosiglitazone on HMGB1 release. Furthermore, the overexpression of PPARγ markedly potentiated the inhibitory effect of rosiglitazone on HMGB1 release. In addition, rosiglitazone inhibited LPS-induced expression of Toll-like receptor 4 signal molecules, suggesting a possible mechanism by which rosiglitazone modulates HMGB1 release. Notably, the administration of rosiglitazone to mice improved survival rates in an LPS-induced animal model of endotoxemia, where reduced levels of circulating HMGB1 were demonstrated. Taken together, these results suggest that PPARs play an important role in the cellular response to inflammation by inhibiting HMGB1 release.

PPARδ Coordinates Angiotensin II-induced Senescence in Vascular Smooth Muscle Cells through PTEN-mediated Inhibition of Superoxide Generation

Background: PPARδ is a ligand-activated transcriptional factor that has been implicated in the vascular homeostasis. Results: Activation of PPARδ significantly attenuated Ang II-induced senescence of VSMCs by up-regulation of PTEN and ensuing modulation of the PI3K/Akt signaling. Conclusion: PPARδ inhibits Ang II-induced senescence of VSMCs via PTEN-mediated inhibition of ROS generation. Significance: PPARδ provides a novel insight into the treatment of atherosclerotic vascular disease. Cellular senescence-associated changes in blood vessels have been implicated in aging and age-related cardiovascular disorders. Here, we demonstrate that peroxisome proliferator-activated receptor (PPAR) δ coordinates angiotensin (Ang) II-induced senescence of human vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly attenuated Ang II-induced generation of superoxides and suppressed senescence of VSMCs. A marked increase in the levels of p53 and p21 induced by Ang II was blunted by the treatment with GW501516. Ligand-activated PPARδ up-regulated expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and suppressed the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Knockdown of PTEN with siRNA abrogated the effects of PPARδ on cellular senescence, on PI3K/Akt signaling, and on generation of ROS in VSMCs treated with Ang II. Finally, administration of GW501516 to apoE-deficient mice treated with Ang II significantly reduced the number of senescent cells in the aorta, where up-regulation of PTEN with reduced levels of phosphorylated Akt and ROS was demonstrated. Thus, ligand-activated PPARδ confers resistance to Ang II-induced senescence by up-regulation of PTEN and ensuing modulation of the PI3K/Akt signaling to reduce ROS generation in vascular cells.

Activation of PPARδ counteracts angiotensin II-induced ROS generation by inhibiting rac1 translocation in vascular smooth muscle cells.

Abstract Angiotensin II (Ang II)-mediated modification of the redox milieu of vascular smooth muscle cells (VSMCs) has been implicated in several pathophysiological processes, including cell proliferation, migration and differentiation. In this study, we demonstrate that the peroxisome proliferator-activated receptor (PPAR) δ counteracts Ang II-induced production of reactive oxygen species (ROS) in VSMCs. Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly reduced Ang II-induced ROS generation in VSMCs. This effect was, however, reversed in the presence of small interfering (si)RNA against PPARδ. The marked increase in ROS levels induced by Ang II was also eliminated by the inhibition of phosphatidylinositol 3-kinase (PI3K) but not of protein kinase C, suggesting the involvement of the PI3K/Akt signalling pathway in this process. Accordingly, ablation of Akt with siRNA further enhanced the inhibitory effects of GW501516 in Ang II-induced superoxide production. Ligand-activated PPARδ also blocked Ang II-induced translocation of Rac1 to the cell membrane, inhibiting the activation of NADPH oxidases and consequently ROS generation. These results indicate that ligand-activated PPARδ plays an important role in the cellular response to oxidative stress by decreasing ROS generated by Ang II in vascular cells.

Ligand-activated PPARδ inhibits UVB-induced senescence of human keratinocytes via PTEN-mediated inhibition of superoxide production

UV radiation-mediated photodamage to the skin has been implicated in premature aging and photoaging-related skin cancer and melanoma. Little is known about the cellular events that underlie premature senescence, or how to impede these events. In the present study we demonstrate that PPARδ (peroxisome-proliferator-activated receptor δ) regulates UVB-induced premature senescence of normal keratinocytes. Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly attenuated UVB-mediated generation of ROS (reactive oxygen species) and suppressed senescence of human keratinocytes. Ligand-activated PPARδ up-regulated the expression of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and suppressed the PI3K (phosphatidylinositol 3-kinase)/Akt pathway. Concomitantly, translocation of Rac1 to the plasma membrane, which leads to the activation of NADPH oxidases and generation of ROS, was significantly attenuated. siRNA (small interfering RNA)-mediated knockdown of PTEN abrogated the effects of PPARδ on cellular senescence, on PI3K/Akt/Rac1 signalling and on generation of ROS in keratinocytes exposed to UVB. Finally, when HR-1 hairless mice were treated with GW501516 before exposure to UVB, the number of senescent cells in the skin was significantly reduced. Thus ligand-activated PPARδ confers resistance to UVB-induced cellular senescence by up-regulating PTEN and thereby modulating PI3K/Akt/Rac1 signalling to reduce ROS generation in keratinocytes.

Ligand-activated PPARδ upregulates α-smooth muscle actin expression in human dermal fibroblasts: A potential role for PPARδ in wound healing

BACKGROUND The phenotypic changes that accompany differentiation of resident fibroblasts into myofibroblasts are important aspects of the wound healing process. Recent studies showed that peroxisome proliferator-activated receptor (PPAR) δ plays a critical role in wound healing. OBJECTIVE To determine whether the nuclear receptor PPARδ can modulate the differentiation of human dermal fibroblasts (HDFs) into myofibroblasts. METHODS These studies were undertaken in primary HDFs using Western blot analyses, small interfering (si)RNA-mediated gene silencing, reporter gene assays, chromatin immunoprecipitation (ChIP), migration assays, collagen gel contraction assays, and real-time PCR. RESULTS Activation of PPARδ by GW501516, a specific ligand of PPARδ, specifically upregulated the myofibroblast marker α-smooth muscle actin (α-SMA) in a time- and concentration-dependent manner. This induction was significantly inhibited by the presence of siRNA against PPARδ, indicating that PPARδ is involved in myofibroblast transdifferentiation of HDFs. Ligand-activated PPARδ increased α-SMA promoter activity in a dual mode by directly binding a direct repeat-1 (DR1) site in the α-SMA promoter, and by inducing expression of transforming growth factor (TGF)-β, whose downstream effector Smad3 interacts with a Smad-binding element (SBE) in another region of the promoter. Mutations in these cis-elements totally abrogated transcriptional activation of the α-SMA gene by the PPARδ ligand; thus both sites represent novel types of PPARδ response elements. GW501516-activated PPARδ also increased the migration and contractile properties of HDFs, as demonstrated by Transwell and collagen lattice contraction assays, respectively. In addition, PPARδ-mediated upregulation of α-SMA was correlated with elevated expression of myofibroblast markers such as collagen I and fibronectin, with a concomitant reduction in expression of the epithelial marker E-cadherin. CONCLUSION PPARδ plays pivotal roles in wound healing by promoting fibroblast-to-myofibroblast differentiation via TGF-β/Smad3 signaling.

Activation of Peroxisome Proliferator-Activated Receptor δ Inhibits Angiotensin II-Induced Activation of Matrix Metalloproteinase-2 in Vascular Smooth Muscle Cells

We investigated the role of peroxisome proliferator-activated receptor (PPAR) δ on angiotensin (Ang) II-induced activation of matrix metalloproteinase (MMP)-2 in vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand for PPARδ, attenuated Ang II-induced activation of MMP-2 in a concentration-dependent manner. GW501516 also inhibited the generation of reactive oxygen species in VSMCs treated with Ang II. A marked increase in the mRNA levels of tissue inhibitor of metalloproteinase (TIMP)-2 and -3, endogenous antagonists of MMPs, was also observed in GW501516-treated VSMCs. These effects were markedly reduced in the presence of siRNAs against PPARδ, indicating that the effects of GW501516 are PPARδ dependent. Among the protein kinases inhibited by GW501516, suppression of phosphatidylinositol 3-kinase/Akt signaling was shown to have the greatest effect on activation of MMP-2 in VSMCs treated with Ang II. Concomitantly, GW501516-mediated inhibition of MMP-2 activation in VSMCs treated with Ang II was associated with the suppression of cell migration to levels approaching those in cells not exposed to Ang II. Thus, activation of PPARδ confers resistance to Ang II-induced degradation of the extracellular matrix by upregulating expression of its endogenous inhibitor TIMP and thereby modulating cellular responses to Ang II in vascular cells.

PPARδ reduces abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E-deficient mice by regulating extracellular matrix homeostasis and inflammatory responses

BACKGROUND Abdominal aortic aneurysm (AAA) is an inflammatory disorder characterized by a localized degradation of connective tissue and apoptosis of vascular smooth muscle cells. This study examined whether the ligand-activated peroxisome proliferator-activated receptor (PPAR) δ can directly antagonize angiotensin II (Ang II)-induced AAA formation in apoE-deficient mice. METHODS AND RESULTS Six-month-old male apoE-deficient mice were infused with Ang II and/or GW501516 (1.44 and 3.3mg/kg/day, respectively) via osmotic mini-pumps. At day 28, aortic size was measured and tissues were collected for analyses. Co-infusion of GW501516, an activator of PPARδ, attenuated both the incidence and the severity of Ang II-induced AAA in apoE-deficient mice. Ligand-activated PPARδ also reduced infiltration of macrophages, resulting in significant decreases in chemotactic proteins such as monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and inducible nitric oxide synthase. The anti-inflammatory effect of GW501516 was associated with the suppression of apoptotic cell death, along with the inhibition of medial smooth muscle cell loss and focal elastin destruction, which leads to a medial dissection and aortic rupture. These ameliorative effects of GW501516 on Ang II-induced aneurysm were correlated with increased expression of extracellular matrix (ECM) proteins, such as types I and III collagen, fibronectin, and elastin, along with the up-regulation of transforming growth factor-β1. In addition, ligand-activated PPARδ also increased the expression of tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3, while it strongly suppressed that of matrix metalloproteinase-2. CONCLUSIONS PPARδ attenuates Ang II-induced AAA formation by regulating ECM homeostasis and inflammatory responses, suggesting a novel strategy for the treatment of AAA.

Peroxisome proliferator-activated receptor δ inhibits Porphyromonas gingivalis lipopolysaccharide-induced activation of matrix metalloproteinase-2 by downregulating NADPH oxidase 4 in human gingival fibroblasts.

We investigated the roles of peroxisome proliferator-activated receptor δ (PPARδ) in Porphyromonas gingivalis-derived lipopolysaccharide (Pg-LPS)-induced activation of matrix metalloproteinase 2 (MMP-2). In human gingival fibroblasts (HGFs), activation of PPARδ by GW501516, a specific ligand of PPARδ, inhibited Pg-LPS-induced activation of MMP-2 and generation of reactive oxygen species (ROS), which was associated with reduced expression of NADPH oxidase 4 (Nox4). These effects were significantly smaller in the presence of small interfering RNA targeting PPARδ or the specific PPARδ inhibitor GSK0660, indicating that PPARδ is involved in these events. In addition, modulation of Nox4 expression by small interfering RNA influenced the effect of PPARδ on MMP-2 activity, suggesting a mechanism in which Nox4-derived ROS modulates MMP-2 activity. Furthermore, c-Jun N-terminal kinase and p38, but not extracellular signal-regulated kinase, mediated PPARδ-dependent inhibition of MMP-2 activity in HGFs treated with Pg-LPS. Concomitantly, PPARδ-mediated inhibition of MMP-2 activity was associated with the restoration of types I and III collagen to levels approaching those in HGFs not treated with Pg-LPS. These results indicate that PPARδ-mediated downregulation of Nox4 modulates cellular redox status, which in turn plays a critical role in extracellular matrix homeostasis through ROS-dependent regulation of MMP-2 activity.