Influence of RRM, RGG and Potential Phosphorylated Sites in Cold-Inducible Protein RBM3 on its Subcellular Localization and Neuroprotective Effects.

BACKGROUND: As a potent mediator of hypothermic neuroprotection, the cold-inducible protein RBM3 is characterized with one RNA-recognition motifs (RRM) and one arginine-glycine-rich (RGG) domain. It is known that these conserved domains are required for nuclear localization in some RNA-binding proteins. However, little is known about the actual role of RRM and RGG domains in subcellular localization of RBM3. METHODS: To clarify it, various mutants of human Rbm3 gene were constructed. Plasmids were transfected into cells and the localization of RBM3 protein and its varias mutants in cells and role in neuroprotection. RESULTS: In human neuroblastoma SH-SY5Y cells, either a truncation of RRM domain (aa 1-86) or RGG domain (aa 87-157) led to an obvious cytoplasmic distribution, compared to a predominant nuclear localization of whole RBM3 protein (aa 1-157). In contrast, mutants in several potential phosphorylated sites of RBM3, including Ser102, Tyr129, Ser147, and Tyr155, did not alter the nuclear localization of RBM3. Similarly, mutants in two Di-RGG motif sites also did not affect the subcellular distribution of RBM3. Lastly, the role of Di-RGG motif in RGG domains was further investigated. The mutant of double arginines in either Di-RGG motif-1 (Arg87/90) or -2 (Arg99/105) exhibited a higher cytoplasmic localization, indicating that both Di-RGG motifs are required for nucleic localization of RBM3. CONCLUSIONS: Our data suggest that RRM and RGG domains are both required for the nuclear localization of RBM3, with two Di-RGG domain being crucial for nucleocytoplasmic shuttling of RBM3.

Neural Cell Adhesion Molecule Regulates Osteoblastic Differentiation Through Wnt/ß-Catenin and PI3K-Akt Signaling Pathways in MC3T3-E1 Cells.

Neural cell adhesion molecule (NCAM) is involved in cell multi-directional differentiation, but its role in osteoblast differentiation is still poorly understood. In the present study, we investigated whether and how NCAM regulates osteoblastic differentiation. We found that NCAM silencing inhibited osteoblast differentiation in pre-osteoblastic MC3T3-E1 cells. The function of NCAM was further confirmed in NCAM-deficient mesenchymal stem cells (MSCs), which also had a phenotype with reduced osteoblastic potential. Moreover, NCAM silencing induced decrease of Wnt/ß-catenin and Akt activation. The Wnt inhibitor blocked osteoblast differentiation, and the Wnt activator recovered osteoblast differentiation in NCAM-silenced MC3T3-E1 cells. We lastly demonstrated that osteoblast differentiation of MC3T3-E1 cells was inhibited by the PI3K-Akt inhibitor. In conclusion, these results demonstrate that NCAM silencing inhibited osteoblastic differentiation through inactivation of Wnt/ß-catenin and PI3K-Akt signaling pathways.

Expression regulation of cold-inducible protein RBM3 by FAK/Src signaling for neuroprotection against rotenone under mild hypothermia.

Mild hypothermia is a well-established technique for alleviating neurological injuries in clinical surgery. RNA-binding protein motif 3 (RBM3) has been identified as a crucial factor in mediating hypothermic neuroprotection, providing its induction as a promising strategy for mimicking therapeutic hypothermia. However, little is known about molecular control of RBM3 and signaling pathways affected by hypothermia. In the present study, human SH-SY5Y neuroblastoma cells were used as a neural cell model. Screening of signaling pathways showed that cold exposure led to inactivation of ERK and AMPK pathways, and activation of FAK and PLCγ pathways, with activities of p38, JNK and AKT pathways moderately changed. Next, various small molecule inhibitors specific to these signaling pathways were applied. Interestingly, only FAK-specific inhibitor exhibited a significant inhibitory effect on hypothermia-induced RBM3 gene transcription and protein expression. Likewise, FAK silencing using siRNA technique significantly abrogated the induction of RBM3 by hypothermia. Moreover, FAK inhibition accounted for an inactivation of Src, a known kinase downstream of FAK. Next, either the silencing of Src by siRNA or its inactivation by a chemical inhibitor, strongly blocked the induction of RBM3 by cooling. Notably, in HEK293 and PC12 cells, FAK/Src activation was also shown to be indispensable for hypothermia-stimulated RBM3 expression. Lastly, the CCK8 and Western blot assays showed that both FAK/Src inacitivation and their knockdown substantially abrogate the neuroprotective effects of mild hypothermia against rotenone in SH-SY5Y cells. These data suggest that FAK/Src signaling axis regulates the transcription of Rbm3 gene and mediates neuroprotective effects of mild hypothermia.

Neural cell adhesion molecule regulates chondrocyte hypertrophy in chondrogenic differentiation and experimental osteoarthritis.

Chondrocyte hypertrophy-like change is an important pathological process of osteoarthritis (OA), but the mechanism remains largely unknown. Neural cell adhesion molecule (NCAM) is highly expressed and involved in the chondrocyte differentiation of mesenchymal stem cells (MSCs). In this study, we found that NCAM deficiency accelerates chondrocyte hypertrophy in articular cartilage and growth plate of OA mice. NCAM deficiency leads to hypertrophic chondrocyte differentiation in both murine MSCs and chondrogenic cells, in which extracellular signal-regulated kinase (ERK) signaling plays an important role. Moreover, NCAM expression is downregulated in an interleukin-1ß-stimulated OA cellular model and monosodium iodoacetate-induced OA rats. Overexpression of NCAM substantially inhibits hypertrophic differentiation in the OA cellular model. In conclusion, NCAM could inhibit hypertrophic chondrocyte differentiation of MSCs by inhibiting ERK signaling and reduce chondrocyte hypertrophy in experimental OA model, suggesting the potential utility of NCAM as a novel therapeutic target for alleviating chondrocyte hypertrophy of OA.

Benzoylaconitine Inhibits Production of IL-6 and IL-8 via MAPK, Akt, NF-κB Signaling in IL-1ß-Induced Human Synovial Cells.

Benzoylaconitine (BAC), the main hydrolysate of aconitine, is a lower toxic monoester type alkaloid considered as the pharmacodynamic constituent in Aconitum species. In this study, the effects and mechanisms of BAC on production of inflammatory cytokines interleukin (IL)-6 and IL-8 were investigated in IL-1ß-stimulated human synovial SW982 cells. The SW982 cells were incubated with BAC (0, 5 and 10 µM) before stimulating with IL-1ß (10 ng/mL). The results revealed that BAC suppressed gene and protein expression of IL-6 and IL-8 induced by IL-1ß. BAC decreased activation of mitogen-activated protein kinase (MAPK) and phosphorylation of Akt. BAC also inhibited degradation of inhibitor of kappaB (IκB)-α, phosphorylation and nuclear transposition of p65 protein. The results demonstrate that BAC exerts an anti-inflammatory effect dependent on MAPK, Akt and nuclear factor-κB (NF-κB) pathways in human synovial cells stimulated with IL-1ß, suggesting that BAC may be exploited as a potential therapeutic agent for rheumatoid arthritis (RA) treatment.

Mild Hypothermia Prevents NO-Induced Cytotoxicity in Human Neuroblastoma Cells Via Induction of COX-2.

The cold-inducible protein RBM3 mediates hypothermic neuroprotection against nitric oxide (NO)-induced cell death. Meanwhile, it is well-known that cyclooxygenase-2 (COX-2) is upregulated by RBM3 in several types of cells; however, it is still unclear whether COX-2 contributes to the neuroprotective effects of mild hypothermia/RBM3 against NO-induced cell death. Using human SH-SY5Y neuroblastoma cells, it was revealed that NO remarkably downregulates the expression of COX-2 at both mRNA and protein levels. When COX-2 was silenced using siRNA technique, cells became more sensitive to NO-induced cell death. Conversely, the overexpression of COX-2 significantly prevented NO-induced cell death in SH-SY5Y cells, indicating a pro-survival role of COX-2. Upon mild hypothermia pre-treatment, COX-2 was notably induced at both mRNA and protein levels; however, COX-2 silencing abrogated hypothermia-related neuroprotection against NO-induced cell death. Furthermore, it was revealed that either silencing or overexpression of RBM3 had no effects on the expression of COX-2 in SH-SY5Y cells. These findings suggest that mild hypothermia could protect neuroblastoma cells against NO-induced cell death by inducing COX-2 in a RBM3-independent manner.

Fengshi Gutong Capsule Attenuates Osteoarthritis by Inhibiting MAPK, NF-κB, AP-1, and Akt Pathways.

Background and purpose: Fengshi Gutong capsule (FSGTC), a traditional herbal formula, has been used clinically in China for the treatment of arthritis. However, the mechanism underlying the therapeutic effects of FSGTC on osteoarthritis (OA) has not been elucidated. The present study investigated the function and mechanisms of FSGTC in rat OA model and interleukin (IL)-1ß-stimulated synovial cells. Materials and methods: Rat OA model was established by intra-articular injection containing 4% papain. IL-1ß-induced SW982 cells were used as an OA cell model. Safranin-O-Fast green (S-O) and hematoxylin-eosin (HE) stainings were used to observe the changes in cartilage morphology. Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR (qPCR) detected the expression of inflammatory cytokines. In addition, molecular mechanisms were analyzed by Western blot in the OA cell model. Results: FSGTC treatment significantly relieved the degeneration of cartilage and reduced the contents of tumor necrosis factor-α (TNF-α) and IL-6 in the serum in papain-induced OA rats. FSGTC also reduced the protein and mRNA levels of IL-6 and IL-8 in IL-1ß-stimulated SW982 cells. Moreover, it inhibited the phosphorylation levels of ERK (extracellular signal-related kinase), JNK (c-Jun N-terminal kinase), p38, Akt (protein kinase B), and c-Jun. It also decreased the extent of IκBα degradation and p65 protein translocation into the nucleus. Conclusion: The current data confirmed the protective effects of FSGTC in the rat and OA cell models. The results suggested that FSGTC reduced the production of inflammatory mediators via restraining the activation of mitogen-activated protein kinases (MAPK), nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and Akt.

Neuroprotective Effects of Proanthocyanidins, Natural Flavonoids Derived From Plants, on Rotenone-Induced Oxidative Stress and Apoptotic Cell Death in Human Neuroblastoma SH-SY5Y Cells.

Proanthocyanidins (PA) are natural flavonoids widely present in many vegetables, fruits, nuts and seeds, and especially in grape seed. In the present study, we examined the neuroprotective effects of PA and the underlying molecular mechanism in rotenone model of Parkinson's disease (PD). We found that pretreatment with PA significantly reduced rotenone-induced oxidative stress in human neuroblastoma SH-SY5Y dopaminergic cells. In addition, PA markedly enhanced cell viability against rotenone neurotoxicity and considerably blocked rotenone-induced activation of caspase-9, caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP), biochemical features of apoptosis. Further study demonstrated that the anti-apoptotic effect of PA was mediated by suppressing p38, JNK, and ERK signaling, and inhibitors of these three signaling pathways reproduced the protective effect of PA separately. In summary, our results demonstrated that PA mitigated rotenone-induced ROS generation and antagonized apoptosis in SH-SY5Y cells by inhibiting p38, JNK, and ERK signaling pathways, and it may provide a new insight of PA in PD therapy.

Cold Shock Induced Protein RBM3 but Not Mild Hypothermia Protects Human SH-SY5Y Neuroblastoma Cells From MPP+-Induced Neurotoxicity.

The cold shock protein RBM3 can mediate mild hypothermia-related protection in neurodegeneration such as Alzheimer's disease. However, it remains unclear whether RBM3 and mild hypothermia provide same protection in model of Parkinson's disease (PD), the second most common neurodegenerative disorder. In this study, human SH-SY5Y neuroblastoma cells subjected to insult by 1-methyl-4-phenylpyridinium (MPP+) served as an in-vitro model of PD. Mild hypothermia (32°C) aggravated MPP+-induced apoptosis, which was boosted when RBM3 was silenced by siRNA. In contrast, overexpression of RBM3 significantly reduced this apoptosis. MPP+ treatment downregulated the expression of RBM3 both endogenously and exogenously and suppressed its induction by mild hypothermia (32°C). In conclusion, our data suggest that cold shock protein RBM3 provides neuroprotection in a cell model of PD, suggesting that RBM3 induction may be a suitable strategy for PD therapy. However, mild hypothermia exacerbates MPP+-induced apoptosis even that RBM3 could be synthesized during mild hypothermia.

Cold-Inducible Protein RBM3 Protects UV Irradiation-Induced Apoptosis in Neuroblastoma Cells by Affecting p38 and JNK Pathways and Bcl2 Family Proteins.

Induced by hypothermia, cold-inducible protein RBM3 (RNA-binding protein motif 3), has been implicated in neuroprotection against various toxic insults such as hypoxia and ischemia. However, whether mild hypothermia and RBM3 prevent neural cells from UV irradiation-elicited apoptosis is unclear. In the present study, human neuroblastoma cell line SH-SY5Y was used as a cell model for neural cell death, and it was demonstrated that mild hypothermia protects SH-SY5Y cells from UV irradiation-induced apoptosis. However, the protective effect of mild hypothermia was abrogated when RBM3 was silenced. Conversely, the overexpression of RBM3 rescued SH-SY5Y cells from UV-induced apoptosis, as indicated by the decreased levels of cleaved caspase-3 and PARP, and increased cell survival. The analysis on the mechanism underlying RBM3-mediated neuroprotection against UV insult showed that RBM3 could substantially block the activation of p38 and JNK signaling pathways. In addition, the overexpression of RBM3 reduced the expression of pro-apoptotic proteins Bax and Bad, leaving the pro-survival protein Bcl-2 unaffected. In conclusion, RBM3 is the key mediator of mild hypothermia-related protection against UV in neuroblastoma cells, and the neuroprotective effect might be exerted through interfering with pro-apoptotic signaling pathways p38 and JNK and regulating pro-apoptotic proteins Bax and Bad.

Anti-inflammatory effects of pitavastatin in interleukin-1ß-induced SW982 human synovial cells.

The present study shows the basis for the anti-inflammatory effects of pitavastatin in interleukin (IL)-1ß-induced human synovial cells. The SW982 cells were pretreated with pitavastatin at different concentrations (5µM and 10µM), followed by IL-1ß (10ng/mL) stimulation. The results showed that pitavastatin inhibited the expression of inflammatory mediators IL-6 and IL-8. Furthermore, pitavastatin inhibited the phosphorylation of p38, extracellular signal-related kinase (ERK), c-jun N-terminal kinase (JNK) and protein kinase B (Akt). It also suppressed the degradation of I kappa B alpha and blocked p65 translocation into the nucleus. These findings suggest that the mechanism underlying the inhibitory effects of pitavastatin on IL-1ß-induced IL-6 and IL-8 release might be mediated by the suppression of mitogen-activated protein kinase (MAPK), Akt, and nuclear factor-κB (NF-κB) signaling pathways. These results may also indicate that pitavastatin may be potentially utilized as an effective therapeutic agent for the treatment of osteoarthritis.

NCAM affects directional lamellipodia formation of BMSCs via ß1 integrin signal-mediated cofilin activity.

The neural cell adhesion molecule (NCAM), a key member of the immunoglobulin-like CAM family, was reported to regulate the migration of bone marrow-derived mesenchymal stem cells (BMSCs). However, the detailed cellular behaviors including lamellipodia formation in the initial step of directional migration remain largely unknown. In the present study, we reported that NCAM affects the lamellipodia formation of BMSCs. Using BMSCs from Ncam knockout mice we found that Ncam deficiency significantly impaired the migration and the directional lamellipodia formation of BMSCs. Further studies revealed that Ncam knockout decreased the activity of cofilin, an actin-cleaving protein, which was involved in directional protrusions. To explore the molecular mechanisms involved, we examined protein tyrosine phosphorylation levels in Ncam knockout BMSCs by phosphotyrosine peptide array analyses, and found that the tyrosine phosphorylation level of ß1 integrin, a protein upstream of cofilin, was greatly upregulated in Ncam-deficient BMSCs. Notably, by blocking the function of ß1 integrin with RGD peptide or ROCK inhibitor, the cofilin activity and directional lamellipodia formation of Ncam knockout BMSCs could be rescued. Finally, we found that the effect of NCAM on tyrosine phosphorylation of ß1 integrin was independent of the fibroblast growth factor receptor. These results indicated that NCAM regulates directional lamellipodia formation of BMSCs through ß1 integrin signal-mediated cofilin activity.

RNA-binding protein RBM3 prevents NO-induced apoptosis in human neuroblastoma cells by modulating p38 signaling and miR-143.

Nitric oxide (NO)-induced apoptosis in neurons is an important cause of neurodegenerative disease in humans. The cold-inducible protein RBM3 mediates the protective effects of cooling on apoptosis induced by various insults. However, whether RBM3 protects neural cells from NO-induced apoptosis is unclear. This study aimed to investigate the neuroprotective effect of RBM3 on NO-induced apoptosis in human SH-SY5Y neuroblastoma cells. Firstly, we demonstrated that mild hypothermia (32 °C) induces RBM3 expression and confers a potent neuroprotective effect on NO-induced apoptosis, which was substantially diminished when RBM3 was silenced by siRNA. Moreover, overexpression of RBM3 exhibited a strong protective effect against NO-induced apoptosis. Signaling pathway screening demonstrated that only p38 inhibition by RBM3 provided neuroprotective effect, although RBM3 overexpression could affect the activation of p38, JNK, ERK, and AKT signaling in response to NO stimuli. Notably, RBM3 overexpression also blocked the activation of p38 signaling induced by transforming growth factor-ß1. Furthermore, both RBM3 overexpression and mild hypothermia abolished the induction of miR-143 by NO, which was shown to mediate the cytotoxicity of NO in a p38-dependent way. These findings suggest that RBM3 protects neuroblastoma cells from NO-induced apoptosis by suppressing p38 signaling, which mediates apoptosis through miR-143 induction.

Hydroxysafflor yellow A inhibits IL-1ß-induced release of IL-6, IL-8, and MMP-1 via suppression of ERK, NF-κB and AP-1 signaling in SW982 human synovial cells.

Hydroxysafflor yellow A (HSYA), the main active ingredient in medical and edible dual purpose plant safflower, is reported to have multiple bioactivities. In the present study, the anti-inflammatory effects of HSYA and the underlying mechanisms were investigated in interleukin (IL)-1ß-induced SW982 human synovial cells. The cells were pretreated with HSYA at various concentrations (2.5, 10 and 40 µM) followed by IL-1ß (10 ng mL-1) stimulation. HSYA significantly inhibited the expression of IL-6, IL-8 and matrix metalloproteinase (MMP)-1 in IL-1ß-stimulated SW982 cells. HSYA also inhibited the phosphorylation of extracellular signal-regulated kinase (ERK), p65 and c-Jun. It also suppressed the degradation of IκBα and blocked p65 translocation into the nucleus. These results indicate that the inhibitory effects of HSYA on IL-1ß-induced IL-6, IL-8 and MMP-1 release might be mediated via suppression of ERK, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) signaling pathways. The present data support the potential role of HSYA as an effective therapeutic agent in osteoarthritis.

Thrombospondin-4 Promotes Neuronal Differentiation of NG2 Cells via the ERK/MAPK Pathway.

NG2-expressing neural progenitors can produce neurons in the central nervous system, providing a potential cell resource of therapy for neurological disorders. However, the mechanism underlying neuronal differentiation of NG2 cells remains largely unknown. In this report, we found that a thrombospondin (TSP) family member, TSP4, is involved in the neuronal differentiation of NG2 cells. When TSP4 was overexpressed, NG2 cells underwent spontaneous neuronal differentiation, as demonstrated by the induction of various neuronal differentiation markers such as NeuN, Tuj1, and NF200, at the messenger RNA and protein levels. In contrast, TSP4 silencing had an opposite effect on the expression of neuronal differentiation markers in NG2 cells. Next, the signaling pathway responsible for TSP4-mediated NG2 cell differentiation was investigated. We found that ERK but not p38 and AKT signaling was affected by TSP4 overexpression. Furthermore, when ERK signaling was blocked by the inhibitor U0126, the neuronal marker expression of NG2 cells was substantially increased. Together, these findings suggested that TSP4 promoted neuronal differentiation of NG2 cells by inhibiting ERK/MAPK signaling, revealing a novel role of TSP4 in cell fate specification of NG2 cells.

PRDM5 promotes the proliferation and invasion of murine melanoma cells through up-regulating JNK expression.

PRDM (PRDI-BF1 and RIZ domain-containing) proteins constitute a family of zinc finger proteins and play important roles in multiple cellular processes by acting as epigenetic modifiers. PRDM5 is a recently identified member of the PRDM family and may function as a tumor suppressor in several types of cancer. However, the role of PRDM5 in murine melanoma remains largely unknown. In our study, effect of PRDM5 on murine melanoma cells was determined and results showed that PRDM5 overexpression significantly promoted proliferation, migration, and invasion of murine melanoma B16F10 cells. Consistently, silencing of PRDM5 expression significantly inhibited proliferation, invasion, and migration of B16F10 cells. In vivo study also showed that PRDM5 silencing significantly inhibited the growth and metastasis of melanoma in mice. PRDM5 was then found to increase the expression and activation of JNK in B16F10 cells. JNK silencing significantly reduced PRDM5-mediated up-regulation of JNK expression and blocked the PRDM5-induced proliferation and invasion of B16F10 cells. To further verify the involvement of JNK signaling in PRDM5-induced progression of B16F10 cells, a specific JNK inhibitor was employed to inhibit the JNK signaling pathway, and results showed that PRDM5-induced proliferation and invasion of B16F10 cells were abolished. We conclude that PRDM5 promotes the proliferation and invasion of murine melanoma cells through up-regulating JNK expression and strategies targeting PRDM5 may be promising for the therapy of melanoma.

[Inhibitory effect of oleanolic acid on inflammatory response in IL-1ß-stimulated human synovial sarcoma SW982 cells].

To study the role of oleanolic acid on interleukin (IL)-1ß-stimulated expression of inflammatory cytokines, and to explore its anti-inflammatory mechanism in SW982 cells, the toxicity of oleanolic acid on SW982 cells was detected by MTT; effects of different concentrations of oleanolic acid(5, 10, 20 µmol·L(-1)) on the expression of inflammatory factors IL-6, IL-8 and matrix metalloproteinase-1 (MMP-1) was tested at protein and m RNA levels. The study was performed in IL-1ß-stimulated SW982 cells together with enzyme-linked immunosorbent assay (ELISA) and real-time fluorescence quantitative PCR (real-time PCR) methods; the influence of oleanolic acid on the phosphorylation of mitogen-activated protein kinase (MAPK), phosphatidyl inositol-3-kinase/Akt (PI3K/Akt) and nuclear transcription factor-κB (NF-κB) signaling pathways related protein was analyzed by Western blot. Results showed that different concentrations of oleanolic acid(≤40 µmol·L(-1)) were almost non-toxicity to SW982 cells; oleanolic acid significantly inhibited the expression of inflammatory factors in a dose-dependent manner; oleanolic acid restrained extracellular signal-related kinase (ERK), p38, c-jun N-terminal kinase (JNK) and Akt protein phosphorylation and IκB-α protein degradation obviously. The inhibition effect of oleanolic acid on inflammatory factors stimulated by IL-1ß may be worked through MAPK, PI3K/Akt and NF-κB signaling pathways.

NF-κB protects human neuroblastoma cells from nitric oxide-induced apoptosis through upregulating biglycan.

Excessive nitric oxide (NO) produced in inflammation may result in oxidative stress, which is closely related to the neurodegenerative diseases and brain damage. Massive NO production can enhance NF-κB activity in various neural cells, but the function of this activation by NO and the target genes transactivated by NF-κB are still largely unknown. In the present study, our results showed sodium nitropruside (SNP), a NO donor, triggered apoptotic cell death and NF-κB activation in human neuroblastoma SH-EP1 cells, and inhibition of NF-κB activation by its super endogenous inhibitor, I-κBαM, sensitized SH-EP1 cells to NO-induced apoptosis. Conversely, NF-κB activation induced by insulin-like growth factor (IGF)-1 antagonizes NO-induced apoptotic cell death in SH-EP1 cells. In addition, cDNA microarray analysis showed biglycan, an extracellular glycoprotein, was up-regulated by NF-κB, and recombinant biglycan protein conferred a protective effect on NF-κB mediated NO-induced apoptotic cell death in SH-EP1 cells. These findings suggest biglycan may serve as a potential target in preventing NO-induced neurodegenerative diseases.

Identification of a novel role of RING finger protein 11 promoting the metastasis of murine melanoma cells.

Melanoma is the leading cause of skin cancer death owing to its highly metastatic nature and resistance to chemotherapy. It may account for 80% of the deaths relating to skin cancers. Once it progressed to metastatic stage, no current effective treatment is available for melanoma. Therefore, in-depth understanding of the mechanism underlying the metastatic process is imperative and would be of great help for improving the treatment of melanoma. Here, wedemonstrate that RING finger protein 11 (RNF11) disruption by insertional mutagenesis impairs the metastatic potential of murine melanoma B16F10 cells. The requirement of RNF11 in the migration of melanoma cells is further confirmed by gene knockdown and overexpression experiments in vitro. Together, our findings suggest a novel role of RNF11 in promoting the metastasis of melanoma cells which may potentially be useful for the treatment of melanoma by developing a new intervention target.

NF-κB regulates caspase-4 expression and sensitizes neuroblastoma cells to Fas-induced apoptosis.

Found in neurons and neuroblastoma cells, Fas-induced apoptosis and accompanied activation of NF-κB signaling were thought to be associated with neurodegenerative diseases. However, the detailed functions of NF-κB activation in Fas killing and the effect of NF-κB activation on its downstream events remain unclear. Here, we demonstrated that agonistic Fas antibody induces cell death in a dose-dependent way and NF-κB signaling is activated as well, in neuroblastoma cells SH-EP1. Unexpectedly, NF-κB activation was shown to be pro-apoptotic, as suggested by the reduction of Fas-induced cell death with either a dominant negative form of IκBα (DN-IκBα) or an IκB kinase-specific inhibitor. To our interest, when analyzing downstream events of NF-κB signaling, we found that DN-IκBα only suppressed the expression of caspase-4, but not other caspases. Vice versa, enhancement of NF-κB activity by p65 (RelA) overexpression increased the expression of caspase-4 at both mRNA and protein levels. More directly, results from dual luciferase reporter assay demonstrated the regulation of caspase-4 promoter activity by NF-κB. When caspase-4 activity was blocked by its dominant negative (DN) form, Fas-induced cell death was substantially reduced. Consistently, the cleavage of PARP and caspase-3 induced by Fas was also reduced. In contrast, the cleavage of caspase-8 remained unaffected in caspase-4 DN cells, although caspase-8 inhibitor could rescue Fas-induced cell death. Collectively, these data suggest that caspase-4 activity is required for Fas-induced cell apoptosis and caspase-4 may act upstream of PARP and caspase-3 and downstream of caspase-8. Overall, we demonstrate that NF-κB can mediate Fas-induced apoptosis through caspase-4 protease, indicating that caspase-4 is a new mediator of NF-κB pro-apoptotic pathway in neuroblastoma cells.