Jesang Ko

C-Reactive Protein Promotes Monocyte Chemoattractant Protein-1—Mediated Chemotaxis Through Upregulating CC Chemokine Receptor 2 Expression in Human Monocytes

Background—Inflammation plays a crucial role in atherosclerosis. An elevated serum C-reactive protein (CRP) level is a strong marker for future atherosclerotic cardiovascular diseases. In addition, recent data suggest that CRP may directly promote atherogenesis. In this study, we investigated whether CRP can directly activate human circulating monocytes. Methods and Results—Incubation of THP-1 monocytes with CRP (10 μg/mL) increased CC chemokine receptor 2 (CCR2) expression at both the protein and transcript levels, which in turn enhanced chemotaxis mediated by monocyte chemoattractant protein-1 (MCP-1) up to 2-fold. The CRP-induced upregulation of CCR2 expression involved binding of CRP to the FcγR, most notably FcγRI, and phospholipase D1 activation. Serum high-sensitivity CRP levels in 52 normocholesterolemic human subjects were positively correlated with CCR2 surface expression on circulating monocytes (r = 0.62, P <0.001) and MCP-1–mediated monocyte chemotaxis (r = 0.53, P <0.001). Conclusions—Elevated blood CRP levels may promote accumulation of monocytes in the atherogenic arterial wall by increasing chemotactic activities of monocytes in response to MCP-1.

HMG-CoA Reductase Inhibition Reduces Monocyte CC Chemokine Receptor 2 Expression and Monocyte Chemoattractant Protein-1–Mediated Monocyte Recruitment In Vivo

Background—The migration of circulating monocytes to the arterial wall during atherogenesis is largely modulated by activation of the CC chemokine receptor 2 (CCR2), a dominant monocyte chemotaxis receptor. The present study investigated whether 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition affects CCR2 gene expression and CCR2-dependent monocyte recruitment. Methods and Results—Competitive reverse transcription-polymerase chain reaction analysis and flow cytometry showed that simvastatin, an HMG-CoA reductase inhibitor, dose-dependently reduced monocyte CCR2 mRNA and protein expression. Treatment of 21 normocholesterolemic men with simvastatin (20 mg/d for 2 weeks) decreased CCR2 protein and mRNA expression in circulating monocytes. Promoter and electrophoretic mobility shift assays showed that simvastatin activated a peroxisome proliferator response element in THP-1 monocytes. Moreover, simvastatin-induced CCR2 downregulation was completely reversed by the synthetic peroxisome proliferator-activated receptor-&ggr; antagonist GW9662. Simvastatin-treated monocytes showed little chemotaxis movement in response to monocyte chemoattractant protein-1 (MCP-1), a specific CCR2 ligand. Treatment of C57/BL6 mice with simvastatin (0.2 &mgr;g/g body weight IP, daily for 1 week) inhibited transmigration of CD80+ monocytes to the MCP-1–injected intraperitoneal space. Moreover, few circulating inflammatory cells from simvastatin-treated Sprague-Dawley rats (0.2 &mgr;g/g body weight IP, daily for 2 weeks) were recruited to the aortic wall of hypercholesterolemic littermates. Conclusions—The inhibition of CCR2/MCP-1–dependent monocyte recruitment by simvastatin may prevent excessive accumulation of monocytes in the arterial wall during atherogenesis.

Expression and Purification of Enzymatically Active Forms of the Human Lysyl Oxidase-like Protein 4

The lysyl oxidase-like protein 4 (LOXL4) is the latest member of the emerging family of lysyl oxidases, several of which were shown to function as copper-dependent amine oxidases catalyzing lysine-derived cross-links in extracellular matrix proteins. LOXL4 contains four scavenger receptor cysteine-rich domains in addition to the characteristic domains of the LOX family, including the copper-binding domain, the cytokine receptor-like domain, and the residues of the lysyl-tyrosyl quinone cofactor. In an effort to assess its amine oxidase activity, we expressed LOXL4 as recombinant forms attached with hexa-histidine residues at the carboxyl terminus by using an Escherichia coli expression system. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis. The purified LOXL4 proteins showed β-aminopropionitrile-inhibitable activity of 0.022-0.032 units/mg toward a nonpeptidyl substrate, benzylamine. These results indicate that LOXL4, with the four scavenger receptor cysteine rich domains, may also function as an active amine oxidase. Availability of the pure and active forms of LOXL4 will be significantly helpful in functional studies related to substrate specificity and crystal structure of this amine oxidase, which should provide significant insights into functional differences within the LOX family members.

Identification and differential expression of novel human cervical cancer oncogene HCCR-2 in human cancers and its involvement in p53 stabilization.

Basic studies of oncogenesis have demonstrated that either the elevated production of particular oncogene proteins or the occurrence of qualitative abnormalities in oncogenes can contribute to neoplastic cellular transformation. The purpose of this study was to identify unique oncogenes that are differentially expressed in human cancers and characterize their functions in tumorigenesis. To discover new putative oncogenes, the differential display RT–PCR method was applied using normal cervical tissues, cervical cancer cell lines, cervical cancer tissues, and metastatic tissues. We identified a new human cervical cancer oncogene HCCR-2 that was overexpressed in various human tumors including leukemia, lymphoma, and carcinomas of the breast, kidney, ovary, stomach, colon, and uterine cervix. Ectopic expression of HCCR-2 resulted in direct tumorigenic conversions of NIH/3T3 and Rat1 fibroblasts. Nude mice injected with NIH/3T3 cells stably transfected with HCCR-2 formed tumors in 4 weeks. The resultant tumors display characteristics of an epithelial carcinoma. In HCCR-2 transfected NCI-H460 cells and RKO cells, stabilization of the p53 tumor suppressor occurred without genetic mutation and correlated with functional impairment, as indicated by the defective induction of p53-induced p21WAF1, MDM2, and bax. These results indicate that HCCR-2 probably represents a new oncogene that is related to tumorigenesis, functioning as a negative regulator of the p53 tumor suppressor.

Angiogenic activity of human CC chemokine CCL15 in vitro and in vivo

CCL15 is a novel human CC chemokine and exerts its biological activities on immune cells through CCR1 and CCR3. Because a number of chemokines induce angiogenesis and endothelial cells express CCR1 and CCR3, we investigated the angiogenic activity of CCL15. Both CCL15(1‐92) and N‐terminal truncated CCL15(25‐92) stimulate the chemotactic endothelial cell migration and differentiation, but CCL15(25‐92) is at least 100‐fold more potent than CCL15(1‐92). Treatment with pertussis toxin (PTX), with anti‐CCR1, or with anti‐CCR3 antibody inhibits the CCL15(25‐92)‐induced endothelial cell migration. CCL15(25‐92) also stimulates sprouting of vessels from aortic rings and mediates angiogenesis in the chick chorioallantoic membrane assay. Our findings demonstrate that CCL15(25‐92) has in vitro and in vivo angiogenic activity, and suggest roles of the chemokine in angiogenesis.

Differential effects of annexins I, II, III, and V on cytosolic phospholipase A2 activity: specific interaction model.

Annexins (ANXs) are a family of proteins with calcium‐dependent phospholipid binding properties. Although inhibition of phospholipase A2 (PLA2) by ANX‐I has been reported, the mechanism is still controversial. Previously we proposed a ‘specific interaction’ model for the mechanism of cytosolic PLA2 (cPLA2) inhibition by ANX‐I [Kim et al., FEBS Lett. 343 (1994) 251–255]. Here we have studied the cPLA2 inhibition mechanism using ANX‐I, N‐terminally deleted ANX‐I (ΔANX‐I), ANX‐II, ANX‐II2P112, ANX‐III, and ANX‐V. Under the conditions for the specific interaction model, ANX‐I, ΔANX‐I, and ANX‐II2P112 inhibited cPLA2, whereas inhibition by ANX‐II and ANX‐III was negligible. Inhibition by ANX‐V was much smaller than that by ANX‐I. The protein–protein interactions between cPLA2 and ANX‐I, ΔANX‐I, and ANX‐II2P112 were verified by immunoprecipitation. We can therefore conclude that inhibition of cPLA2 by specific interaction is not a general function of all ANXs, and is rather a specific function of ANX‐I. The results are consistent with the specific interaction model.

Lysophosphatidylcholine up-regulates CXCR4 chemokine receptor expression in human CD4 T cells

Oxidized low‐density lipoprotein (OxLDL) is an inflammatory modulator in the atherosclerotic plaque. We examined the effect of lysophosphatidylcholine (lysoPC), a main phospholipid component of OxLDL, on inflammatory responses in human CD4 T cells. We found that lysoPC dose‐ and time‐dependently increased expression of CXCR4, the chemokine receptor on CD4 T cells. This increase was inhibited by caffeic acid phenethyl ester or SN50, nuclear factor‐κB inhibitors, and also by suppression of G2A expression, the specific receptor for lysoPC, using antisense oligonucleotide. lysoPC enhanced CD4 T cell chemotaxis in response to stromal cell‐derived factor‐1 (SDF‐1), the exclusive ligand for CXCR4. lysoPC also enhanced SDF‐1‐stimulated production of inflammatory cytokines interleukin‐2 and interferon‐γ by CD4 T cells activated by anti‐CD3 immunoglobulin G. In conclusion, this study demonstrates that lysoPC directly modulates inflammatory responses in human CD4 T cells. The data suggest that the presence of lysoPC and SDF‐1 in atherosclerotic lesions may trigger inflammatory responses mediated by CD4 T cells, which may play an important role in progression of atherosclerosis.

Human LZIP binds to CCR1 and differentially affects the chemotactic activities of CCR1-dependent chemokines

Signaling molecules that bind to chemokine receptors should play key roles in regulation of cell migration induced by chemokines. To characterize the CCR1‐mediated cellular signal transduction mechanism, we used the yeast two‐hybrid system to identify a cellular ligand for CCR1. LZIP, which has been known as a transcription factor in various cell types, was identified as a CCR1 binding protein. Although the ability of LZIP to bind DNA is possibly what allows it to function as a transcription factor, its detailed function and participation in chemotaxis have not been established. We found that LZIP binds to CCR1 based on results of a mammalian two‐ hybrid assay and immunoprecipitation experiments. The 21‐260 residues of LZIP were essential for interaction with CCR1. Results from a chemotaxis assay using LZIP transfected cells showed that LZIP enhanced Lkn‐1‐induced chemotaxis, whereas the chemotactic activities induced by other CC chemokines that bind to CCR1, including MIP‐1α, RANTES, or HCC‐4, were not affected by LZIP overexpression. These data indicate that LZIP binds to CCR1 and that the interaction between CCR1 and LZIP participates in regulation of Lkn‐1‐dependent cell migration without affecting the chemotactic activities of other CC chemokines that bind to CCR1.

Transgenic mouse model for breast cancer: Induction of breast cancer in novel oncogene HCCR-2 transgenic mice

Transgenic mice containing novel oncogene HCCR-2 were generated to analyse the phenotype and to characterize the role of HCCR-2 in cellular events. Mice transgenic for HCCR-2 developed breast cancers and metastasis. The level of p53 in HCCR-2 transgenic mice was elevated in most tissues including breast, brain, heart, lung, liver, stomach, kidney, spleen, and lymph node. We examined whether stabilized p53 is functional in HCCR-2 transgenic mice. Defective induction of p53 responsive genes including p21WAF1, MDM2, and bax indicates that stabilized p53 in HCCR-2 transgenic mice exists in an inactive form. These results suggest that HCCR-2 represents an oncoprotein that is related to breast cancer development and regulation of the p53 tumor suppressor.

Leukotactin-1/CCL15-induced chemotaxis signaling through CCR1 in HOS cells

Leukotactin‐1 (Lkn‐1)/CCL15 is a recently cloned CC‐chemokine that binds to the CCR1 and CCR3. Although Lkn‐1 has been known to function as a chemoattractant for neutrophils, monocytes and lymphocytes, its cellular mechanism remains unclear. To understand the mechanism of Lkn‐1‐induced chemotaxis signaling, we examined the chemotactic activities of human osteogenic sarcoma cells expressing CCR1 in response to Lkn‐1 using inhibitors of signaling molecules. Inhibitors of Gi/Go protein, phospholipase C (PLC) and protein kinase Cδ (PKCδ) inhibited the chemotactic activity of Lkn‐1 indicating that Lkn‐1‐induced chemotaxis signal is transduced through Gi/Go protein, PLC and PKCδ. The activities of PLC and PKCδ were also enhanced by Lkn‐1 stimulation. Chemotactic activity of Lkn‐1 was inhibited by the treatment of cycloheximide and actinomycin D suggesting that newly synthesized proteins are needed for chemotaxis. Nuclear factor‐κB (NF‐κB) inhibitor reduced chemotactic activity of Lkn‐1. DNA binding activity of NF‐κB was also enhanced by Lkn‐1 stimulation. These results suggest that Lkn‐1 transduces the signal through Gi/Go protein, PLC, PKCδ, NF‐κB and newly synthesized proteins for chemotaxis.