Soo Hyuk Kim

Ubiquitin ligase CHIP induces TRAF2 proteasomal degradation and NF‐κB inactivation to regulate breast cancer cell invasion

Transcriptional factor nuclear factor‐kappaB (NF‐κB) plays a crucial role in human breast cancer cell invasion and metastasis. The carboxyl terminus of Hsc70‐interacting protein (CHIP) is a U‐box‐type ubiquitin ligase that induces ubiquitination and proteasomal degradation of its substrate proteins. In this study, we investigated the role of CHIP in the NF‐κB pathway in the invasion of MDA‐MB‐231 cells, a highly aggressive breast cancer cell line. We showed that overexpression of CHIP significantly inhibits the invasion of the MDA‐MB‐231 cells. The overexpression of CHIP suppressed expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase‐9 (MMP‐9) in MDA‐MB‐231 cells. Moreover, CHIP strongly inhibited the nuclear localization and the transcriptional activity of NF‐κB. The activation of the IkappaB kinase complex (IKK) was also blocked by CHIP overexpression. Importantly, CHIP overexpression resulted in a significant decrease in the level of TNF receptor‐associated factor 2 (TRAF2), an upstream key player in the NF‐κB pathway. However, the level of TRAF2 was restored after treatment with a proteasome inhibitor, MG‐132. Moreover, CHIP overexpression promoted the ubiquitination of TRAF2. We also found cell invasion significantly decreased in cells transfected with TRAF2 small interfering RNA (siRNA). In contrast, when CHIP expression was suppressed by siRNA in poorly invasive MCF‐7 cells, cell invasion significantly increased in conjunction with enhanced NF‐κB activation and TRAF2 levels. Taken together, these results suggest that CHIP regulates NF‐κB‐mediated cell invasion via the down‐regulation of TRAF2. J. Cell. Biochem. 112: 3612–3620, 2011.

Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.

Retraction: The Effect of Soluble RAGE on Inhibition of Angiotensin II-Mediated Atherosclerosis in Apolipoprotein E Deficient Mice

Background The cross talk between RAGE and angiotensin II (AngII) activation may be important in the development of atherosclerosis. Soluble RAGE (sRAGE), a truncated soluble form of the receptor, acts as a decoy and prevents the inflammatory response mediated by RAGE activation. In this study, we sought to determine the effect of sRAGE in inhibiting AngII-induced atherosclerosis in apolipoprotein E knockout mice (Apo E KO). Methods and Results 9 week old Apo E KO mice were infused subcutaneously with AngII (1 µg/min/kg) and saline for 4 weeks using osmotic mini-pumps. The mice were divided into 4 groups 1. saline infusion and saline injection; 2. saline infusion and sRAGE injection; 3. AngII infusion and saline injection; 4. AngII infusion and sRAGE injection. Saline or 0.5 µg, 1 µg, to 2 µg/day/mouse of sRAGE were injected intraperitoneally daily for 28 days. We showed that atherosclerotic plaque areas in the AngII-infused Apo E KO mice and markers of inflammation such as RAGE, ICAM-1, VCAM-1, and MCP-1 were increased in aorta compared to that of the Apo E KO mice. However, the treatment of 0.5 µg, 1 µg, and 2 µg of sRAGE in AngII group resulted in the dose-dependent decrease in atherosclerotic plaque area. We also demonstrated that sRAGE decreased RAGE expression level as well as inflammatory cytokines and cell adhesion molecules in AngII or HMGB1 treated-rat aorta vascular smooth muscle cells. Conclusion The results demonstrated that partical blockade of RAGE activation by sRAGE prevent AngII -induced atherosclerosis. Therefore these results suggested that first, RAGE activation may be important in mediating AngII-induced atherogenesis, and second, AngII activation is a major pathway in the development of atherosclerosis. Taken together, results from this study may provide the basis for future anti- atherosclerotic drug development mediated through RAGE activation.

Protective effect of survivin in Doxorubicin-induced cell death in h9c2 cardiac myocytes.

Background and Objectives Apoptosis has been known to be an important mechanism of doxorubicin-induced cardiotoxicity. Survivin, which belongs to the inhibitor of apoptosis protein family, is associated with apoptosis and alteration of the cardiac myocyte molecular pathways. Therefore, we investigated the anti-apoptotic effect and cellular mechanisms of survivin using a protein delivery system in a doxorubicin-induced cardiac myocyte injury model. Materials and Methods We constructed a recombinant survivin which was fused to the protein transduction domain derived from HIV-TAT protein. In cultured H9c2 cardiac myocytes, TAT-survivin (1 µM) was added for 1 hour prior to doxorubicin (1 µM) treatment for 24 hours. Cell viability and apoptosis were evaluated by 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, caspase-3 activity, and terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay. We measured the expression levels of several apoptosis-related signal proteins. Results The survivin level was significantly reduced in a dose dependent manner up to 1 µM of doxorubicin in concentration. Purified recombinant TAT-survivin protein was efficiently delivered to H9c2 cardiac myocytes, and its transduction showed an anti-apoptotic effect, demonstrated by reduced caspase-3 activity and the apoptotic index, concomitantly with increased cell viability against doxorubicin injury. The phosphorylation of p38 mitogen-activated protein (MAP) kinase and the release of Smac from mitochondria were suppressed and the expression levels of Bcl-2 and cAMP response element-binding protein (CREB), the transcription factor of Bcl-2, were recovered following TAT-survivin transduction, indicating that survivin had an anti-apoptotic effect against doxorubicin injury. Conclusion Our results suggest that survivin has a potentially cytoprotective effect against doxorubicin-induced cardiac myocyte apoptosis through mechanisms that involve a decrease in the phosphorylation of p38 MAP kinase, mitochondrial Smac release, and increased expression of Bcl-2 and CREB.

C-reactive protein induces p53-mediated cell cycle arrest in H9c2 cardiac myocytes

C-reactive protein (CRP) is one of the most important biomarker for cardiovascular diseases. Recent studies have shown that CRP affects cell survival, differentiation and apoptosis. However, the effect of CRP on the cell cycle has not been studied yet. We investigated the cell cycle alterations and cellular mechanisms induced by CRP in H9c2 cardiac myocytes. Flow cytometry analysis showed that CRP-treated H9c2 cells displayed cell cycle arrest in G0/G1 phase. CRP treatment resulted in a significant reduction in the levels of CDK4, CDK6 and cyclin D1 in a concentration-dependent manner. Interestingly, CRP caused an increase in the p53 accumulation and its phosphorylation on Ser15, leading to induce p21 upregulation. Treatment with a specific p53 inhibitor, PFT-α restored the levels of CDK4 and CDK6. A significant increase of ERK1/2 phosphorylation level was detected in CRP-treated cells. Furthermore, pretreatment of a specific ERK inhibitor resulted in decreased p53 phosphorylation and p21 induction. ERK inhibitor pretreatment induced significant restoration of protein levels of CDK4 and CDK6, leading to re-entry into the cell cycle. In addition, increased phosphorylation of p53 and ERK induced by CRP was considerably reversed by Fc gamma receptor IIIa (FcγRIIIa) knock-down using siRNA. FcγRIIIa siRNA transfection also restored the levels of cell cycle proteins. Our study has provided the first proposal on the novel insights into how CRP directly affects cell cycle in cells.

C-Reactive Protein Inhibits Survivin Expression via Akt/mTOR Pathway Downregulation by PTEN Expression in Cardiac Myocytes

C-reactive protein (CRP) is one of the most important biomarkers for arteriosclerosis and cardiovascular disease. Recent studies have shown that CRP affects cell cycle and inflammatory process in cardiac myocytes. Survivin is also involved in cardiac myocytes replication and apoptosis. Reduction of survivin expression is associated with less favorable cardiac remodeling in animal models. However, the effect of CRP on survivin expression and its cellular mechanism has not yet been studied. We demonstrated that treatment of CRP resulted in a significant decrease of survivin protein expression in a concentration-dependent manner in cardiac myocytes. The upstream signaling proteins of survivin, such as Akt, mTOR and p70S6K, were also downregulated by CRP treatment. In addition, CRP increased the protein and mRNA levels of PTEN. The siRNA transfection or specific inhibitor treatment for PTEN restored the CRP-induced downregulation of Akt/mTOR/p70S6K pathway and survivin protein expression. Moreover, pretreatment with a specific p53 inhibitor decreased the CRP-induced PTEN expression. ERK-specific inhibitor also blocked the p53 phosphorylation and PTEN expression induced by CRP. Our study provides a novel insight into CRP-induced downregulation of survivin protein expression in cardiac myocytes through mechanisms that involved in downregulation of Akt/mTOR/p70S6K pathway by expression of PTEN.

PPARα agonists inhibit inflammatory activation of macrophages through upregulation of β-defensin 1

BACKGROUND Effects of peroxisome proliferator-activated receptor alpha (PPARα) agonists on cardiovascular outcome have been controversial. Although these agents primarily affect lipoprotein metabolism, their pleiotropic anti-inflammatory effect is one of the potential anti-atherosclerotic mechanisms. This study aimed to evaluate the effect of fenofibrate and gemfibrozil on inflammation in macrophages and reveal pathways these agents may affect. METHODS AND RESULTS The two PPARα agonists inhibited secretion of CXCL2, TNF-α, IL-6, activation of p65 of NF-κB, ERK, and TLR4 expression. These changes occurred simultaneously with upregulation and secretion of β-defensin 1, an inflammation-modulating peptide. To demonstrate the role of β-defensin 1, it was knocked-down by target-specific siRNA. The effects of PPARα agonists on TLR4 expression and chemokine secretion were obviously abrogated with this treatment. In experiments investigating whether β-defensin 1 acts extracellularly, inflammatory chemokines decreased significantly after the addition of recombinant β-defensin 1 or conditioned media to cells. In experiments designed to clarify if the effects of the two agents are PPARα-dependent, induction of mRNA and secretion β-defensin 1 and inhibition of chemokine release were clearly reduced with GW6471, a PPARα blocker. CONCLUSIONS Our results reveal the pathways by which fenofibrate and gemfibrozil inhibit LPS-induced inflammatory activation of macrophages. This study elucidated a novel anti-inflammatory mechanism that acts through PPARα, β-defensin 1, and TLR4 pathways.

Matrix metalloproteinase‐1 induces cleavage of exogenous alphab‐crystallin transduced by a cell‐penetrating peptide

Cell‐penetrating peptides (CPPs), including TAT‐CPP, have been used to deliver exogenous proteins into living cells. Although a number of proteins fused to TAT‐CPP can be delivered into various cells, little is known about the proteolytic cleavage of TAT‐fusion proteins in cells. In this study, we demonstrate that a small heat shock protein (sHSP), alphaB‐crystallin (αB‐crystallin), delivered by TAT‐CPP is susceptible to proteolytic cleavage by matrix metalloproteinase‐1 (MMP‐1) in cardiac myoblast H9c2 cells. Recombinant TAT‐αB‐crystallin was efficiently transduced into H9c2 cells. For a few hours following protein transduction, generation of a 14‐kDa fragment, a cleavage band of TAT‐αB‐crystallin, increased in a time‐dependent manner. This fragment was observed only in detergent‐insoluble fractions. Interestingly, treatment with MMP inhibitors blocked the cleavage of TAT‐αB‐crystallin. In test tubes, recombinant MMP‐1 processed TAT‐αB‐crystallin to generate the major cleavage fragment 14‐kDa, as observed in the cells treated with TAT‐αB‐crystallin. The N‐terminal sequences of the 14‐kDa fragment were identified as Leu‐Arg‐Ala‐Pro‐Ser‐Trp‐Phe, indicating that this fragment is generated by cleavage at Phe54‐Leu55 of αB‐crystallin. The MMP‐1‐selective inhibitor abolished the production of 14‐kDa fragments in cells. In addition, the cleaved fragment of TAT‐αB‐crystallin was significantly reduced in cells transfected with MMP‐1 siRNA. Moreover, the enzymatic activity of MMP‐1 was markedly increased in TAT‐αB‐crystallin‐treated cells. TAT‐αB‐crystallin has a cytoprotective effect on H9c2 cells under hypoxic insult, moreover, MMP‐1‐selective inhibitor treatment led to even increased cell viability. These results suggest that MMP‐1 is responsible for proteolytic cleavage of TAT‐αB‐crystallin during its intracellular transduction in H9c2 cells. J. Cell. Biochem. 112: 2454–2462, 2011.