Jeong-Woo Kang

Interaction network mapping among IL-32 isoforms.

IL-32 has been studied for its pleiotropic effects ranging from host immune responses to cell differentiation. Although several IL-32 isoforms have been characterized for their effects on cells, the roles of the others remain unclear. We previously reported that IL-32δ interacted with IL-32β and inhibited IL-32β-mediated IL-10 production. Thus, we performed comprehensive analyses to reveal more interactions between IL-32 isoforms in this study. We screened the interactions of 81 combinations of nine IL-32 isoforms by using a yeast two-hybrid assay, which identified 13 heterodimeric interactions. We verified these results by using reciprocal immunoprecipitation assays and reconfirmed 10 interactions, and presented the interaction network map between IL-32 isoforms. Our data suggest that IL-32 may have diverse intracellular effects through the interactions with its different isoforms.

Intracellular Interaction of Interleukin (IL)-32α with Protein Kinase Cϵ (PKCϵ) and STAT3 Protein Augments IL-6 Production in THP-1 Promonocytic Cells

Background: IL-32α is known to interact with FAK1, and IL-32α overexpression in chronic myeloid leukemia cells increases natural killer cell-mediated killing. Results: IL-32α interacted with PKCϵ and STAT3, mediated STAT3 phosphorylation, and thereby augmented IL-6 production. Conclusion: IL-32α elevated IL-6 production through interaction with PKCϵ and STAT3. Significance: The interaction of IL-32α with PKCϵ and STAT3 reveals a new intracellular mediatory role of IL-32α. IL-32α is known as a proinflammatory cytokine. However, several evidences implying its action in cells have been recently reported. In this study, we present for the first time that IL-32α plays an intracellular mediatory role in IL-6 production using constitutive expression systems for IL-32α in THP-1 cells. We show that phorbol 12-myristate 13-acetate (PMA)-induced increase in IL-6 production by IL-32α-expressing cells was higher than that by empty vector-expressing cells and that this increase occurred in a time- and dose-dependent manner. Treatment with MAPK inhibitors did not diminish this effect of IL-32α, and NF-κB signaling activity was similar in the two cell lines. Because the augmenting effect of IL-32α was dependent on the PKC activator PMA, we tested various PKC inhibitors. The pan-PKC inhibitor Gö6850 and the PKCϵ inhibitor Ro-31-8220 abrogated the augmenting effect of IL-32α on IL-6 production, whereas the classical PKC inhibitor Gö6976 and the PKCδ inhibitor rottlerin did not. In addition, IL-32α was co-immunoprecipitated with PMA-activated PKCϵ, and this interaction was totally inhibited by the PKCϵ inhibitor Ro-31-8220. PMA-induced enhancement of STAT3 phosphorylation was observed only in IL-32α-expressing cells, and this enhancement was inhibited by Ro-31-8220, but not by Gö6976. We demonstrate that IL-32α mediated STAT3 phosphorylation by forming a trimeric complex with PKCϵ and enhanced STAT3 localization onto the IL-6 promoter and thereby increased IL-6 expression. Thus, our data indicate that the intracellular interaction of IL-32α with PKCϵ and STAT3 promotes STAT3 binding to the IL-6 promoter by enforcing STAT3 phosphorylation, which results in increased production of IL-6.

Luteolin 8-C-β-fucopyranoside inhibits invasion and suppresses TPA-induced MMP-9 and IL-8 via ERK/AP-1 and ERK/NF-κB signaling in MCF-7 breast cancer cells.

Matrix metalloproteinase 9 (MMP-9) and interleukin-8 (IL-8) play major roles in tumor progression and invasion of breast cancer cells. The present study was undertaken to investigate the inhibitory mechanism of cell invasion by luteolin 8-C-β-fucopyranoside (named as LU8C-FP), a C-glycosylflavone, in human breast cancer cells. We investigated whether LU8C-FP would inhibit MMP-9 activation and IL-8 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 breast cancer cells. LU8C-FP suppressed TPA-induced MMP-9 and IL-8 secretion and mRNA expression via inhibition of the MAPK signaling pathway and down-regulation of nuclear AP-1 and NF-κB. TPA-induced phosphorylation of ERK 1/2 was suppressed by LU8C-FP, whereas JNK and p38 MAPK phosphorylation were unaffected. In addition, LU8C-FP blocked the ERK 1/2 pathways following expression of MMP-9 and IL-8. These results suggest LU8C-FP may function to suppress invasion of breast cancer cells through the ERK/AP-1 and ERK/NF-κB signaling cascades.

Interleukin-32δ interacts with IL-32β and inhibits IL-32β-mediated IL-10 production

There is growing evidence for multifunctional properties of IL‐32. We previously demonstrated that IL‐32β upregulates IL‐10 production through the association with PKCδ. In this study, we examined the effects of other IL‐32 isoforms on IL‐10 production. We found that IL‐32δ decreased IL‐10 production and investigated the inhibitory mechanism of IL‐32δ. We showed that IL‐32δ suppressed IL‐32β binding to PKCδ by interacting with IL‐32β. The inhibitory effect of IL‐32δ on IL‐32β association with PKCδ was further verified by immuno‐fluorescence staining. The co‐localization of IL‐32β and PKCδ around the nuclear membrane was disrupted by IL‐32δ. Our data therefore indicate that IL‐32δ plays an inhibitory role against IL‐32β function, which also suggests that IL‐32 may be regulated by its own isoform.

Interleukin (IL)-32β-mediated CCAAT/Enhancer-binding Protein α (C/EBPα) Phosphorylation by Protein Kinase Cδ (PKCδ) Abrogates the Inhibitory Effect of C/EBPα on IL-10 Production

Background: IL-32β promotes IL-10 production in myeloid cells. Results: IL-32β-mediated C/EBPα serine 21 phosphorylation by PKCδ induced the dissociation of C/EBPα from IL-10 promoter, thereby promoting IL-10 production. Conclusion: IL-32β suppressed the inhibitory effect of C/EBPα on IL-10 production by mediating C/EBPα serine 21 phosphorylation by PKCδ. Significance: Our data suggest that IL-32β functions as an intracellular regulator of IL-10 production. We previously reported that IL-32β promotes IL-10 production in myeloid cells. However, the underlying mechanism remains elusive. In this study, we demonstrated that IL-32β abrogated the inhibitory effect of CCAAT/enhancer-binding protein α (C/EBPα) on IL-10 expression in U937 cells. We observed that the phosphorylation of C/EBPα Ser-21 was inhibited by a PKCδ-specific inhibitor, rottlerin, or IL-32β knockdown by siRNA and that IL-32β shifted to the membrane from the cytosol upon phorbol 12-myristate 13-acetate treatment. We revealed that IL-32β suppressed the binding of C/EBPα to IL-10 promoter by using ChIP assay. These data suggest that PKCδ and IL-32β may modulate the effect of C/EBPα on IL-10 expression. We next demonstrated by immunoprecipitation that IL-32β interacted with PKCδ and C/EBPα, thereby mediating C/EBPα Ser-21 phosphorylation by PKCδ. We showed that IL-32β suppressed the inhibitory effect of C/EBPα on IL-10 promoter activity. However, the IL-10 promoter activity was reduced to the basal level by rottlerin treatment. When C/EBPα serine 21 was mutated to glycine (S21G), the inhibitory effect of C/EBPα S21G on IL-10 promoter activity was not modulated by IL-32β. Taken together, our results show that IL-32β-mediated C/EBPα Ser-21 phosphorylation by PKCδ suppressed C/EBPα binding to IL-10 promoter, which promoted IL-10 production in U937 cells.

Novel PPARγ partial agonists with weak activity and no cytotoxicity; identified by a simple PPARγ ligand screening system.

Peroxisome proliferator-activated receptors (PPARs) are the transcriptional factor that regulate glucose and lipid homeostasis and widely well-known as molecular targets for improvement of metabolic disorder. Because major transcriptional activity of PPARs depends on their proper ligands, the studies for PPAR ligands have been continuously developed. We previously reported the simple enzyme-linked immunosorbent assay (ELISA) systems to screen PPAR ligands and a chemical library including flavonoid derivatives have applied to these systems. In this study, we introduce two compounds (KU16476 and KU28843) identified as PPARγ partial agonists by a screening ELISA for PPARγ ligand. KU16476 and KU28843 significantly increased binding between PPARγ and SRC-1 in a simple ELISA system. Co-activator recruiting-induced abilities of two compounds were less than that of indomethacin, a well-known PPARγ agonist. To determine whether these compounds would be PPARγ partial agonists, each candidate with indomethacin were applied to a simple ELISA based on binding between PPARγ and SRC-1. Cotreatment with indomethacin significantly increased binding between PPARγ and SRC-1 than treatment of indomethacin or candidate alone. Two compounds had no considerable cytotoxicities, induced partial adipogenesis, and accumulated lipid droplets in 3T3-L1 fibroblast. Also, these two compounds enhanced expression of PPARγ-mediated genes such as aP2 and UCP-2. By docking study, we confirmed that two compounds bound well to the active site of PPARγ with hydrophobic interactions. We suggest that two compounds identified by a simple ELISA system can be PPARγ partial agonists. These PPARγ partial agonists and these studies to find out novel PPARγ agonists may contribute to drug development against metabolic disorders.

IL-32θ negatively regulates IL-1β production through its interaction with PKCδ and the inhibition of PU.1 phosphorylation

It has been well known that IL‐32 exerts pro‐inflammatory effects on the various inflammatory diseases in clinical studies. Here, we confirmed that IL‐32θ, a new isoform of IL‐32, decreased the phorbol 12‐myristate 13‐acetate (PMA)‐induced IL‐1β expression in THP‐1 human myelomonocyte. We previously reported that the IL‐32 isoforms control expressions of other cytokines via novel PKCs. Likewise, IL‐32θ interacted with PKCδ, and consequently inhibited PKCδ‐mediated phosphorylation of PU.1. Moreover, IL‐32θ attenuated the localization of PU.1 into the IL‐1β promoter region. These findings reveal that IL‐32θ reduces PKCδ‐mediated phosphorylation of PU.1, resulting in attenuation of IL‐1β production.

IL-32θ downregulates CCL5 expression through its interaction with PKCδ and STAT3.

Interleukin-32 (IL-32) exists in several isoforms and plays an important role in inflammatory response. Recently, we identified a new isoform, IL-32θ, and performed a microarray analysis to identify IL-32θ-regulated genes in THP-1 myelomonocytic cells. Upon stimulating IL-32θ-expressing THP-1 cells with phorbol myristate acetate (PMA), we found that the CCL5 transcript level was significantly reduced. We confirmed the downregulation of CCL5 protein expression by using an enzyme-linked immunosorbent assay (ELISA). Because STAT3 phosphorylation on Ser727 by PKCδ is reported to suppress CCL5 protein expression, we examined whether IL-32θ-mediated STAT3 Ser727 phosphorylation occurs through an interaction with PKCδ. In this study, we first demonstrate that IL-32θ interacts with PKCδ and STAT3 using co-immunoprecipitation (Co-IP) and pulldown assay. Moreover, STAT3 was rarely phosphorylated on Ser727 in the absence of IL-32θ, leading to the binding of STAT3 to the CCL5 promoter. These results indicate that IL-32θ, through its interaction with PKCδ, downregulates CCL5 expression by mediating the phosphorylation of STAT3 on Ser727 to render it transcriptionally inactive. Therefore, similar to what we have reported for IL-32α and IL-32β, our data from this study suggests that the newly identified IL-32θ isoform also acts as an intracellular modulator of inflammation.

IL-32α down-regulates β2 integrin (CD18) expression by suppressing PU.1 expression in myeloid cells

Myeloid-specific CD18 associates with CD11 and plays a critical role in leukocyte adhesion to the endothelium. In this study, we observed that CD18 expression was decreased by IL-32α in THP-1 and K562 cells upon PMA stimulation, and investigated the mechanism by which IL-32α down-regulated CD18 expression. We found that IL-32α suppressed the expression of PU.1, a major transcription factor for CD18. Because we previously demonstrated that IL-32α mediated STAT3 S727 phosphorylation by PKCε, and STAT3 regulates PU.1 expression, we performed time-course analyses of STAT3 S727 phosphorylation and found that IL-32α induces prolonged phosphorylation of STAT3 S727 until 72h after PMA stimulation. The expression pattern of C/EBPα, another transcriptional regulator of PU.1, was not affected by IL-32α. In addition, we showed that STAT3 binding to the PU.1 promoter was suppressed by IL-32α. Thus, we examined the relatedness among these factors and found that IL-32α-mediated STAT3 S727 phosphorylation induced C/EBPα association. When STAT3 was mutated at S727 to proline (S727P), the mutant STAT3 S727P did not interact with C/EBPα. We further demonstrated that only the intact STAT3 interacted with the basic leucine zipper region of C/EBPα. The PU.1 promoter was activated by co-expression of STAT3 and IL-32α upon PMA stimulation. However, the promoter activity was inhibited with STAT3 and C/EBPα co-expression. Therefore, our data suggest that IL-32α-mediated STAT3 S727 phosphorylation induced C/EBPα association, which inhibited PU.1 expression, and then resulted in the down-regulation of CD18 expression.

Naringenin Derivative Diethyl (5,4'-dihydroxy flavanone-7-yl) Phosphate Inhibits Cell Growth and Induces Apoptosis in A549 Human Lung Cancer Cells

Anti-cancer effects of naringenin derivative diethyl (5,4′-dihydroxy flavanone-7-yl) phosphate were evaluated in human lung cancer cells. The effect of diethyl (5,4′-dihydroxy flavanone-7-yl) phosphate (dEdHF-7-p) on A549 cell viability was measured using MTS assay and cell counting. Morphological changes were detected using phase-contrast microscopy. Apoptosis was analyzed using Hoechst staining. The influence of dEdHF-7-p on cell cycle distribution was determined using propidium iodide (PI) staining, and protein expression was determined by Western blot analysis. A newly synthesized naringenin derivative dEdHF-7-p suppressed cell growth of A549 though mechanisms including inhibition of cell cycle and increased apoptosis. Apoptotic and cell cycle modulators were changed by dEdHF-7-p in A549 cells; cyclins, ppRB, and antiapoptotic factor Bcl-2 were down-regulated, whereas apoptotic factor Bax and cyclin-dependent kinase inhibitors p21 and p53 were enhanced, thereby releasing cytochrome c into the cytosol of dEdHF-7-p -treated-A549 cells. dEdHF-7-p treatment processed caspases-3/-8/-9 and cleavage of poly ADP-ribose polymerase. The dEdHF-7-p treatment enhanced Fas expression and decreased expression of cell survival factors such as PI3K and p-Akt in a dose-dependent manner. Taken together, dEdHF-7-p induces apoptosis by inhibiting the PI3K/Akt survival signaling pathway and modulating mitochondria-emanated intrinsic and Fas extrinsic pathways in A549 cells.