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.

CDCP1 (CUB domain containing protein 1) is a potential urine-based biomarker in the diagnosis of low-grade urothelial carcinoma.

Urine-based cytology is non-invasive and widely used for clinical diagnosis of urothelial carcinoma (UC), but its sensitivity is less than 40% for low-grade UC detection. As such, there is a need for new diagnostic and prognostic biomarkers of UC. CUB domain containing protein 1 (CDCP1) is a type I transmembrane glycoprotein highly expressed in various cancers. Using tissue array analysis, we demonstrated that CDCP1 expression in UC patients (n = 133), especially in those with low-grade UC, was significantly higher than in 16 normal persons. In addition, CDCP1 expression in urinary UC cells could also be detected by using immunocytochemistry method (n = 11). Furthermore, in 5637-CD cells, overexpression of CDCP1 affected the expression of epithelial mesenchymal transition-related markers and increased matrix metalloproteinase 2 expression and migration ability. Conversely, the knockdown of CDCP1 in T24 cells had the opposite effects. Using specific inhibitors, we demonstrated the involvement of c-Src/PKCδ signaling in the CDCP1-regulated migration of UC. In conclusion, our data suggest that CDCP1 contributes to the malignant progression of UC and may have the potential as a urine-based biomarker for detecting low-grade UC. However, a cohort study needs to be conducted.

Gemcitabine-induced Gli-dependent activation of hedgehog pathway resists to the treatment of urothelial carcinoma cells.

Patients with urothelial carcinoma (UC) experience gemcitabine resistance is a critical issue. The role of hedgehog pathway in the problem was explored. The expressions of phospho-AKTser473, phospho-GSK3ßser9 and Gli2 were up-regulated in gemcitabine-resistant NTUB1 (NGR) cells. Without hedgehog ligands, Gli proteins can be phosphorylated by GSK3ß kinase to inhibit their downstream regulations. Furthermore, the GSK3ß kinase can be phosphorylated by AKT at its Ser9 residue to become an inactive kinase. Therefore, overexpression of AKT1, Flag-GSKS9D (constitutively inactive form) or active Gli2 (GLI2ΔN) in NTUB1 cells could activate Gli2 pathway to enhance migration/invasion ability and increase gemcitabine resistance, respectively. Conversely, overexpression of Flag-GSKS9A (constitutively active form) or knockdown of Gli2 could suppress Gli2 pathway, and then reduce gemcitabine resistance in NGR cells. Therefore, we suggest gemcitabine-activated AKT/GSK3ß pathway can elicit Gli2 activity, which leads to enhanced migration/invasion ability and resistance to gemcitabine therapy in UC patients. The non-canonical hedgehog pathway should be evaluated in the therapy to benefit UC patients.

New Templated Ostwald Ripening Process of Mesostructured FeOOH for Third-Harmonic Generation Bioimaging.

Emerging advances in iron oxide nanoparticles exploit their high magnetization for various applications, such as bioseparation, hyperthermia, and magnetic resonance imaging. In contrast to their excellent magnetic performance, the harmonic generation and luminescence properties of iron oxide nanoparticles have not been thoroughly explored, thus limiting their development as a tool in photomedicine. In this work, a seed/growth-inspired synthesis is developed combined with primary mineralization and a ligand-assisted secondary growth strategy to prepare mesostructured α-FeOOH nanorods (NRs). The sub-wavelength heterogeneity of the refractive index leads to enhanced third-harmonic generation (THG) signals under near-infrared excited wavelengths at 1230 nm. The as-prepared NRs exhibit an 11-fold stronger THG intensity compared to bare α-FeOOH NRs. Using these unique nonlinear optical properties, it is demonstrated that mesostructured α-FeOOH NRs can serve as biocompatible and nonbleaching contrast agents in THG microscopy for long-term labeling of cells as well as in angiography in vivo by modifying lectin to enhance the binding efficiency to the glycocalyx layers on the wall of blood vessels. These results provide a new insight into Fe-based nanoplatforms capable of emitting coherent light as molecular probes in optical microscopy, thus establishing a complementary microscopic imaging method for macroscopic magnetic imaging systems.

Metformin promotes apoptosis in hepatocellular carcinoma through the CEBPD-induced autophagy pathway.

Metformin, as an AMP-activated protein kinase (AMPK) activator, can activate autophagy. A study showed that metformin decreased the risk of hepatocellular carcinoma (HCC) in diabetic patients. However, the detailed mechanism in the metformin-mediated anticancer effect remains an open question. Transcription factor CCAAT/enhancer-binding protein delta (CEBPD) has been suggested to serve as a tumor suppressor and is responsive to multiple anticancer drugs in HCC. In this study, we found that CEBPD and autophagy are involved in metformin-induced cell apoptosis in Huh7 cells. The underlying mechanisms in this process included a reduction in Src-mediated CEBPD protein degradation and an increase in CEBPD-regulated LC3B and ATG3 gene transcription under metformin treatment. We also found that AMPK is involved in metformin-induced CEBPD expression. Combined treatment with metformin and rapamycin can enhance autophagic cell death through the AMPK-dependent and AMPK-independent pathway, respectively. Taken together, we provide a new insight and therapeutic approach by targeting autophagy in the treatment of HCC.

Histone deacetylase inhibitor trichostatin A resensitizes gemcitabine resistant urothelial carcinoma cells via suppression of TG-interacting factor.

Gemcitabine and cisplatin (GC) has been widely used for advanced and metastatic urothelial carcinoma (UC). However, resistance to this remedy has been noticed. We have demonstrated that increase of TG-interacting factor (TGIF) in specimens is associated with worse prognosis of upper tract UC (UTUC) patients. The roles of TGIF in the gemcitabine resistance of UC were explored. Specimens of 23 locally advanced/advanced stage UTUC patients who received GC systemic chemotherapy after radical nephroureterectomy were collected to evaluate the alterations of TGIF in the resistance to the remedy by using immunohistochemistry. In vitro characterizations of mechanisms mediating TGIF in gemcitabine resistance were conducted by analyzing NTUB1 cells and their gemcitabine-resistant subline, NGR cells. Our results show that increased TGIF is significantly associated with chemo-resistance, poor progression-free survival, and higher cancer-related deaths of UTUC patients. Higher increases of TGIF, p-AKT(Ser473) and invasive ability were demonstrated in NGR cells. Overexpression of TGIF in NTUB1 cells upregulated p-AKT(Ser473) activation, enhanced migration ability, and attenuated cellular sensitivity to gemcitabine. Knockdown of TGIF in NGR cells downregulated p-AKT(Ser473) activation, declined migration ability, and enhanced cellular sensitivity to gemcitabine. In addition, histone deacetylases inhibitor trichostatin A (TSA) inhibited TGIF, p-AKT(Ser473) expression and migration ability. Synergistic effects of gemcitabine and TSA on NGR cells were also demonstrated. Collectively, TGIF contributes to the gemcitabine resistance of UC via AKT activation. Combined treatment with gemcitabine and TSA might be a promising therapeutic remedy to improve the gemcitabine resistance of UC.

Transforming growth factor ß-interacting factor-induced malignant progression of hepatocellular carcinoma cells depends on superoxide production from Nox4.

Hepatocellular carcinoma (HCC) is one of the most deadly malignancies worldwide because of its high recurrence rate, high metastatic potential, and resistance to drugs. Elucidation of the mechanisms underlying malignancy in HCC is needed to improve diagnosis, therapy, and prognosis. Previously, we showed that transforming growth factor ß-interacting factor (TGIF) antagonizes arsenic trioxide-induced apoptosis of HepG2 cells and is associated with poor prognosis and progression of urothelial carcinoma in patients after radical nephroureterectomy. To determine whether TGIF plays a role in HCC tumorigenesis, we compared the expression of TGIF, its downstream targets, and reactive oxygen species levels between HCC HepG2 cells and the more invasive SK-Hep1 cells. Superoxide production, phosphorylation of c-Src(Y416) and AKT(S473), and expression of TGIF and NADPH oxidase (Nox) were higher in invasive SK-Hep1 cells than in HepG2 cells. TGIF-overexpressing HepG2 xenograft tumors markedly promoted tumor growth and metastasis to the lungs. Overexpression of TGIF in HepG2 cells increased superoxide production from Nox4, matrix metalloproteinase expression, invadopodia formation, and cellular migration/invasion ability. Conversely, knockdown of TGIF in SK-Hep1 cells attenuated these processes. Using gene knockdown and pharmacological inhibitors, we demonstrate that c-Src/AKT is the upstream signaling that regulates TGIF-induced Nox4 activation and subsequent superoxide production. Taken together, our results implicate TGIF as a potential biomarker for prognosis and target for clinical therapy in patients with advanced HCC.

TG-interacting factor transcriptionally induced by AKT/FOXO3A is a negative regulator that antagonizes arsenic trioxide-induced cancer cell apoptosis.

Arsenic trioxide (ATO) is a multi-target drug approved by the Food and Drug Administration as the first-line chemotherapeutic agent for the treatment of acute promyelocytic leukemia. In addition, several clinical trials are being conducted with arsenic-based drugs for the treatment of other hematological malignancies and solid tumors. However, ATO's modest clinical efficacy on some cancers, and potential toxic effects on humans have been reported. Determining how best to reduce these adverse effects while increasing its therapeutic efficacy is obviously a critical issue. Previously, we demonstrated that the JNK-induced complex formation of phosphorylated c-Jun and TG-interacting factor (TGIF) antagonizes ERK-induced cyclin-dependent kinase inhibitor CDKN1A (p21(WAF1/CIP1)) expression and resultant apoptosis in response to ATO in A431 cells. Surprisingly, at low-concentrations (0.1-0.2 µM), ATO increased cellular proliferation, migration and invasion, involving TGIF expression, however, at high-concentrations (5-20 µM), ATO induced cell apoptosis. Using a promoter analysis, TGIF was transcriptionally regulated by ATO at the FOXO3A binding site (-1486 to -1479bp) via the c-Src/EGFR/AKT pathway. Stable overexpression of TGIF promoted advancing the cell cycle into the S phase, and attenuated 20 µM ATO-induced apoptosis. Furthermore, blockage of the AKT pathway enhanced ATO-induced CDKN1A expression and resultant apoptosis in cancer cells, but overexpression of AKT1 inhibited CDKN1A expression. Therefore, we suggest that TGIF is transcriptionally regulated by the c-Src/EGFR/AKT pathway, which plays a role as a negative regulator in antagonizing ATO-induced CDKN1A expression and resultant apoptosis. Suppression of these antagonistic effects might be a promising therapeutic strategy toward improving clinical efficacy of ATO.

TG-interacting factor mediates arsenic-induced malignant transformation of keratinocytes via c-Src/EGFR/AKT/FOXO3A and redox signalings.

Inorganic arsenic is well known as a carcinogen in human beings. Chronic exposure to inorganic arsenic increases risks of developing some cancers and non-carcinogenic diseases, such as skin lesions in humans. However, the modes of action are not well elucidated. In the present study, HaCaT cells, an immortalized non-tumorigenic human keratinocyte, were continuously exposed to low-dose trivalent arsenic (arsenic trioxide, 0.1 and 0.2 µM) for at least 4 weeks. We proved that low-dose arsenic could stimulate malignant transformation of HaCaT cells, including increase of cellular proliferation, epithelial-to-mesenchymal transition markers alteration, matrix metalloproteinases activation, invadopodia formation, migration/invasion activities, and anchorage-independent growth. Surprisingly, low-dose arsenic could also transcriptionally increase TG-interacting factor (TGIF) expression via c-Src/EGFR/AKT/FOXO3A signaling involving superoxide production from NADPH oxidase. Moreover, stable overexpression of TGIF could also induce malignant transformation of HaCaT cells. Knockdown of TGIF with its specific shRNA abolished the arsenic-induced effects. Taken together, we suggest that TGIF plays an important role in low-dose arsenic-induced malignant transformation of HaCaT cells, which is regulated by c-Src/EGFR/AKT/FOXO3A pathway and redox signaling.

Involvement of TG-interacting factor in microglial activation during experimental traumatic brain injury.

Traumatic brain injury (TBI) is a complex injury involving several physiological alterations, potentially leading to neurological impairment. Previous mouse studies using high-density oligonucleotide array analysis have confirmed the upregulation of transforming growth-interacting factor (TGIF) mRNA in TBI. TGIF is a transcriptional corepressor of transforming growth factor beta (TGF-ß) signaling which plays a protective role in TBI. However, the functional roles of TGIF in TBI are not well understood. In this study, we used confocal microscopy after immunofluorescence staining to demonstrate the increase of TGIF levels in the activated microglia of the pericontusional cortex of rats with TBI. Intracerebral knockdown of TGIF in the pericontusional cortex significantly downregulated TGIF expression, attenuated microglial activation, reduced the volume of damaged brain tissue, and facilitated recovery of limb motor function. Collectively, our results indicate that TGIF is involved in TBI-induced microglial activation, resulting in secondary brain injury and motor dysfunction. This study investigated the roles of transforming growth-interacting factor (TGIF) in a traumatic brain injury (TBI)-rat model. We demonstrated the increase of TGIF levels in the activated microglia of the pericontusional cortex of rats with TBI. Intracerebral knockdown of TGIF in the pericontusional cortex of the TBI rats significantly attenuated micoglial activation, reduced the volume of damaged brain tissue, and facilitated recovery of limb motor function. We suggest that inhibition of TGIF might provide a promising therapeutic strategy for TBI.

Hypothermia may attenuate ischemia/reperfusion-induced cardiomyocyte death by reducing autophagy.

OBJECTIVE: We sought to assess the effect of therapeutic hypothermia on the autophagy that occurred in ischemia-reperfused (IR) H9c2 cardiomyocytes. METHODS: In control studies, the H9c2 cells at a density of 1 × 10(5) per culture dish in six-well plate were exposed to normoxic culture medium at 37 °C for 12h. All assays contained appropriate controls and were performed in triplicate and repeated on three separately initiated cultures. In hypothermia-treated group, the ischemic and hypoxic cells were maintained in a 32 °C incubation. The trypan blue exclusion method was used to assess the cell viability. Autophagy was evaluated by determining both the microtubule-associated protein 1 light chain 3 [LC3] levels and punctuate distribution of the autophagic vesicle associated form [LC3-II]. RESULTS: The results were mean ± standard error of mean of triplicates. The viable cell percentage for control group, IR group, and IR group treated with hypothermia at the start of ischemia, or reperfusion were 100% ± 9%, 20% ± 1%, 32% ± 3%, and 41% ± 3%, respectively. The cell death in I/R H9c2 cells was positively associated with increased LC3 levels and punctuate distribution of (LC3-II). Mild hypothermia adopted at the start of ischemia or reperfusion significantly reduced both the cell death and the autophagy in H9c2 cells. CONCLUSION: Our data indicate that in H9c2, IR stimulates cell autophagy and causes cell death, which can be attenuated by mild hypothermia. Our results, if further confirmed in vivo, may have important clinical implications during IR injury.

Role of macrophage CCAAT/enhancer binding protein delta in the pathogenesis of rheumatoid arthritis in collagen-induced arthritic mice.

BACKGROUND: The up-regulation of CCAAT/enhancer binding protein delta (CEBPD) has frequently been observed in macrophages in age-associated disorders, including rheumatoid arthritis (RA). However, the role of macrophage CEBPD in the pathogenesis of RA is unclear. METHODOLOGY AND PRINCIPAL FINDINGS: We found that the collagen-induced arthritis (CIA) score and the number of affected paws in Cebpd(-/-) mice were significantly decreased compared with the wild-type (WT) mice. The histological analysis revealed an attenuated CIA in Cebpd(-/-) mice, as shown by reduced pannus formation and greater integrity of joint architecture in affected paws of Cebpd(-/-) mice compared with WT mice. In addition, immunohistochemistry analysis revealed decreased pannus proliferation and angiogenesis in Cebpd(-/-) mice compared with WT mice. CEBPD activated in macrophages played a functional role in promoting the tube formation of endothelial cells and the migration and proliferation of synoviocytes. In vivo DNA binding assays and reporter assays showed that CEBPD up-regulated CCL20, CXCL1, IL23A and TNFAIP6 transcripts through direct binding to their promoter regions. CCL20, IL23A, CXCL1 and TNFAIP6 contributed to the migration and proliferation of synoviocytes, and the latter two proteins were involved in tube formation of endothelial cells. Finally, two anti-inflammatory chemicals, inotilone and rosmanol, reduced the expression of CEBPD and its downstream targets and mitigated the above phenomena. CONCLUSIONS AND SIGNIFICANCE: Collectively, our findings suggest that CEBPD and its downstream effectors could be biomarkers for the diagnosis of RA and potentially serve as therapeutic targets for RA therapy.

Overexpression of TG-interacting factor is associated with worse prognosis in upper urinary tract urothelial carcinoma.

Prognostic outcome prediction would be useful for the treatment of patients with upper urinary tract urothelial carcinoma (UC). However, its prognostic biomarkers are not well established so far. According to the results of analysis of 168 human upper urinary tract UC specimens, overexpressed TG-interacting factor (TGIF) in nuclei of tumor tissues is significantly correlated with poor progression-free survival and higher cancer-related death. When both TGIF and p21 expression are altered, these patients had an even worse prognosis than those with one or no marker altered. Furthermore, to elucidate the role of TGIF in the progression of UC, overexpression of TGIF in RT4 or TSGH8301 cells was performed, and the results revealed that TGIF can significantly increase migration/invasion ability, matrix metalloproteinase expression, and invadopodia formation via the phosphatidylinositol 3-kinase-AKT pathway. In contrast, knockdown of TGIF with its specific short hairpin RNA inhibited the invasion ability of T24 cells. Besides, TGIF could inhibit p21(WAF/CIP1) expression, up-regulate cyclin D1 expression, and phosphorylate retinoblastoma to promote G1-S transition and cellular proliferation. In conclusion, we demonstrated that TGIF contributes to the progression of urothelial carcinoma via the phosphatidylinositol 3-kinase-AKT pathway. It may serve as an attractive therapeutic or prognostic target for selected patients with upper urinary tract UC.

TG-interacting factor-induced superoxide production from NADPH oxidase contributes to the migration/invasion of urothelial carcinoma.

Urothelial carcinoma (UC) of the bladder is the fourth most common cancer and the ninth leading cause of death from cancer among men in the United States. However, higher recurrence, resistance to therapy, and poor diagnostic/prognostic biomarkers of UC prompt us to identify novel targets to improve the clinical applications. TG-interacting factor (TGIF), a transcriptional corepressor to modulate the TGF-ß signaling, is associated with various types of human cancer. In the present study, we found that cellular migration activity, reactive oxygen species production, AKT(S473) phosphorylation, TGIF, and p67(phox) expression were higher in invasive T24 cells than in noninvasive RT4 cells. In addition, overexpression of TGIF in RT4 cells enhanced cellular migration/invasion ability; it involved NADPH oxidase 2 (Nox2)/p67(phox) complex activation, reactive oxygen species production, and AKT(S473) phosphorylation. In contrast, the migration/invasion ability of T24 cells was suppressed by the knockdown of TGIF or p67(phox), respectively. Overexpression of AKT1 could increase cellular superoxide production and invasion. Moreover, by using the PI3K/AKT inhibitor wortmannin or shRNA of AKT1, the TGIF-induced Nox activation and superoxide production were significantly inhibited. Accordingly, we suggest that PI3K/AKT signaling mediates TGIF-induced Nox2/p67(phox) complex activation and the resultant superoxide production which reinforces the PI3K/AKT signaling to promote the cellular migration/invasion ability of UC.

NADPH oxidase-produced superoxide mediates EGFR transactivation by c-Src in arsenic trioxide-stimulated human keratinocytes.

Arsenic is a well-known poison and carcinogen in humans. However, it also has been used to effectively treat some human cancers and non-carcinogenic ailments. Previously, we demonstrated in keratinocytes that arsenic trioxide (ATO)-induced p21(WAF1/CIP1) (p21) expression leading to cellular cytotoxicity through the c-Src/EGFR/ERK pathway and generation of reactive oxygen species (ROS). In this study, we found that EGFR-Y845 and EGFR-Y1173 could be phosphorylated by ATO. Using confocal microscopy and flow cytometry, we found that pretreatment with apocynin, DPI, and tiron could remove ATO-induced ROS production. Furthermore, to increase NADPH oxidase activity, ATO could induce cytosolic p67(phox) expression and translocation to membrane. In addition, knockdown of p67(phox) could abolish ATO-induced ROS production. Therefore, we suggest that NADPH oxidase-produced superoxide was a major source of ATO-induced ROS production. Conversely, ATO-induced NADPH oxidase activation and superoxide generation could be inhibited by the c-Src inhibitor PP1, but not by the EGFR inhibitor PD153035. In addition, overexpression of c-Src as well as treatment with ATO could stimulate EGFR-Y845/ERK phosphorylation, p21 expression, and cellular arrest/apoptosis, which could be attenuated by pretreatment with apocynin or knockdown of p67(phox). Collectively, we suggest that NADPH oxidase was involved in the ATO-induced arrest/apoptosis of keratinocytes, which was regulated by c-Src activation.

Suppression of TG-interacting factor sensitizes arsenic trioxide-induced apoptosis in human hepatocellular carcinoma cells.

HCC (hepatocellular carcinoma) is among the most common and lethal cancers worldwide with a poor prognosis mainly due to a high recurrence rate and chemotherapy resistance. ATO (arsenic trioxide) is a multi-target drug that has been effectively used as an anticancer drug in acute promyelocytic leukaemia. However, a Phase II trial involving patients with HCC indicates that the use of arsenic as a single agent is not effective against HCC. TGIF (TG-interacting factor) is a transcriptional co-repressor that interferes with TGF-ß (transforming growth factor-ß) signalling which plays a growth-inhibitory role in HCC. In the present study, we demonstrated that ATO induced hepatocellular apoptosis via TGF-ß/Smad signalling and led to downstream induction of p21(WAF1/CIP1) (p21). However, ATO could also induce TGIF expression via a post-transcriptional regulation mechanism to antagonize this effect. Using a biotin-labelled RNA probe pull-down assay and in vivo RNA immunoprecipitation analysis, we identified that HuR (human antigen R) bound to the TGIF mRNA 3'-UTR (3'-untranslated region) and prevented it from degradation. ATO treatment increased the interaction between HuR and TGIF mRNA, and reduction of HuR expression inhibited ATO-induced TGIF expression. Moreover, the EGFR (epidermal growth factor receptor)/PI3K (phosphoinositide 3-kinase)/Akt pathway was shown to mediate the post-transcriptional regulation of TGIF in response to ATO. Finally, we also demonstrated that the down-regulation of TGIF could sensitize ATO-induced HepG2 cell apoptosis. Collectively, we propose that the EGFR/PI3K/Akt pathway may regulate the post-transcriptional regulation of TGIF expression to antagonize ATO-induced apoptosis in HCC. Blockage of the PI3K/Akt pathway or TGIF expression combined with ATO treatment may be a promising strategy for HCC therapy.

Involvement of glycogen synthase kinase-3ß in arsenic trioxide-induced p21 expression.

Arsenic trioxide (ATO) has been effectively used as a therapeutic agent to treat acute promyelocytic leukemia and solid tumors, via induction of cell cycle arrest or apoptosis. In our previous studies, we suggest that c-Jun might act as an adapter to regulate p21(WAF1/CIP1) (p21) expression in response to ATO. Therefore, how to regulate the c-Jun to bind to the p21 promoter was further elucidated. It has been reported that glycogen synthase kinase-3ß (GSK-3ß) can phosphorylate the C-terminus (Ser243) of c-Jun to decrease its protein stability and DNA-binding ability and can also increase the degradation of p21 in resting condition or under ultraviolet irradiation. Therefore, we hypothesized that ATO-induced p21 expression might be through the inhibition of GSK-3ß. Using the DNA affinity precipitation assay, ATO could dephosphorylate the C-terminus (Ser243) of c-Jun to enhance its binding to the p21 promoter and resultant p21 expression. ATO, as well as LiCl (GSK-3ß inhibitor), could induce GSK-3ß(Ser9) phosphorylation and p21 expression in a time- and dose-dependent manner. Constitutively active GSK-3ß, FlagGSKCA, and constitutively inactive GSK-3ß, FlagGSKCI, were constructed to further confirm the involvement of GSK-3ß in the ATO-induced p21 expression. However, the stability of p21 protein was increased by ATO, but not LiCl treatment using cycloheximide. Furthermore, ATO-induced GSK-3ß(Ser9) phosphorylation was through the ERK pathway, but not the PI3K/Akt pathway. We suggest that, taken together, ATO-induced ERK phosphorylation could inhibit GSK-3ß activity to dephosphorylate the C-terminus (Ser243) of c-Jun to increase p21 expression and resultant cell death.

TBK1-associated protein in endolysosomes (TAPE) is an innate immune regulator modulating the TLR3 and TLR4 signaling pathways.

The innate immune system elicits the first wave of immune responses against pathogen infection. Its operational modes are complex and have yet to be defined. Here, we report the identification of an innate immune regulator termed TAPE (TBK1-associated protein in endolysosomes), previously known as CC2D1A/Freud-1/Aki-1, which modulates the TLR3 and TLR4 pathways. We found that TAPE activated the TBK1, NF-κB, and ERK pathways leading to IFN-ß and inflammatory cytokine induction. TAPE was shown to colocalize with endosomal marker Rab5 and lysosomal marker LAMP1 in mammalian cells, suggesting that TAPE resided in endolysosomes. Knockdown of TAPE selectively impaired the TLR3 and endocytic TLR4 pathways to IFN-ß induction. Furthermore, TAPE interacted and synergized with Trif to activate IFN-ß. TAPE knockdown failed to block Trif-mediated IFN-ß induction, whereas Trif knockdown impaired the TLR3 and TAPE cooperation on IFN-ß induction, suggesting that TAPE acts upstream of Trif. Together, our data demonstrate a central role for TAPE in linking TLR3 and TLR4 to innate immune defenses at an early step.

Blockage of JNK pathway enhances arsenic trioxide-induced apoptosis in human keratinocytes.

Arsenic is well known as a carcinogen predisposing humans to some severe diseases and also as an effective medicine for treating acute promyelocytic leukemia, syphilis, and psoriasis. Multiple active mechanisms, including cell cycle arrest and apoptosis, have been proposed in therapy; however, the opposing effects of arsenic remain controversial. Our previous study found that arsenic trioxide (ATO)-induced activation of p21(WAF1/CIP1) (p21) led to A431 cell death through the antagonistic effects of the signaling of ERK1/2 and JNK1. In the current study, the inhibitory effects of JNK1 on ATO-induced p21 expression were explored. Over-expression of JNK1 in A431 cells could inhibit p21 expression, which was associated with HDAC1 and TGIF. Using the GST pull-down assay and fluorescence resonance energy transfer analysis, N-terminal domain (amino acids 1-108) of TGIF, critical to its binding with c-Jun, was found. Using reporter assays, requirement of the C-terminal domain (amino acids 138-272) of TGIF to suppress ATO-induced p21 expression was observed. Thus, the domains of TGIF that carried out its inhibitory effects on p21 were identified. Finally, treatment with JNK inhibitor SP600125 could enhance ATO-induced apoptosis of HaCaT keratinocytes by using flow cytometry.