Man Sub Kim

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.

Wogonin induces apoptosis by suppressing E6 and E7 expressions and activating intrinsic signaling pathways in HPV-16 cervical cancer cells

Wogonin is a flavonoid compound extracted from Scutellaria baicalensis and is well known as a benzodiazepine receptor ligand with anxiolytic effects. Many recent studies have demonstrated that wogonin modulates angiogenesis, proliferation, invasion, and tumor progress in various cancer tissues. We further explored the mechanism of action of wogonin on cervical cancer cells that contain or lack human papillomavirus (HPV) DNA. Wogonin was cytotoxic to HPV 16 (+) cervical cancer cells, SiHa and CaSki, but not to HPV-negative cells. We demonstrated that wogonin induced apoptosis by suppressing the expressions of the E6 and E7 viral oncogenes in HPV-infected cervical cancer CaSki and SiHa cells. The modulation of p53 and protein retinoblastoma (pRb) were also triggered by the suppression of E6 and E7 expressions. However, p53 was not altered in HPV-negative cervical cancer C33A cells. Moreover, wogonin modulated the mitochondrial membrane potential and the expression of pro- and anti-apoptotic factors such as Bax and Bcl-2. Wogonin also provoked the cleavage of caspase-3, caspase-9, and poly ADP ribose polymerase. After transfection of siRNAs to target E6 and E7, additional restoration of p53 and pRb was not induced, but processing of caspases and PARP was increased compared with wogonin treatment alone. Together, our findings demonstrated that wogonin effectively promotes apoptosis by downregulating E6 and E7 expressions and promoting intrinsic apoptosis in human cervical cancer cells.

Antiobesity Effects of a Sulfur Compound Thiacremonone Mediated via Down‐regulation of Serum Triglyceride and Glucose Levels and Lipid Accumulation in the Liver of db/db Mice

Garlic is widely used as a spice. Garlic extracts exert anticancer and antiinflammatory effects, but its antiobesity efficacy studies have produced conflicting results. The antiobesity effects of thiacremonone, a sulfur compound isolated from garlic, was evaluated in obese db/db mice. Thiacremonone was orally administrated to mice for 3 weeks. The thiacremonone‐treated db/db mice showed a loss of body weight and decrease in blood triglyceride and glucose levels compared with the control mice. Histological analysis further revealed that thiacremonone significantly decreased lipid accumulation in the fatty livers of treated db/db mice. It was observed that GLUT‐4 expression and glucose uptake were up‐regulated by thiacremonone in 3T3‐L1 adipocytes. Thiacremonone treatment also suppressed expression levels of acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FAS), which are involved in lipid metabolism, in the liver of db/db mice. In addition, thiacremonone enhanced peroxisome proliferator‐activated receptor γ (PPARγ) expression in the fatty liver. Taken together, these results suggest that thiacremonone may play a vital role in improving the management of obesity and related metabolic syndromes via inhibition of lipid accumulation. Copyright

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.

6-O-Veratroyl catalpol suppresses pro-inflammatory cytokines via regulation of extracellular signal-regulated kinase and nuclear factor-κB in human monocytic cells.

The compound 6-O-veratroyl catalpol (6-O) is a bioactive iridoid glucoside that was originally isolated from Pseudolysimachion rotundum var. subintegrum. It has been demonstrated that catapol derivative iridoid glucosides including 6-O, possess anti-inflammatory activity in carragenan-induced paw edema mouse model as well as bronchoalveolar lavage fluid of ovalbumin-induced mouse model. In the present study, we investigated whether 6-O modulates inflammatory responses in THP-1 monocytic cells stimulated with phorbol12-myristate-13-acetate (PMA). Our data showed that 6-O inhibited PMA induced interleukin (IL)-1β and tumor necrosis factor (TNF)-α expression in THP-1 monocytic cells. Mechanistic studies revealed that 6-O suppressed the activity of protein kinase C (PKC), which further resulted in downstream inactivation of extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) inflammatory pathway. The results implied that 6-O may protect against inflammatory responses that could be a potential compound in treating inflammatory diseases.

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.

Luteolin 8-C-β-fucopyranoside downregulates IL-6 expression by inhibiting MAPKs and the NF-κB signaling pathway in human monocytic cells

Numerous studies have been suggested that derivatives can improve the effects of original substances. Therefore, we made luteolin derivative luteolin 8-C-β-fucopyranoside (LU8C-FP) for better anti-inflammatory and anti-cancer effects. In a previous study, we demonstrated that LU8C-FP inhibits invasion of human breast cancer cells via suppression of matrix metalloproteinase 9 and IL-8, which play major roles in tumor progression and cancer cell invasion. Various stimuli trigger inflammatory responses by inducing pro-inflammatory cytokines and chemokines in THP-1 cells. IL-6 induces inflammation via inducing various cytokines and appears to be a potential mediator of inflammatory diseases. Here, we investigated the precise mechanism by which LU8C-FP inhibited phorbol 12-myristate 13-acetate-induced IL-6 mRNA and protein expression. We showed LU8C-FP downregulated IL-6 expression by inhibiting mitogen-activated protein kinases and the nuclear factor-kappaB signaling pathway in human monocytic cells. Furthermore, LU8C-FP exerts less cytotoxicity than luteolin and also it has specific inhibitory effect on IL-6 expression. However, luteolin has a variety of inhibitory effects on pro-inflammatory cytokines and chemokines. Our in vitro studies may provide valuable information leading to the use of LU8C-FP to treat inflammatory diseases caused by IL-6.