Paraoxonase-2 agonist vutiglabridin promotes autophagy activation and mitochondrial function to alleviate non-alcoholic steatohepatitis.

BACKGROUND AND PURPOSE: Only limited therapeutic agents have been developed for non-alcoholic steatohepatitis (NASH). Glabridin, a promising anti-obesity candidate, has only limited druggability due to its low in vivo chemical stability and bioavailability. Therefore, we developed vutiglabridin (VUTI), which is based on a glabridin backbone, and investigated its mechanism of action in treating NASH in animal models. EXPERIMENTAL APPROACH: Anti-NASH effects of VUTI were determined in in vitro fatty liver models, spheroids of primary human hepatocytes and L02 normal liver cell lines. To identify VUTI possible cellular target/s, biotin-labelled VUTI was synthesized and underwent chemical proteomic analysis. Further, the evaluation of VUTI therapeutic efficacy was carried out using an amylin-NASH and high-fat (HF) diet-induced obese (DIO) mouse models. This was carried out using transcriptomic, lipidomic and proteomic analyses of the livers from the amylin-NASH mouse model. KEY RESULTS: VUTI treatment markedly reduces hepatic steatosis, fibrosis and inflammation by promoting lipid catabolism, activating autophagy and improving mitochondrial dysfunction, all of which are hallmarks of effective NASH treatment. The cellular target of VUTI was identified as paraoxonase 2 (PON2), a newly proposed protein target for the treatment of NASH, VUTI enhanced PON2 activity. The results using PON2 knockdown cells demonstrated that PON2 is important for VUTI- activation of autophagy, promoting mitochondrial function, decreasing oxidative stress and alleviating lipid accumulation under lipotoxic condition. CONCLUSION AND IMPLICATIONS: Our data demonstrated that VUTI is a promising therapeutic for NASH. Targeting PON2 may be important for improving liver function in various immune-metabolic diseases including NASH.

NF-κB/p53-activated inflammatory response involves in diquat-induced mitochondrial dysfunction and apoptosis.

Inflammation generated by environmental toxicants including pesticides could be one of the factors underlying neuronal cell damage in neurodegenerative diseases. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in PC12 cells treated with diquat. We found that diquat induced apoptosis, as demonstrated by the activation of caspases and nuclear condensation, inhibition of mitochondrial complex I activity, and decreased ATP level in PC12 cells. Diquat also reduced the dopamine level, indicating that cell death induced by diquat is due to cytotoxicity of dopaminergic neuronal components in these cells. Exposure of PC12 cells to diquat led to the production of reactive oxygen species (ROS), and the antioxidant N-acetyl-cystein attenuated the cytotoxicity of caspase-3 pathways. These results demonstrate that diquat-induced apoptosis is involved in mitochondrial dysfunction through production of ROS. Furthermore, diquat increased expression of cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α) via inflammatory stimulation. Diquat induced nuclear accumulation of NF-κB and p53 proteins. Importantly, an inhibitor of NF-κB nuclear translocation blocked the increase of p53. Both NF-κB and p53 inhibitors also blocked the diquat-induced inflammatory response. Pretreatment of cells with meloxicam, a COX-2 inhibitor, also blocked apoptosis and mitochondrial dysfunction. These results represent a unique molecular characterization of diquat-induced cytotoxicity in PC12 cells. Our results demonstrate that diquat induces cell damage in part through inflammatory responses via NF-κB-mediated p53 signaling. This suggests the potential to generate mitochondrial damage via inflammatory responses and inflammatory stimulation-related neurodegenerative disease.

PGAM5 regulates PINK1/Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunction.

Mitochondrial dynamics and mitophagy are critical processes for regulating mitochondrial homeostasis. Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein that plays crucial roles in apoptosis and necroptosis, but the roles of PGAM5 in mitochondrial dynamics and mitophagy remain unclear. In this study, we investigated the role of PGAM5 in carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced mitochondrial damage and the correlation between mitochondrial dynamics and mitophagy using SH-SY5Y cells. We found that CCCP decreased mitochondrial membrane potential, resulting in mitochondrial dysfunction. CCCP increased PGAM5, dynamin-related protein 1 (DRP1), and optic atrophy 1 (OPA1) expression of the mitochondrial fraction in a time-dependent manner. Knockdown of PGAM5 inhibited DRP1 translocation without a change in OPA1 expression in CCCP-treated cells. Furthermore, knockdown of PGAM5 and DRP1 significantly blocked the increase of PTEN-induced putative protein kinase 1 (PINK1) and Parkin expression in the mitochondrial fraction of CCCP-treated cells. Interestingly, CCCP did not alter PINK1/Parkin expression in the mitochondrial fraction of OPA1 knockdown cells. Inhibiting mitophagy by PGAM5 knockdown accelerated CCCP-induced apoptosis. CCCP treatment also results in PINK1 stabilization on the mitochondrial membrane, which subsequently increases Parkin recruitment from the cytosol to abnormal mitochondria. In addition, we found that CCCP increased the level of mitochondrial LC3II, indicating that Parkin recruitment of PINK1 is a result of mitophagy. We propose that activation of PGAM5 is associated with DRP1 recruitment and PINK1 stabilization, which contribute to the modulation of mitophagy in CCCP-treated cells with mitochondrial dysfunction. In conclusion, we demonstrated that PGAM5 regulates PINK1-Parkin-mediated mitophagy, which can exert a neuroprotective effect against CCCP-induced apoptosis.

Opa-interacting protein 5 modulates docetaxel-induced cell death via regulation of mitophagy in gastric cancer.

Damage to mitochondria induces mitophagy, a cellular process that is gaining interest for its therapeutic relevance to a variety of human diseases. However, the mechanism underlying mitochondrial depolarization and clearance in mitophagy remains poorly understood. We previously reported that mitochondria-induced cell death was caused by knockdown of Neisseria gonorrhoeae opacity-associated-interacting protein 5 in gastric cancer. In this study, we show that Neisseria gonorrhoeae opacity-associated-interacting protein 5 loss and gain of function modulates mitophagy induced by treatment with docetaxel, a chemotherapy drug for gastric cancer. The activation of mitophagy by Neisseria gonorrhoeae opacity-associated-interacting protein 5 overexpression promoted cell survival, preventing docetaxel-induced mitochondrial clearance. Conversely, short interfering RNA-mediated knockdown of Neisseria gonorrhoeae opacity-associated-interacting protein 5 accelerated docetaxel-induced apoptosis while increasing mitochondrial depolarization, reactive oxygen species, and endoplasmic reticulum stress and decreasing adenosine triphosphate production. We also found that the mitochondrial outer membrane proteins mitofusin 2 and phosphatase and tensin homolog-induced putative kinase 1 colocalized with Neisseria gonorrhoeae opacity-associated-interacting protein 5 in mitochondria and that mitofusin 2 knockdown altered Neisseria gonorrhoeae opacity-associated-interacting protein 5 expression. These findings indicate that Neisseria gonorrhoeae opacity-associated-interacting protein 5 modulates docetaxel-induced mitophagic cell death and therefore suggest that this protein comprises a potential therapeutic target for gastric cancer treatment.

Cystatin SN inhibits auranofin-induced cell death by autophagic induction and ROS regulation via glutathione reductase activity in colorectal cancer.

This corrects the article DOI: 10.1038/cddis.2017.100.

Cystatin SN inhibits auranofin-induced cell death by autophagic induction and ROS regulation via glutathione reductase activity in colorectal cancer.

Cystatin SN (CST1) is a specific inhibitor belonging to the cystatin superfamily that controls the proteolytic activities of cysteine proteases such as cathepsins. Our previous study showed that high CST1 expression enhances tumor metastasis and invasiveness in colorectal cancer. Recently, auranofin (AF), a gold(I)-containing thioredoxin reductase 1 (TrxR1) inhibitor, has been used clinically to treat rheumatoid arthritis. AF is a proteasome-associated deubiquitinase inhibitor and can act as an anti-tumor agent. In this study, we investigated whether CST1 expression induces autophagy and tumor cell survival. We also investigated the therapeutic effects of AF as an anti-tumor agent in colorectal cancer (CRC) cells. We found that CRC cells expressing high levels of CST1 undergo increased autophagy and exhibit chemotherapeutic resistance to AF-induced cell death, while those expressing low levels of CST1 are sensitive to AF. We also observed that knockdown of CST1 in high-CST1 CRC cells using CST1-specific small interfering RNAs attenuated autophagic activation and restored AF-induced cell mortality. Conversely, the overexpression of CST1 increased autophagy and viability in cells expressing low levels of CST1. Interestingly, high expression of CST1 attenuates AF-induced cell death by inhibiting intracellular reactive oxygen species (ROS) generation, as demonstrated by the fact that the blockage of ROS production reversed AF-induced cell death in CRC cells. In addition, upregulation of CST1 expression increased cellular glutathione reductase (GR) activity, reducing the cellular redox state and inducing autophagy in AF-treated CRC cells. These results suggest that high CST1 expression may be involved in autophagic induction and protects from AF-induced cell death by inhibition of ROS generation through the regulation of GR activity.

Clearance of Damaged Mitochondria Through PINK1 Stabilization by JNK and ERK MAPK Signaling in Chlorpyrifos-Treated Neuroblastoma Cells.

Mitochondrial quality control and clearance of damaged mitochondria through mitophagy are important cellular activities. Studies have shown that PTEN-induced putative protein kinase 1 (PINK1) and Parkin play central roles in triggering mitophagy; however, little is known regarding the mechanism by which PINK1 modulates mitophagy in response to reactive oxygen species (ROS)-induced stress. In this study, chlorpyrifos (CPF)-induced ROS caused mitochondrial damage and subsequent engulfing of mitochondria in double-membrane autophagic vesicles, indicating that clearance of damaged mitochondria is due to mitophagy. CPF treatment resulted in PINK1 stabilization on the outer mitochondrial membrane and subsequently increased Parkin recruitment from the cytosol to the abnormal mitochondria. We found that PINK1 physically interacts with Parkin in the mitochondria of CPF-treated cells. Furthermore, a knockdown of PINK1 strongly inhibited the LC3-II protein level by blocking Parkin recruitment. This indicates that CPF-induced mitophagy is due to PINK1 stabilization in mitochondria. We observed that PINK1 stabilization was selectively regulated by ROS-mediated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling activation but not p38 signaling. In the mitochondria of CPF-exposed cells, pretreatment with specific inhibitors of JNK and ERK1/2 significantly decreased PINK1 stabilization and Parkin recruitment and blocked the LC3-II protein level. Specifically, JNK and ERK1/2 inhibition also dramatically blocked the interaction between PINK1 and Parkin. Our results demonstrated that PINK1 regulation plays a critical role in CPF-induced mitophagy. The simple interpretation of these results is that JNK and ERK1/2 signaling regulates PINK1/Parkin-dependent mitophagy in the mitochondria of CPF-treated cells. Overall, this study proposes a novel molecular regulatory mechanism of PINK1 stabilization under CPF exposure.

mTOR inhibition by rapamycin protects against deltamethrin-induced apoptosis in PC12 Cells.

The autophagy pathway can be induced and upregulated in response to intracellular reactive oxygen species (ROS). In this study, we explored a novel pharmacotherapeutic approach involving the regulation of autophagy to prevent deltamethrin (DLM) neurotoxicity. We found that DLM-induced apoptosis in PC12 cells, as demonstrated by the activation of caspase-3 and -9 and by nuclear condensation. DLM treatment significantly decreased dopamine (DA) levels in PC12 cells. In addition, we observed that cells treated with DLM underwent autophagic cell death, by monitoring the expression of LC3-II, p62, and Beclin-1. Exposure of PC12 cells to DLM led to the production of ROS. Treatment with N-acetyl cysteine (NAC) effectively blocked both apoptosis and autophagy. In addition, mitogen-activated protein kinase (MAPK) inhibitors attenuated apoptosis as well as autophagic cell death. We also investigated the modulation of DLM-induced apoptosis in response to autophagy regulation. Pretreatment with the autophagy inducer, rapamycin, significantly enhanced the viability of DLM-exposed cells, and this enhancement of cell viability was partially due to alleviation of DLM-induced apoptosis via a decrease in levels of cleaved caspase-3. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), significantly increased DLM toxicity in these cells. Our results suggest that DLM-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against DLM-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 109-121, 2017.

PPAR-γ activation attenuates deltamethrin-induced apoptosis by regulating cytosolic PINK1 and inhibiting mitochondrial dysfunction.

Central events in the mitochondrial-dependent cell death pathway include the disruption of mitochondrial membrane potential, which causes the release of apoptogenic molecules leading to cell death. Based on the cytotoxic mechanism of deltamethrin (DLM), we examined the neuroprotective mechanisms of rosiglitazone (RGZ), which is against DLM-induced neuronal cell death. In this study, we found that DLM induces apoptosis in SH-SY5Y cells as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, neuronal cell death in response to DLM was due to mitochondrial dependent-apoptosis pathways since DLM increased cytochrome c release into the cytosol and activated caspase-9. DLM exposure reduced PINK1 expression, and pretreatment with RGZ significantly reduced cytochrome c release and caspase-9 activation. RGZ also attenuated the reduction of complex I activity, mitochondrial membrane potential, and ATP levels. Pretreatment with RGZ significantly enhanced PINK1 expression in DLM-exposed cells. In addition, RGZ increased cytosolic PINK1 by inhibiting mitochondrial translocation of PINK1. Interestingly, RGZ fails to rescue DLM-induced mitochondrial dysfunction both in PINK1 knockdown and PPAR-γ antagonist treated cells. Results from this study suggest that RGZ exerts anti-apoptotic effects against DLM-induced cytotoxicity by attenuation of mitochondrial dysfunction through cytosolic PINK1-dependent signaling pathways.

IL-32θ inhibits monocytic differentiation of leukemia cells by attenuating expression of transcription factor PU.1.

PU.1 is a key transcription factor regulating the myeloid differentiation. PU.1-induced monocytic differentiation into macrophage is also important for blood cancer development. Therefore, we chose THP-1 monocytic leukemia cells to investigate the function of a recently discovered IL-32θ. Genetic analyses identified differences in the sequences of IL-32θ and IL-32ß. Using previously established cell lines that stably express IL-32θ and IL-32ß and cell lines transiently expressing IL-32θ, we observed that expression of IL-32θ inhibited phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation in both THP-1 and HL-60 cells. IL-32θ also suppressed expression of the macrophage cell surface markers, CD11b, CD18, and CD36. Interestingly, expression of IL-32ß or IL-32θ had no effect on the expression levels of cell cycle related factors. As a result, we concluded that these isoforms did not contribute to PMA-induced cell cycle arrest. IL-32θ was found to modulate expression of PU.1, a transcription factor necessary for myeloid lineage commitment. Transient expression of PU.1 in THP-1/IL-32θ cells rescued the observed differentiation defect. Additionally, transient expression of both CCAAT-enhancer-binding protein α (C/EBPα) and PU.1 in THP-1/IL-32θ cells exhibited synergistic effects in rescuing the differentiation defect. These observations indicate that intracellular IL-32θ inhibits the differentiation of monocytes into macrophages by attenuating PU.1 expression.

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-32ß, our data from this study suggests that the newly identified IL-32θ isoform also acts as an intracellular modulator of inflammation.

Interleukin-32α downregulates the activity of the B-cell CLL/lymphoma 6 protein by inhibiting protein kinase Cε-dependent SUMO-2 modification.

A proinflammatory cytokine IL-32 acts as an intracellular mediator. IL-32α interacts with many intracellular molecules, but there are no reports of interaction with a transcriptional repressor BCL6. In this study, we showed that PMA induces an interaction between IL-32α, PKCε, and BCL6, forming a trimer. To identify the mechanism of the interaction, we treated cells with various inhibitors. In HEK293 and THP-1 cell lines, treatment with a pan-PKC inhibitor, PKCε inhibitor, and PKCδ inhibitor decreased BCL6 and IL-32α protein expression. MAPK inhibitors and classical PKC inhibitor did not decrease PMA-induced BCL6 and IL-32α protein expression. Further, the pan-PKC inhibitor and PKCε inhibitor disrupted PMA-induced interaction between IL-32α and BCL6. These data demonstrate that the intracellular interaction between IL-32α and BCL6 is induced by PMA-activated PKCε. PMA induces post-translational modification of BCL6 by conjugation to SUMO-2, while IL-32α inhibits. PKCε inhibition eliminated PMA-induced SUMOylation of BCL6. Inhibition of BCL6 SUMOylation by IL-32α affected the cellular function and activity of the transcriptional repressor BCL6 in THP-1 cells. Thus, we showed that IL-32α is a negative regulator of the transcriptional repressor BCL6. IL-32α inhibits BCL6 SUMOylation by activating PKCε, resulting in the modulation of BCL6 target genes and cellular functions of BCL6.

Interleukin-32α modulates promyelocytic leukemia zinc finger gene activity by inhibiting protein kinase Cɛ-dependent sumoylation.

Interleukin-32 (IL-32) is a proinflammatory cytokine. However, there is growing evidence that IL-32 also plays a mediatory role intracellularly. In this study, we present evidence that IL-32α modifies and inhibits promyelocytic leukemia zinc finger (PLZF), a sequence-specific transcriptional regulator that regulates the expression of a subset of interferon (IFN)-stimulated genes (ISGs). We screened IL-32α-interacting proteins in a human spleen cDNA library using the yeast two-hybrid assay, and investigated the functional relevance of the interaction between IL-32α and PLZF. We demonstrated that IL-32α interacts with protein kinase C (PKC)δ and PKCɛ in a phorbol 12-myristate 13-acetate (PMA) dependent way, and that PKCɛ regulates the interaction of IL-32α with PLZF. We verified the involvement of PKCɛ in the interaction between these proteins by using various PKC inhibitors. PLZF is known to be modified by small ubiquitin-like modifier (SUMO)-1, but it is unclear whether SUMO-2 conjugation of PLZF occurs. We showed that IL-32α inhibited SUMO-2-conjugation of PLZF. Further, we demonstrated that sumoylated PLZF decreased when IL-32α was co-expressed. PKCɛ affected the sumoylation of PLZF only in the presence of IL-32α because PKC inhibitor treatment did not reduce PLZF sumoylation in the absence of IL-32α. We finally investigated whether IL-32α-mediated inhibition of PLZF sumoylation affected the transcriptional activity of PLZF, and demonstrated that the inhibition of sumoylation of PLZF by IL-32α down-regulated ISGs induced by PLZF. Together, our data suggest that IL-32α associates with PLZF and PKCɛ, and then inhibits PLZF sumoylation, resulting in suppression of the transcriptional activity of PLZF.

IL-32θ negatively regulates IL-1ß 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-1ß 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-1ß promoter region. These findings reveal that IL-32θ reduces PKCδ-mediated phosphorylation of PU.1, resulting in attenuation of IL-1ß production.

Luteolin induces intrinsic apoptosis via inhibition of E6/E7 oncogenes and activation of extrinsic and intrinsic signaling pathways in HPV-18-associated cells.

Luteolin, a flavonoid extracted from a number of plants with recognized anticancer, anti-inflammatory and anti-oxidative activities, inhibits angiogenic processes and modulates multidrug resistance. However, the efficacy and mechanisms of action of this flavonoid agent are still undergoing study. In order to elucidate whether luteolin exhibits an anticancer effect in cervical cancer cells, HeLa cells were incubated with luteolin and apoptosis was assessed by observing nuclear morphological changes, and performing Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Cell cycle analysis, western blotting, RT-PCR and mitochondrial membrane potential measurements were also carried out. Luteolin showed a significant dose-dependent cytotoxic effect only in human papillomavirus (HPV)-positive cervical cancer cells, when compared to its effect on HPV-negative cervical cancer C33A cells. Expression levels of human papilloma virus E6 and E7 oncogenes were suppressed, those of related factors pRb and p53 were recovered and E2F5 was increased by luteolin treatment. Furthermore, luteolin enhanced the expression of death receptors and death receptor downstream factors such as Fas/FasL, DR5/TRAIL and FADD in HeLa cells, and activated caspase cascades. In particular, luteolin enhanced the activity of caspase-3 and -8 in a dose-dependent manner. Activation of caspase-3 induced caspase-8 activity and vice versa. Luteolin also induced mitochondrial membrane potential collapse and cytochrome c release, and inhibited Bcl-2 and Bcl-xL expression. In conclusion, luteolin exerts anticarcinogenic activity through inhibition of E6 and E7 expression and cross-activation of caspase-3 and -8. Taken together, these results suggest that luteolin induces inactivation of HPV-18 oncogene expression and apoptosis by activating the intrinsic and extrinsic pathways.

IL-32α down-regulates ß2 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.

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-32ß and inhibited IL-32ß-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.

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.

Interleukin (IL)-32ß-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.

We previously reported that IL-32ß promotes IL-10 production in myeloid cells. However, the underlying mechanism remains elusive. In this study, we demonstrated that IL-32ß 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-32ß knockdown by siRNA and that IL-32ß shifted to the membrane from the cytosol upon phorbol 12-myristate 13-acetate treatment. We revealed that IL-32ß suppressed the binding of C/EBPα to IL-10 promoter by using ChIP assay. These data suggest that PKCδ and IL-32ß may modulate the effect of C/EBPα on IL-10 expression. We next demonstrated by immunoprecipitation that IL-32ß interacted with PKCδ and C/EBPα, thereby mediating C/EBPα Ser-21 phosphorylation by PKCδ. We showed that IL-32ß 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-32ß. Taken together, our results show that IL-32ß-mediated C/EBPα Ser-21 phosphorylation by PKCδ suppressed C/EBPα binding to IL-10 promoter, which promoted IL-10 production in U937 cells.

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

There is growing evidence for multifunctional properties of IL-32. We previously demonstrated that IL-32ß 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-32ß binding to PKCδ by interacting with IL-32ß. The inhibitory effect of IL-32δ on IL-32ß association with PKCδ was further verified by immuno-fluorescence staining. The co-localization of IL-32ß and PKCδ around the nuclear membrane was disrupted by IL-32δ. Our data therefore indicate that IL-32δ plays an inhibitory role against IL-32ß function, which also suggests that IL-32 may be regulated by its own isoform.