Il-Chul Kim

Wnt-7a Causes Loss of Differentiated Phenotype and Inhibits Apoptosis of Articular Chondrocytes via Different Mechanisms

Although regulation of chondrogenesis and cartilage development by Wnt signaling is well established, the function of Wnt in the maintenance and destruction of cartilage remains largely unknown. Here we investigated the involvement and regulatory mechanisms of Wnt signaling in cartilage destruction. We found that interleukin-1β, the primary pro-inflammatory cytokine involved in cartilage destruction, induces expression of Wnt-5a and -7a in primary culture articular chondrocytes. The level of β-catenin was also increased in chondrocytes of arthritic cartilage, suggesting the association of Wnt/β-catenin signaling with arthritic cartilage destruction. In addition, our results show that Wnt-7a induces dedifferentiation and inhibits NO-induced apoptosis of primary culture articular chondrocytes. Wnt-7a induces dedifferentiation of articular chondrocytes by stimulating transcriptional activity of β-catenin, whereas NO-induced apoptosis is inhibited via the activation of cell survival signaling, such as phosphatidylinositol 3-kinase and Akt, which block apoptotic signaling cascade. Our results collectively suggest that Wnt-7a is associated with cartilage destruction by regulating the maintenance of differentiation status and the apoptosis of articular chondrocytes via different mechanisms.

Identification of a serodiagnostic antigen, legumain, by immunoproteomic analysis of excretory-secretory products of Clonorchis sinensis adult worms.

Clonorchis sinensis, the Chinese liver fluke, is the causative agent of clonorchiasis as well as liver and biliary diseases. The excretory‐secretory products (ESPs) of the parasites play important roles in host–parasite interactions. In this study, we have investigated the proteome of ESPs obtained from C. sinensis adult worms. Although the full genome database of C. sinensis is not yet available, we have successfully identified 62 protein spots using 2‐DE‐based mass analysis and EST database of C. sinensis. The proteins identified include detoxification enzymes, such as glutathione S‐transferase and thioredoxin peroxidase, myoglobin and a number of cysteine proteases that are expressed abundantly. In order to identify potential targets for the diagnosis and therapy of clonorchiasis, we conducted immunoblot analysis of the ESPs proteome using the sera obtained from clonorchiasis patients and identified legumains and cysteine proteases as antigens present in the ESPs. Although the cysteine proteases were previously reported to elicit antigenicity, the legumains are found herein for the first time as a serological antigen of C. sinensis. To confirm these findings, we expressed recombinant legumain in Escherichia coli and verified that recombinant legumain also functions as a potent antigen against the sera of clonorchiasis patients. Our results illustrate the validity of immuno‐proteomic approaches in the identification of serodiagnostic antigens in the parasites.

Downregulation of p53 by phosphatase of regenerating liver 3 is mediated by MDM2 and PIRH2.

AIMS The phosphatase of regenerating liver (PRL) family is related to tumorigenesis and metastasis in various cancer types. Its overexpression increases cell motility and proliferation via the downregulation of p21 expression. In a previous study, we reported that PRL-1 downregulates p53 and is a target gene of p53. In this study, we investigated whether a member of the PRL family, PRL-3, could regulate p53 like PRL-1 in cancer cells. MAIN METHODS To elucidate the role of PRL-3 in regulating p53 in cancer cells, we used a cell culture system to measure protein level, transcriptional level, apoptosis or localization. KEY FINDINGS We determined that PRL-3 overexpression reduced the activity of the p21 and p53 reporters. Additionally, the levels of endogenous and exogenous p53 protein were reduced in cells transiently expressing PRL-3, whereas the ablation of PRL-3 by siRNA increased levels of the p53 protein. The downregulation of p53 by PRL-3 inhibited p53-mediated apoptosis. However, the phosphatase-dead mutant C104S, prenylated-site mutant C170S, and C104S/C170S PRL-3 evidenced minimal effects on the downregulation of p53 protein as compared with wild-type PRL-3. Further examinations revealed that PRL-3 expression reduced the stability of p53 by inducing the transcription of p53 induced protein with a RING-H2 domain (PIRH2) through early growth response (EGR) and by increasing the phosphorylation of mouse double minute 2 (MDM2), and then both negatively regulated p53. SIGNIFICANCE These findings demonstrated that PRL-3, like PRL-1, can negatively regulate p53 via the activation of PIRH2 and MDM2 in cancer cells.

Retinoic acid inhibits chondrogenesis of mesenchymal cells by sustaining expression of N‐cadherin and its associated proteins

Retinoic acid (RA) is a well‐known regulator of chondrocyte phenotype. RA inhibits chondrogenic differentiation of mesenchymal cells and also causes loss of differentiated chondrocyte phenotype. The present study investigated the mechanisms underlying RA regulation of chondrogenesis. RA treatment in chondrifying mesenchymal cells did not affect precartilage condensation, but blocked progression from precartilage condensation to cartilage nodule formation. This inhibitory effect of RA was independent of protein kinase C and extracellular signal‐regulated protein kinase, which are positive and negative regulators of cartilage nodule formation, respectively. The progression from precartilage condensation to cartilage nodule requires downregulation of N‐cadherin expression. However, RA treatment caused sustained expression of N‐cadherin and its associated proteins including α‐ and β‐catenin suggesting that modulation of expression of these molecules is associated with RA‐induced inhibition of chondrogenesis. This hypothesis was supported by the observation that disruption of the actin cytoskeleton by cytochalasin D (CD) blocks RA‐induced sustained expression of cell adhesion molecules and overcomes RA‐induced inhibition of chondrogenesis. Taken together, our results suggest RA inhibits chondrogenesis by stabilizing cell‐to‐cell interactions at the post‐precartilage condensation stage. J. Cell. Biochem. 89: 837–847, 2003.

Retinoic acid inhibits adipogenesis via activation of Wnt signaling pathway in 3T3-L1 preadipocytes.

Although retinoic acid (RA) is well known to inhibit the differentiation of 3T3-L1 cells into adipocytes both in vivo and in vitro, its molecular mechanism is not fully understood. In this report, we investigate the inhibitory mechanism of adipocyte differentiation by RA in 3T3-L1 cells. Because both RA and Wnt are known to inhibit adipogenesis at a common step involving the inhibition of PPAR-γ expression, we focused on the crosstalk between these two signaling pathways. We found that RA treatment resulted in a dramatic inhibition of adipogenesis, especially at an early phase of differentiation, and led to increased β-catenin protein expression. Moreover, RA enhances the transcriptional activity of β-catenin as well as Wnt gene expression during adipogenesis. Taken together, the present study demonstrated that Wnt/β-catenin signaling may be associated with the RA-induced suppression of adipogenesis and may cooperatively inhibit adipocyte differentiation.

IRF-2 regulates NF-κB activity by modulating the subcellular localization of NF-κB

Nuclear Factor-kappa B (NF-kappaB) is a transcription factor essential to the control of cell proliferation, survival, differentiation, immune response, and inflammation. Constitutive NF-kappaB activation has been observed in a broad variety of solid tumors and hematological malignancies, which suggests that NF-kappaB signaling may perform a critical role in the development of human cancers. Interferon regulatory factor-2 (IRF-2), an antagonistic transcriptional repressor of IRF-1, evidences oncogenic potential, but little is currently known regarding the mechanism underlying the oncogenic activities of IRF-2. In this study, we report that IRF-2 recruits RelA/p65 transcription factors into the nucleus via physical interaction. While the nuclear recruitment of RelA by IRF-2 augments TNFalpha-induced NF-kappaB dependent transcription, the N-terminal truncated mutant form of IRF-2 inhibits the nuclear localization of RelA, and thus interferes with NF-kappaB activation. Furthermore, the knockdown of IRF-2 by IRF-2 siRNA attenuates TNFalpha-induced NF-kappaB dependent transcription by inhibiting the nuclear localization of RelA. Thus, these results show that IRF-2 regulates NF-kappaB activity via the modulation of NF-kappaB subcellular localization.

Roles of interferon‐gamma and its target genes in schizophrenia: Proteomics‐based reverse genetics from mouse to human

A decreased production of interferon gamma (IFNG) has been observed in acute schizophrenia. In order to explore the possible relationship between IFNG and schizophrenia, we attempted to analyze the differentially expressed proteins in the brains of interferon‐gamma knockout (Ifng‐KO) mice. Five upregulated and five downregulated proteins were identified with 2D gels and MALDI‐TOF/TOF MS analyses in Ifng‐KO mouse brain. Of the identified proteins, we focused on creatine kinase brain (CKB) and triose phosphate isomerase 1 (TPI1). Consistent with the proteomic data, reverse transcriptase‐mediated PCR, immunoblotting, and immunohistochemistry analyses confirmed that the levels of gene expressions of Ckb and Tpi1 were downregulated and upregulated, respectively. When we analyzed the genetic polymorphisms of the single nucleotide polymorphisms (SNPs) of their human orthologous genes in a Korean population, the promoter SNPs of CKB and TPI1 were weakly associated with schizophrenia. In addition, IFNG polymorphisms were associated with schizophrenia. These results suggest that IFNG and proteins affected by IFNG may play a role in the pathogenesis of schizophrenia.

hnRNP L inhibits CD44 V10 exon splicing through interacting with its upstream intron

CD44 is a complex cell adhesion molecule that mediates communication and adhesion between adjacent cells as well as between cells and the extracellular matrix. CD44 pre-mRNA produces various mRNA isoforms through alternative splicing of 20 exons, among which exons 1-5 (C1-C5) and 16-20 (C6-C10) are constant exons, whereas exons 6-15 (V1-V10) are variant exons. CD44 V10 exon has important roles in breast tumor progression and Hodgkin lymphoma. Here we show that increased expression of hnRNP L inhibits V10 exon splicing of CD44 pre-mRNA, whereas reduced expression of hnRNP L promotes V10 exon splicing. In addition, hnRNP L also promotes V10 splicing of endogenous CD44 pre-mRNA. Through mutation analysis, we demonstrate that the effects of hnRNP L on V10 splicing are abolished when the CA-rich sequence on the upstream intron of V10 exon is disrupted. However, hnRNP L effects are stronger if more CA-repeats are provided. Furthermore, we show that hnRNP L directly contacts the CA-rich sequence. Importantly, we provide evidences that hnRNP L inhibits U2AF65 binding on the upstream Py tract of V10 exon. Our results reveal that hnRNP L is a new regulator for CD44 V10 exon splicing.

SUMOylated IRF-1 shows oncogenic potential by mimicking IRF-2

Interferon regulatory factor-1 (IRF-1) is an interferon-induced transcriptional activator that suppresses tumors by impeding cell proliferation. Recently, we demonstrated that the level of SUMOylated IRF-1 is elevated in tumor cells, and that SUMOylation of IRF-1 attenuates its tumor-suppressive function. Here we report that SUMOylated IRF-1 mimics IRF-2, an antagonistic repressor, and shows oncogenic potential. To demonstrate the role of SUMOylated IRF-1 in tumorigenesis, we used SUMO-IRF-1 recombinant protein. Stable expression of SUMO-IRF-1 in NIH3T3 cells resulted in focus formation and anchorage-independent growth in soft agar. Inoculation of SUMO-IRF-1-transfected cells into athymic nude mice resulted in tumor formation and infiltration of adipose tissues. Finally, we demonstrated that SUMO-IRF-1 transforms NIH3T3 cells in a dose-dependent manner suggesting that SUMOylated IRF-1 may act as an oncogenic protein in tumor cells.

Novel antibacterial application of photovoltaic Cu2SnS3 (CTS) nanoparticles

In the present work, Cu2SnS3 (CTS) nanoparticles were synthesized using the hot injection method. The structural, compositional and optical properties of the nanoparticles were evaluated using the X-ray diffraction (XRD), Raman spectroscopy, transmission electron microscopy (TEM) and UV-visible spectroscopy techniques, respectively. The XRD and Raman studies confirmed the formation of monoclinic CTS nanoparticles. The TEM and UV-visible spectroscopy studies indicated the formation of spherical nanoparticles in the size range of 10–12 nm with a band gap of 1.24 eV. The CTS nanoparticles were tested for antibacterial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria strains by the agar-well diffusion method. The antibacterial activity of the nanoparticles was identified by determining the zone of inhibition (ZOI) and minimal inhibitor concentration (MIC). The study results reveal that the synthesized nanoparticles possessed excellent antibacterial activity against both bacterial strains, indicating the potential of the material for further research in biocidal applications. Finally, a plausible mechanism of antibacterial activity of the nanoparticles is proposed.