TG-interacting factor 1 regulates mitotic clonal expansion during adipocyte differentiation.

Obesity is one of the significant health challenges in the world and is highly associated with abnormal adipogenesis. TG-interacting factor 1 (TGIF1) is essential for differentiating murine adipocytes and human adipose tissue-derived stem cells. However, the mode of action needs to be better elucidated. To investigate the roles of TGIF1 in differentiation in-depth, CRISPR/Cas9 knockout technology was performed to generate TGIF1-silenced preadipocytes. The absence of TGIF1 in 3 T3-F442A preadipocytes abolished lipid accumulation throughout the differentiation using Oil Red O staining. Conversely, we established 3 T3-F442A preadipocytes stably expressing TGIF1 and doxycycline-inducible TGIF1 in TGIF1-silenced 3 T3-F442A preadipocytes. Remarkably, the induction of TGIF1 by doxycycline during the initial differentiation phase successfully promoted lipid accumulation in TGIF1-silenced 3 T3-F442A cells. We further explored the mechanisms of TGIF1 in early differentiation. We demonstrated that TGIF1 promoted the mitotic clonal expansion via upregulation of CCAAT/enhancer-binding proteins ß expression, interruption with peroxisome proliferators activated receptor γ downstream regulation, and inhibition of p27kip1 expression. In conclusion, we strengthen the pivotal roles of TGIF1 in early differentiation, which might contribute to resolving obesity-associated metabolic syndromes.

Phosphorylation of TG-interacting factor 1 at carboxyl-terminal sites in response to insulin regulates adipocyte differentiation.

TG-interacting factor 1 (TGIF1) contributes to the differentiation of murine white preadipocyte and human adipose tissue-derived stem cells; however, its regulation is not well elucidated. Insulin is a component of the adipogenic cocktail that induces ERK signaling. TGIF1 phosphorylation and sustained stability in response to insulin were reduced through the use of specific MEK inhibitor U0126. Mutagenesis at T235 or T239 residue of TGIF1 in preadipocytes led to dephosphorylation of TGIF1. The reduced TGIF1 stability resulted in an increase in p27kip1 expression, a decrease in phosphorylated Rb expression and cellular proliferation, and a reduced accumulation of lipids compared to the TGIF1-overexpressed cells. These findings highlight that insulin/ERK-driven phosphorylation of the T235 or T239 residue at TGIF1 is crucial for adipocyte differentiation.

Inactivation of pentraxin 3 suppresses M2-like macrophage activity and immunosuppression in colon cancer.

BACKGROUND: The tumor microenvironment is characterized by inflammation-like and immunosuppression situations. Although cancer-associated fibroblasts (CAFs) are among the major stromal cell types in various solid cancers, including colon cancer, the interactions between CAFs and immune cells remains largely uncharacterized. Pentraxin 3 (PTX3) is responsive to proinflammatory cytokines and modulates immunity and tissue remodeling, but its involvement in tumor progression appears to be context-dependent and is unclear. METHODS: Open-access databases were utilized to examine the association of PTX3 expression and the fibroblast signature in colon cancer. Loss-of-function assays, including studies in tamoxifen-induced Ptx3 knockout mice and treatment with an anti-PTX3 neutralizing antibody (WHC-001), were conducted to assess the involvement of PTX3 in colon cancer progression as well as its immunosuppressive effect. Finally, bioinformatic analyses and in vitro assays were performed to reveal the downstream effectors and decipher the involvement of the CREB1/CEBPB axis in response to PTX3 and PTX3-induced promotion of M2 macrophage polarization. RESULTS: Clinically, higher PTX3 expression was positively correlated with fibroblasts and inflammatory response signatures and associated with a poor survival outcome in colon cancer patients. Blockade of PTX3 significantly reduced stromal cell-mediated tumor development. The decrease of the M2 macrophage population and an increase of the cytotoxic CD8+ T-cell population were observed following PTX3 inactivation in allografted colon tumors. We further revealed that activation of cyclic AMP-responsive element-binding protein 1 (CREB1) mediated the PTX3-induced promotion of M2 macrophage polarization. CONCLUSIONS: PTX3 contributes to stromal cell-mediated protumor immunity by increasing M2-like macrophage polarization, and inhibition of PTX3 with WHC-001 is a potential therapeutic strategy for colon cancer.

Astrocytic Cebpd Regulates Pentraxin 3 Expression to Promote Fibrotic Scar Formation After Spinal Cord Injury.

Astroglial-fibrotic scars resulted from spinal cord injury affect motor and sensory function, leading to paralysis. In particular, the fibrotic scar is a main barrier that disrupts neuronal regeneration after spinal cord injury. However, the association between astrocytes and fibrotic scar formation is not yet understood. We have previously demonstrated that the transcriptional factor Cebpd contributes to astrogliosis, which promotes glial scar formation after spinal cord injury. Herein, we show that fibrotic scar formation was decreased in the epicenter region in Cebpd-/- mice after contusive spinal cord injury and astrocytic Cebpd promoted fibroblast migration through secretion of Ptx3. Furthermore, the expression of Mmp3 was increased under recombinant protein Ptx3 treatment in fibroblasts by observing microarray data, resulting in fibroblast migration. In addition, regulation of Mmp3 occurs through the NFκB signaling pathway by using an irreversible inhibitor of IκBα phosphorylation in pretreated fibroblasts. Of note, we used the synthetic peptide RI37, which blocks fibroblast migration and decreases fibroblast Mmp3 expression in IL-1ß-treated astrocyte conditioned media. Collectively, our data suggest that fibroblast migration can be affected by astrocytic Cebpd through the Ptx3/NFκB/Mmp3 axis pathway and that the RI37 peptide may act as a therapeutic medicine to inhibit fibrotic scar formation after spinal cord injury.

Pentraxin 3 regulates tyrosine kinase inhibitor-associated cardiomyocyte contraction and mitochondrial dysfunction via ERK/JNK signalling pathways.

BACKGROUND: Hepatocellular carcinoma (HCC) patients suffer varying degrees of heart dysfunction after tyrosine kinase inhibitor (TKI) treatment. Interestingly, HCC patients often have higher levels of pentraxin 3 (PTX3), and PTX3 inhibition was found to improve left ventricular dysfunction in animal models. OBJECTIVES: We sought to assess the therapeutic potential of PTX3 inhibition on TKI-associated cardiotoxicity. METHODS: We used a human embryonic stem cell line, RUES2, to generate cardiomyocyte cultures (RUES2-CM) for functional testing. We also assessed heart function and PTX3 expression levels in 16 HCC patients who received TKI treatment, 3 HCC patients who did not receive TKIs, and 7 healthy volunteers. RESULTS: Significantly higher PTX3 expression was noted in HCC patients with TKI treatment versus those without, and 38% of male and 33% of female patients had QTc prolongation after TKI treatment. Treatment of cardiomyocyte cultures with sorafenib also increased PTX3 expression and induced cytoskeletal remodelling, contraction reduction, sodium current inhibition, and mitochondrial respiratory dysfunction. PTX3 colocalised with CD44 in cardiomyocytes, and cardiomyocyte contraction, mitochondrial respiratory function, and regular cytoskeletal and apoptotic protein expression were restored with PTX3 inhibition. CD44 knockdown confirmed PTX3/CD44 signalling. These results suggest a possible mechanism in which sorafenib treatment increases PTX3 expression, thereby resulting in reduced extracellular signal-regulated kinase (ERK) 1/2 expression that affects cardiomyocyte contraction, while also activating c-Jun N-terminal kinase (JNK) downstream pathways to disrupt mitochondrial respiration and trigger apoptosis. CONCLUSIONS: TKI-induced cardiotoxicity may be partly mediated by the upregulation of PTX3, and thus PTX3 inhibition has potential as a therapeutic strategy.

Pentraxin 3 Facilitates Shrimp-Allergic Responses in IgE-Activated Mast Cells.

Background: Since food avoidance is currently the only way to prevent allergic reactions to shrimp, a better understanding of molecular events in the induction and progression of allergy, including food allergy, is needed for developing strategies to inhibit allergic responses. Pentraxin 3 (PTX3) is rapidly produced directly from inflammatory or damaged tissues and is involved in acute immunoinflammatory responses. However, the role of PTX3 in the development of immediate IgE-mediated shrimp allergy remains unknown. Methods: Wild-type BALB/c mice were immunized intraperitoneally and were challenged with shrimp extract. Serum IgE and PTX3 levels were analyzed. RBL-2H3 cells were stimulated with either dinitrophenyl (DNP) or serum of shrimp-allergic mice, and markers of degranulation, proinflammatory mediators, and phosphorylation of signal proteins were analyzed. We further examined the effect of PTX3 in shrimp extract-induced allergic responses in vitro and in vivo. Results: Mice with shrimp allergy had increased PTX3 levels in the serum and small intestine compared with healthy mice. PTX3 augmented degranulation, the production of proinflammatory mediators, and activation of the Akt and MAPK signaling pathways in mast cells upon DNP stimulation. Furthermore, the expression of transcription factor CCAAT/enhancer-binding protein delta (CEBPD) was elevated in PTX3-mediated mast cell activation. Finally, the PTX3 inhibitor RI37 could attenuate PTX3-induced degranulation, proinflammatory mediator expression, and phosphorylation of the Akt and MAPK signaling. Conclusions: The results suggested that PTX3 can facilitate allergic responses. Our data provide new insight to demonstrate that PTX3 is a cause of allergic inflammation and that RI37 can serve as a therapeutic agent in shrimp allergy.

Blockade of the pentraxin 3/CD44 interaction attenuates lung injury-induced fibrosis.

BACKGROUND: Fibrosing interstitial lung diseases (fILD) are potentially fatal with limited therapeutic options and no effective strategies to reverse fibrogenesis. Myofibroblasts are chief effector cells in fibrosis that excessively deposit collagen in the pulmonary interstitium and lead to progressive impairment of gaseous exchange. METHODS: Plasma and lung specimens from patients with fILD were applied for detecting pentraxin 3 (PTX3) abundance by ELISA and Immunohistochemistry. Masson's trichrome and Sirius red stains and hydroxyproline assay were performed for assessing collagen accumulation in the lungs of bleomycin-exposed conditional Ptx3-deficient and PTX3-neutralizing antibody (αPTX3i)-treated mice. Downstream effectors including signaling pathways and fibrotic genes were examined for assessing CD44-involved PTX3-induced fibrosis in HFL1 and primary mouse fibroblasts. RESULTS: PTX3 was upregulated in the lungs and plasma of bleomycin-exposed mice and correlated with disease severity and adverse outcomes in fILD patients. Decreased collagen accumulation, attenuation of alveolar fibrosis and fibrotic markers, and improved lung function were observed in bleomycin-exposed conditional Ptx3-deficient mice. PTX3 activates lung fibroblasts to differentiate towards migrative and highly collagen-expressing myofibroblasts. Lung fibroblasts with CD44 inactivation attenuated the PI3K-AKT1, NF-κB, and JNK signaling pathways and fibrotic markers. αPTX3i mimic-based therapeutic studies demonstrated abrogation of the migrative fibroblast phenotype and myofibroblast activation in vitro. Notably, αPTX3i inhibited lung fibrosis, reduced collagen deposition, increased mouse survival, and improved lung function in bleomycin-induced pulmonary fibrosis. CONCLUSIONS: The present study reveals new insights into the involvement of the PTX3/CD44 axis in fibrosis and suggests PTX3 as a promising therapeutic target in fILD patients.

Microfluidic aptasensor POC device for determination of whole blood potassium.

An integrated microfluidic Au nanoparticle (AuNP) aptasensor device is proposed for monitoring the concentration of potassium (K+) ions in the bloodstream of patients with chronic kidney disease (CKD). In the proposed detection device, the AuNPs in the AuNP/aptamer complex are displaced by the serum K+ ions and react with NaCl to produce a color change in the detection region from which the K+ ion concentration is then inversely derived. The microfluidic device comprises two main components, namely an AuNP aptasensor PMMA (Poly(methyl methacrylate))/paper-microchip and a colorimetric analysis system for the quantitative detection of K+ ion concentration in whole blood. The functions of PMMA/paper microchips include reagent storage, K+ ion/aptamer reaction, and separation of serum from whole blood samples (blood filter). Experimental results show that the microfluidic device provides a linear response over the K+ ion concentration in range of 0.05-9 mM in artificial serum and has a detection limit (LOD) of 0.01 mM. Moreover, the detection results obtained for the 137 whole blood and 287 serum samples of CKD patients are very consistent (R2 = 0.968 and R2 = 0.980) with the measurement results obtained using an ion-selective electrodes (ISE) method. Results confirm that the current microfluidic aptasensor device provides a highly-sensitive and convenient method for performing the point-of-care (POC) monitoring of the whole blood K+ ion concentration.

Disruption of the pentraxin 3/CD44 interaction as an efficient therapy for triple-negative breast cancers.

Due to the heterogeneity and high frequency of genome mutations in cancer cells, targeting vital protumour factors found in stromal cells in the tumour microenvironment may represent an ideal strategy in cancer therapy. However, the regulation and mechanisms of potential targetable therapeutic candidates need to be investigated. An in vivo study demonstrated that loss of pentraxin 3 (PTX3) in stromal cells significantly decreased the metastasis and growth of cancer cells. Clinically, our results indicate that stromal PTX3 expression correlates with adverse prognostic features and is associated with worse survival outcomes in triple-negative breast cancer (TNBC). We also found that transforming growth factor beta 1 (TGF-ß1) induces PTX3 expression by activating the transcription factor CCAAT/enhancer binding protein delta (CEBPD) in stromal fibroblasts. Following PTX3 stimulation, CD44, a PTX3 receptor, activates the downstream ERK1/2, AKT and NF-κB pathways to specifically contribute to the metastasis/invasion and stemness of TNBC MDA-MB-231 cells. Two types of PTX3 inhibitors were developed to disrupt the PTX3/CD44 interaction and they showed a significant effect on attenuating growth and restricting the metastasis/invasion of MDA-MB-231 cells, suggesting that targeting the PTX3/CD44 interaction could be a new strategy for future TNBC therapies.

Biological significance of MYC and CEBPD coamplification in urothelial carcinoma: Multilayered genomic, transcriptional and posttranscriptional positive feedback loops enhance oncogenic glycolysis.

BACKGROUND AND PURPOSE: The aim of this study is to decipher the underlying mechanisms of CCAAT/enhancer-binding protein delta (CEBPD)-enhanced glycolysis as well as the biological significance of CEBPD and MYC coamplification in urothelial carcinoma (UC). METHODS: In vitro analyses were conducted to examine the effects of altered CEBPD or MYC expression on UC cells. The in vivo effects of CEBPD overexpression in a high-glucose environment on tumour growth were investigated in xenografted induced diabetic severe combined immunodeficiency/beige mice. Data mining was used to cross-validate the associations between CEBPD and MYC copy number and transcriptional expression, quantitative reverse transcription-polymerase chain reaction, immunohistochemistry, chromogenic in situ hybridization, and in situ hybridization targeting microRNA were performed on 635 UC patient samples and xenograft samples. UC patient survival in relation to diabetes was validated by using the National Health Insurance Research Database. RESULTS: CEBPD and MYC coamplification (29.6%) occurred at a high frequency, MYC expression promoted chromosomal instability, facilitating CEBPD copy number gain and expression. CEBPD promoted glucose uptake and lactate production by upregulating SLC2A1 and HK2, leading to mitochondrial fission, increased extracellular acidification rate and decreased oxygen consumption rate to fuel cell growth. CEBPD upregulated HK2 expression through multiple regulation pathways including MYC stabilization, suppression of FBXW7 transactivation and MYC-independent transcriptional suppression of hsa-miR-429. Clinical and xenografted experiments confirmed the growth advantage of CEBPD in relation to glucose metabolic dysregulation and the significant correlations between the expression of these genes. CONCLUSIONS: We confirmed that CEBPD has an oncogenic role in UC by activating AKT signalling and initiating metabolic reprogramming from mitochondrial oxidative phosphorylation to glycolysis to satisfy glucose addiction. These novel CEBPD- and MYC-centric multilayered positive feedback loops enhance cancer growth that could complement theranostic approaches.

CPAP enhances and maintains chronic inflammation in hepatocytes to promote hepatocarcinogenesis.

Chronic and persistent inflammation is a well-known carcinogenesis promoter. Hepatocellular carcinoma (HCC) is one of the most common inflammation-associated cancers; most HCCs arise in the setting of chronic inflammation and hepatic injury. Both NF-κB and STAT3 are important regulators of inflammation. Centrosomal P4.1-associated protein (CPAP), a centrosomal protein that participates primarily in centrosome functions, is overexpressed in HCC and can increase TNF-α-mediated NF-κB activation and IL-6-induced STAT3 activation. A transgenic (Tg) mouse model with hepatocyte-specific CPAP expression was established to investigate the physiological role of CPAP in hepatocarcinogenesis. Obvious inflammatory cell accumulation and fatty change were observed in the livers of CPAP Tg mice. The alanine aminotransferase (ALT) level and the expression levels of inflammatory genes, such as IL-6, IL-1ß and TNF-α, were higher in CPAP Tg mice than in wild type (WT) mice. High-dose/short-term treatment with diethylnitrosamine (DEN) increased the ALT level, proinflammatory gene expression levels, and STAT3 and NF-κB activation in CPAP Tg mice; low-dose/long-term DEN treatment induced more severe liver tumor formation in CPAP Tg mice than in WT mice. CPAP can increase the expression of chemokine (C-C motif) ligand 16 (CCL-16), an important chemotactic cytokine, in human hepatocytes. CCL-16 expression is positively correlated with CPAP and TNF-α mRNA expression in the peritumoral part of HCC. In summary, these results suggest that CPAP may promote hepatocarcinogenesis through enhancing the inflammation pathway via increasing the expression of CCL-16.

MCL1 participates in leptin-promoted mitochondrial fusion and contributes to drug resistance in gallbladder cancer.

Obesity is a risk factor for gallbladder cancer (GBC) development, and it correlates with shorter overall survival. Leptin, derived from adipocytes, has been suggested to contribute to the growth of cancer cells; however, the detailed mechanism of leptin in GBC drug resistance remains uninvestigated. In this study, our finding that patients with GBC with a higher BMI were associated with increased GBC risks, including shortened survival, is clinically relevant. Moreover, obese NOD/SCID mice exhibited a higher circulating concentration of leptin, which is associated with GBC growth and attenuated gemcitabine efficacy. We further revealed that leptin can inhibit gemcitabine-induced GBC cell death through myeloid cell leukemia 1 (MCL1) activation. The transcription factor C/EBP δ (CEBPD) is responsive to activated STAT3 (pSTAT3) and contributes to MCL1 transcriptional activation upon leptin treatment. In addition, MCL1 mediates leptin-induced mitochondrial fusion and is associated with GBC cell survival. The findings in this study suggest the involvement of the pSTAT3/CEBPD/MCL1 axis in leptin-induced mitochondrial fusion and survival and provide a potentially new therapeutic target to improve the efficacy of gemcitabine in patients with GBC.

Fibroblast CEBPD/SDF4 axis in response to chemotherapy-induced angiogenesis through CXCR4.

Cancer-associated fibroblasts (CAFs) play an essential role in supporting cancer progression. However, the details and consequent effects in response to the communication between CAFs and angiogenesis remain largely uninvestigated, especially in anticancer drug treatments. We found that cisplatin and 5-fluorouracil could induce fibroblast differentiation toward myofibroblasts via CCAAT/enhancer-binding protein delta (CEBPD) and consequently promote proliferation, migration, and in vitro tube formation of vascular endothelial cells and angiogenesis in vivo. Stromal-cell-derived factor 4 (SDF4) is responsive to anticancer drugs via CEBPD activation in CAFs and contributes to create a permissive environment for tumor cell angiogenesis and promotion of distant metastasis. Importantly, we demonstrated that SDF4 interacts with CXCR4 to trigger VEGFD expression through the activation of the ERK1/2 and p38 pathways in endothelial cells. Taken together, our novel findings support that SDF4 can be a therapeutic target in inhibition of angiogenesis for chemotherapy drug-administrated cancer patients.

CCAAT/Enhancer-binding protein delta mediates glioma stem-like cell enrichment and ATP-binding cassette transporter ABCA1 activation for temozolomide resistance in glioblastoma.

Glioblastoma (GBM) is the most aggressive brain tumor and relapses after chemo- or radiotherapy in a short time. The anticancer drug temozolamide (TMZ) is commonly used for GBM treatment, but glioma stem-like cells (GSCs) often lead to drug resistance and therapeutic failure. To date, the mechanism of GSC formation in TMZ-treated GBM remains largely unknown. CCAAT/Enhancer-binding protein delta (CEBPD) is an inflammation-responsive transcription factor and is proposed to be oncogenic in the context of drug resistance, prompting us to clarify its role in TMZ-resistant GBM. In this study, we first found that the CEBPD protein levels in GBM patients were significantly increased and further contributed to TMZ resistance by promoting GSC formation. Accordingly, the protein levels of stemness transcription factors, namely, SRY-box transcription factor 2 (SOX2), octamer-binding transcription factor 4 (OCT4), NANOG, and ATP-binding cassette subfamily A member 1 (ABCA1), were increased in GSCs and TMZ-treated GBM cells. Increased binding of CEBPD to promoter regions was observed in GSCs, indicating the direct regulation of these GSC-related genes by CEBPD. In addition, an ABCA1 inhibitor increased the caspase 3/7 activity of TMZ-treated GSCs, suggesting that TMZ efflux is controlled by ABCA1 activity and that the expression levels of the ABCA1 gene are an indicator of the efficiency of TMZ treatment. Together, we revealed the mechanism of CEBPD-mediated GSC drug resistance and proposed ABCA1 inhibition as a potential strategy for the treatment of TMZ-resistant GBM.

Metformin Resensitizes Sorafenib-Resistant HCC Cells Through AMPK-Dependent Autophagy Activation.

Despite the activation of autophagy may enable residual cancer cells to survive and allow tumor relapse, excessive activation of autophagy may eventually lead to cell death. However, the details of the association of autophagy with primary resistance in hepatocellular carcinoma (HCC) remain less clear. In this study, cohort analysis revealed that HCC patients receiving sorafenib with HBV had higher mortality risk. We found that high epidermal growth factor receptor (EGFR) expression and activity may be linked to HBV-induced sorafenib resistance. We further found that the resistance of EGFR-overexpressed liver cancer cells to sorafenib is associated with low activity of AMP-activated protein kinase (AMPK) and CCAAT/enhancer binding protein delta (CEBPD) as well as insufficient autophagic activation. In response to metformin, the AMPK/cAMP-response element binding protein (CREB) pathway contributes to CEBPD activation, which promotes autophagic cell death. Moreover, treatment with metformin can increase sorafenib sensitivity through AMPK activation in EGFR-overexpressed liver cancer cells. This study suggests that AMPK/CEBPD-activated autophagy could be a potent strategy for improving the efficacy of sorafenib in HCC patients.

CPAP promotes angiogenesis and metastasis by enhancing STAT3 activity.

Centrosomal P4.1-associated protein (CPAP) is overexpressed in hepatocellular carcinoma (HCC) and positively correlated with recurrence and vascular invasion. Here, we found that CPAP plays an important role in HCC malignancies. Functional characterization indicated that CPAP overexpression increases tumor growth, angiogenesis, and metastasis ex vivo and in vivo. In addition, overexpressed CPAP contributes to sorafenib resistance. Mechanical investigation showed that the expression level of CPAP is positively correlated with activated STAT3 in HCC. CPAP acts as a transcriptional coactivator of STAT3 by directly binding with STAT3. Interrupting the interaction between CPAP and STAT3 attenuates STAT3-mediated tumor growth and angiogenesis. Overexpression of CPAP upregulates several STAT3 target genes such as IL-8 and CD44 that are involved in angiogenesis, and CPAP mRNA expression is positively correlated with the levels of both mRNAs in HCC. Knocked-down expression of CPAP impairs IL-6-mediated STAT3 activation, target gene expression, cell migration, and invasion abilities. IL-6/STAT3-mediated angiogenesis is significantly increased by CPAP overexpression and can be blocked by decreased expression of IL-8. Our findings not only shed light on the importance of CPAP in HCC malignancies, but also provide potential therapeutic strategies for inhibiting the angiogenesis pathway and treating metastatic HCC.

Hepatitis B virus X protein (HBx) enhances centrosomal P4.1-associated protein (CPAP) expression to promote hepatocarcinogenesis.

BACKGROUND: Our previous report suggested that centrosomal P4.1-associated protein (CPAP) is required for Hepatitis B virus (HBV) encoded non-structure protein X (HBx)-mediated nuclear factor kappa light chain enhancer of activated B cells (NF-κB) activation. CPAP is overexpressed in HBV-associated hepatocellular carcinoma (HCC); however, the interaction between CPAP and HBx in HBV-HCC remains unclear. METHODS: The mRNA expression of CPAP and HBx was analyzed by quantitative-PCR (Q-PCR). NF-κB transcriptional activity and CPAP promoter activity were determined using a reporter assay in Huh7 and Hep3B cells. Immunoprecipitation (IP) and in situ proximal ligation assay (PLA) were performed to detect the interaction between CPAP and HBx. Chromatin-IP was used to detect the association of cAMP response element binding protein (CREB) and HBx with the CPAP promoter. Cell proliferation was measured using cell counting kit CCK-8, Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) incorporation, and clonogenic assays. The tumorigenic effects of CPAP were determined using xenograft animal models. RESULTS: HBx can transcriptionally up-regulate CPAP via interacting with CREB. Overexpressed CPAP directly interacted with HBx to promote HBx-mediated cell proliferation and migration; SUMO modification of CPAP was involved in interacting with HBx. Knocked-down expression of CPAP decreased the HBx-mediated tumorigenic effects, including cytokines secretion. Interestingly, overexpressed CPAP maintained the HBx protein stability in an NF-κB-dependent manner; and the expression levels of CPAP and HBx were positively correlated with the activation status of NF-κB in HCC. Increased expression of CPAP and CREB mRNAs existed in the high-risk group with a lower survival rate in HBV-HCC. CONCLUSION: The interaction between CPAP and HBx can provide a microenvironment to facilitate HCC development via enhancing NF-κB activation, inflammatory cytokine production, and cancer malignancies. This study not only sheds light on the role of CPAP in HBV-associated HCC, but also provides CPAP as a potential target for blocking the hyper-activated NF-κB in HCC.

Hepatoma-derived growth factor supports the antiapoptosis and profibrosis of pancreatic stellate cells.

Pancreatic cancer is refractory and is characterized by extensively surrounding and intratumor fibrotic reactions that are contributed by activated pancreatic stellate cells (PSCs). Herein, we show that CCAAT/enhancer-binding protein δ (CEBPD) responds to transforming growth factor-ß1 (TGF-ß1) through reciprocal loop regulation and that activated hypoxia inducible factor-1α (HIF-1α) further contributes to the upregulation of the hepatoma-derived growth factor (HDGF) gene. Secreted HDGF contributes to the antiapoptosis of PSCs and consequently leads to the synthesis and deposition of extracellular matrix proteins for stabilizing PSC/pancreatic cancer cell (PCC) tumor foci. This result agrees with the observation that severe stromal growth positively correlated with stromal HDGF and CEBPD expression in pancreatic cancer specimens. Collectively, the identification of the TGF-ß1-activated CEBPD/HIF-1α/HDGF axis provides new insights into novel discoveries of HDGF in the antiapoptosis and profibrosis of PSCs and the outgrowth of PCCs.

Astrocytic CCAAT/Enhancer-binding protein delta contributes to reactive oxygen species formation in neuroinflammation.

Excessive reactive oxygen species (ROS) can form an oxidative stress and an associated neuroinflammation. However, the contribution of astrocytes to ROS formation, the cause of the resistance of astrocytes to oxidative stress, and the consequences on neurons remain largely uninvestigated. The transcription factor CCAAT/enhancer-binding protein delta (CEBPD) is highly expressed in astrocytes and has been suggested to contribute to the progress of Alzheimer's disease (AD). In this study, we found that ROS formation and expression of p47phox and p67phox, subunits of NADPH oxidase, were increased in AppTg mice but attenuated in AppTg/Cebpd-/- mice. Cebpd can up-regulate p47phox and p67phox transcription via a direct binding on their promoters, which results in an increase in intracellular oxidative stress. In addition, Cebpd also up-regulated Cu/Zn superoxide dismutase (Sod1) in astrocytes. Inactivation of Sod1 increased the sensitization to oxidative stress, which provides a reason for the resistance of astrocytes in an oxidative stress environment. Taken together, the study first revealed and dissected the involvement of astrocytic Cebpd in the promotion of oxidative stress and the contribution of CEBPD to the resistance of astrocytes in an oxidative stress environment.

Bortezomib-induced miRNAs direct epigenetic silencing of locus genes and trigger apoptosis in leukemia.

MicroRNAs (miRNAs) have been suggested to repress transcription via binding the 3'-untranslated regions of mRNAs. However, the involvement and details of miRNA-mediated epigenetic regulation, particularly in targeting genomic DNA and mediating epigenetic regulation, remain largely uninvestigated. In the present study, transcription factor CCAAT/enhancer binding protein delta (CEBPD) was responsive to the anticancer drug bortezomib, a clinical and highly selective drug for leukemia treatment, and contributed to bortezomib-induced cell death. Interestingly, following the identification of CEBPD-induced miRNAs, we found that miR-744, miR-3154 and miR-3162 could target CpG islands in the 5'-flanking region of the CEBPD gene. We previously demonstrated that the Yin Yang 1 (YY1)/polycomb group (PcG) protein/DNA methyltransferase (DNMT) complex is important for CCAAT/enhancer binding protein delta (CEBPD) gene inactivation; we further found that Argonaute 2 (Ago2) interacts with YY1 and binds to the CEBPD promoter. The miRNA/Ago2/YY1/PcG group protein/DNMT complex linked the inactivation of CEBPD and genes adjacent to its 5'-flanking region, including protein kinase DNA-activated catalytic polypeptide (PRKDC), minichromosome maintenance-deficient 4 (MCM4) and ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2), upon bortezomib treatment. Moreover, we revealed that miRNA binding is necessary for YY1/PcG group protein/DNMT complex-mediated epigenetic gene silencing and is associated with bortezomib-induced methylation on genomic DNA. The present study successfully characterized the interactions of the miRNA/Ago2/YY1/PcG group protein/DNMT complex and provided new insights for miRNA-mediated epigenetic regulation in bortezomib-induced leukemic cell arrest and cell death.