CCAAT/enhancer-binding protein-β participates in oxidized LDL-enhanced proliferation in 3T3-L1 cells

Increased circulating oxidized LDL (oxLDL) have been found in obese subjects. Obesity is characterized by an excess of fat mass resulting from an increase in adipocyte number and size. The generation of new adipocytes is a tightly controlled process where multiple factors acting in a signaling cascade follow a precise temporal expression pattern; oxLDL appear to have a role in the impairment of this process. The purpose of this study was to examine the effects of oxLDL on the mechanisms involved in the proliferative stage of the differentiation process in 3T3-L1 cells. After hormonal induction, 3T3-L1 cells undergo approximately two rounds of mitotic clonal expansion (MCE), a process required for adipogenesis. CCAAT/enhancer-binding protein β (C/EBPβ) is immediately expressed after induction, and plays a crucial role in MCE, but its expression must decrease to allow preadipocytes to mature into adipocytes. We found that, in the presence of stimuli to differentiate, oxLDL induced a higher proliferation rate in this cell line, associated with a sustained up-regulation of C/EBPβ, which remained activated inside the nucleus for several days. RNAi-mediated knockdown of C/EBPβ 24 h after oxLDL treatment counteracted the increase in proliferation rate. Both C/EBPβ expression and proliferation processes appear to be influenced by cAMP/protein kinase A (PKA) and extracellular signal-regulated kinases1/2 (ERK1/2) pathways. OxLDL treatment led to increased levels of cAMP, and to a strong, prolonged phosphorylation of ERK1/2 and C/EBPβ. The addition of cAMP and PKA inhibitors, SQ22536 and H-89, respectively, reduced proliferation only in oxLDL-treated cells, whereas the addition of ERK1/2 inhibitor U0126 blocked proliferation in both control and oxLDL-treated cells. C/EBPβ nuclear expression and DNA-binding activity were reduced by U0126, under all tested conditions. These findings show that the altered expression pattern of C/EBPβ is involved in the increase in the number of proliferating cells induced by oxLDL, in hormone-stimulated 3T3-L1 cells.     

Dehydrocostuslactone suppresses angiogenesis in vitro and in vivo through inhibition of Akt/GSK-3β and mTOR signaling pathways

The traditional Chinese medicine component dehydrocostuslactone (DHC) isolated from Saussurea costus (Falc.) Lipschitz, has been shown to have anti-cancer activity. Angiogenesis is an essential process in the growth and progression of cancer. In this study, we demonstrated, for the first time, the anti-angiogenic mechanism of action of DHC to be via the induction of cell cycle progression at the G0/G1 phase due to abrogation of the Akt/glycogen synthase kinase-3β (GSK-3β)/cyclin D1 and mTOR signaling pathway. First, we demonstrated that DHC has an anti-angiogenic effect in the matrigel-plug nude mice model and an inhibitory effect on human umbilical vein endothelial cell (HUVEC) proliferation and capillary-like tube formation in vitro. DHC caused G0/G1 cell cycle arrest, which was associated with the down-regulation of cyclin D1 expression, leading to the suppression of retinoblastoma protein phosphorylation and subsequent inhibition of cyclin A and cdk2 expression. With respect to the molecular mechanisms underlying the DHC-induced cyclin D1 down-regulation, this study demonstrated that DHC significantly inhibits Akt expression, resulting in the suppression of GSK-3β phosphorylation and mTOR expression. These effects are capable of regulating cyclin D1 degradation, but they were significantly reversed by constitutively active myristoylated (myr)-Akt. Furthermore, the abrogation of tube formation induced by DHC was also reversed by overexpression of Akt. And the co-treatment with LiCl and DHC significantly reversed the growth inhibition induced by DHC. Taken together, our study has identified Akt/GSK-3β and mTOR as important targets of DHC and has thus highlighted its potential application in angiogenesis-related diseases, such as cancer.     

Activation of neutral-sphingomyelinase,MAPKs, and p75 NTR-mediating caffeic acid phenethyl ester–induced apoptosis in C6 glioma cells

Background

Caffeic acid phenethyl ester (CAPE), a component of propolis, is reported to possess anti-inflammatory, anti-bacterial, anti-viral, and anti-tumor activities. Previously, our laboratory demonstrated the in vitro and in vivo bioactivity of CAPE and addressed the role of p53 and the p38 mitogen-activated protein kinase (MAPK) pathway in regulating CAPE-induced apoptosis in C6 glioma cells.

Results

C6 cancer cell lines were exposed to doses of CAPE; DNA fragmentation and MAPKs and NGF/P75NTR levels were then determined. SMase activity and ceramide content measurement as well as western blotting analyses were performed to clarify molecular changes. The present study showed that CAPE activated neutral sphingomyelinase (N-SMase), which led to the ceramide-mediated activation of MAPKs, including extracellular signal-regulated kinase (ERK), Jun N-terminus kinase (JNK), and p38 MAPK. In addition, CAPE increased the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75NTR). The addition of an N-SMase inhibitor, GW4869, established that NGF/p75NTR was the downstream target of N-SMase/ceramide. Pretreatment with MAPK inhibitors demonstrated that MEK/ERK and JNK acted upstream and downstream, respectively, of NGF/p75NTR. Additionally, CAPE-induced caspase 3 activation and poly [ADP-ribose] polymerase cleavage were reduced by pretreatment with MAPK inhibitors, a p75NTR peptide antagonist, or GW4869.

Conclusions

Taken together, N-SMase activation played a pivotal role in CAPE-induced apoptosis by activation of the p38 MAPK pathway and NGF/p75NTR may explain a new role of CAPE induced apoptosis in C6 glioma.     

Mitochondrial free fatty acid β-oxidation supports oxidative phosphorylation and proliferation in cancer cells

Oxidative phosphorylation (OxPhos) is functional and sustains tumor proliferation in several cancer cell types. To establish whether mitochondrial β-oxidation of free fatty acids (FFAs) contributes to cancer OxPhos functioning, its protein contents and enzyme activities, as well as respiratory rates and electrical membrane potential (ΔΨm) driven by FFA oxidation were assessed in rat AS-30D hepatoma and liver (RLM) mitochondria. Higher protein contents (1.4–3 times) of β-oxidation (CPT1, SCAD) as well as proteins and enzyme activities (1.7–13-times) of Krebs cycle (KC: ICD, 2OGDH, PDH, ME, GA), and respiratory chain (RC: COX) were determined in hepatoma mitochondria vs. RLM. Although increased cholesterol content (9-times vs. RLM) was determined in the hepatoma mitochondrial membranes, FFAs and other NAD-linked substrates were oxidized faster (1.6–6.6 times) by hepatoma mitochondria than RLM, maintaining similar ΔΨm values. The contents of β-oxidation, KC and RC enzymes were also assessed in cells. The mitochondrial enzyme levels in human cervix cancer HeLa and AS-30D cells were higher than those observed in rat hepatocytes whereas in human breast cancer biopsies, CPT1 and SCAD contents were lower than in human breast normal tissue. The presence of CPT1 and SCAD in AS-30D mitochondria and HeLa cells correlated with an active FFA utilization in HeLa cells. Furthermore, the β-oxidation inhibitor perhexiline blocked FFA utilization, OxPhos and proliferation in HeLa and other cancer cells. In conclusion, functional mitochondria supported by FFA β-oxidation are essential for the accelerated cancer cell proliferation and hence anti-β-oxidation therapeutics appears as an alternative promising approach to deter malignant tumor growth.     

GSK-3β promotes PA-induced apoptosis through changing β-arrestin 2 nucleus location in H9c2 cardiomyocytes

Palmitic acid (PA), a type of saturated fatty acids, induces cardiovascular diseases by causing cardiomyocyte apoptosis with unclear mechanisms. Akt participates in PA-induced cardiomyocyte apoptosis. GSK-3β is a substrate of Akt, we investigated its role in PA-induced apoptosis. We reveal that PA inhibits GSK-3β phosphorylation accompanied by inactivation of Akt in H9c2 cardiomyocytes. We also reveal that inhibition the activity of GSK-3β by its inhibitor LiCl or knockdown by siRNA significantly attenuates PA-induced cardiomyocyte apoptosis, this suggesting that GSK-3β plays a pro-apoptotic role. To detect its downstream factors, we analyzed the roles of JNK, p38 MAPK and β-arrestin 2 (β-Arr2). Here, we report that GSK-3β regulate PA-induced cardiomyocyte apoptosis by affecting the distribution of β-Arr2. PA diminishes the protein level of β-Arr2 and changes its distribution from nucleus to cytoplasm. Either inhibition of β-Arr2 by its siRNA or overexpression of its protein level by transfection of β-Arr2 full-length plasmid promotes PA-induced cardiomyocyte apoptosis, which remind us to focus on the changes of its location. β-Arr2 siRNA decreased the background level of β-Arr2 in nucleus in normal H9c2 cells. Overexpression of β-Arr2 increased cytoplasm level of β-Arr2 as PA did. While LiCl, the inhibitor of GSK-3β decreased PA-induced apoptosis, accompany with increased nucleus level of β-Arr2. Then we concluded that GSK-3β is closely associated with cardiomyocyte apoptosis induced by PA, it performs its pro-apoptotic function by affecting the location of β-Arr2. LiCl inhibits PA-induced cardiomyocyte apoptosis, which might provide novel therapeutic for cardiovascular diseases induced by metabolic syndrome.     

TGF-β regulates β-catenin signaling and osteoblast differentiation in human mesenchymal stem cells

Human adult bone marrow-derived skeletal stem cells a.k.a mesenchymal stem cells (hMSCs) have been shown to be precursors of several different cellular lineages, including osteoblast, chondrocyte, myoblast, adipocyte, and fibroblast. Several studies have shown that cooperation between transforming growth factor β (TGF-β) and Wnt/β-catenin signaling pathways plays a role in controlling certain developmental events and diseases. Our previous data showed that agents like TGF-β, cooperation with Wnt signaling, promote chondrocyte differentiation at the expense of adipocyte differentiation in hMSCs. In this study, we tested mechanisms by which TGF-β activation of β-catenin signaling pathway and whether these pathways interact during osteoblast differentiation of hMSCs. With selective small chemical kinase inhibitors, we demonstrated that TGF-β1 requires TGF-β type I receptor ALK-5, Smad3, phosphoinositide 3-kinases (PI3K), and protein kinase A (PKA) to stabilize β-catenin, and needs ALK-5, PKA, and JNK to inhibit osteoblastogenesis in hMSCs. Knockdown of β-catenin with siRNA stimulated alkaline phosphatase activity and antagonized the inhibitory effects of TGF-β1 on bone sialoprotein (BSP) expression, suggested that TGF-β1 cooperated with β-catenin signaling in inhibitory of osteoblastogenesis in hMSCs. In summary, TGF-β1 activates β-catenin signaling pathway via ALK-5, Smad3, PKA, and PI3K pathways, and modulates osteoblastogenesis via ALK5, PKA, and JNK pathways in hMSCs; the interaction between TGF-β and β-catenin signaling supports the view that β-catenin signaling is a mediator of TGF-β's effects on osteoblast differentiation of hMSCs.     

Cilostazol induces mitochondrial fatty acid β-oxidation in C2C12 myotubes

Cilostazol is a drug licensed for the treatment of intermittent claudication. Its main action is to elevate intracellular levels of cyclic monophosphate (cAMP) by inhibiting the activity of type III phosphodiesterase, a cAMP-degrading enzyme. The effects of cilostazol on fatty acid oxidation (FAO) are as yet unknown. In this study, we report that cilostazol can elevate complete FAO and decrease both triacylglycerol (TAG) accumulation and TAG secretion. This use of cilostazol treatment increases expression of PGC-1α and, subsequently, its target genes, such as ERRα, NOR1, CD36, CPT1, MCAD, and ACO. Expression of these factors is linked to fatty acid β-oxidation but this effect is inhibited by H-89, a specific inhibitor of the PKA/CREB pathway. Importantly, knockdown of PGC-1α using siRNA abolished the effects of cilostazol in fatty acid oxidation (FAO) and TAG metabolism. These findings suggested that the PKA/CREB/PGC-1α pathway plays a critical role in cilostazol-induced fatty acid oxidation and TAG metabolism.     

Diverse regulation of AKT and GSK-3β by O-GlcNAcylation in various types of cells

Protein kinase B (AKT) and glycogen synthase kinase-3β (GSK-3β) are major components of insulin-AKT signaling that plays crucial roles in various types of tissue. Recent studies found that these two kinases are modified posttranslationally by O-GlcNAcylation. Here, we demonstrate that O-GlcNAcylation regulated phosphorylation/activation of AKT and GSK-3β in different manners in kidney HEK-293FT cells, but did not affect these two kinases in hepatic HepG2 cells. In neuronal cells, O-GlcNAcylation regulated phosphorylation of AKT negatively, but had no effect on GSK-3β. These results suggest protein-specific and cell type-specific regulation of AKT and GSK-3β by O-GlcNAcylation. Therefore, studies on the roles of AKT and GSK-3β O-GlcNAcylation should be done in a tissue- and cell type-specific manner.     

Smurf2 induces degradation of GSK-3β and upregulates β-catenin in chondrocytes: A potential mechanism for Smurf2-induced degeneration of articular cartilage

We have previously demonstrated that Smurf2 is highly expressed in human osteoarthritis (OA) tissue, and overexpression of Smurf2 under the control of the type II collagen promoter (Col2a1) induces an OA-like phenotype in aged Col2a1-Smurf2 transgenic mice, suggesting that Smurf2 is located upstream of a signal cascade which initiates OA development. However, the factors downstream of Smurf2 in this signal cascade and how Smurf2-induced OA is initiated are largely unknown. In this study, we further characterized the phenotypic changes in Col2a1-Smurf2 transgenic and WT articular cartilage from the postnatal stage to adulthood. We found that the articular cartilage degeneration occurring at the cartilage surface in 6 month-old Col2a1-Smurf2 transgenic mice progressed from an expanded hypertrophic domain in the basal layer of the deep articular cartilage at 2.5 weeks of age, which may lead to an accelerated calcification and ectopic ossification of this region at 1 month of age, and aggregation and maturation of articular chondrocytes in the middle and deep zones at 2 months and 4.5 months of age, respectively. Furthermore, we discovered that ectopically expressed Smurf2 interacted with GSK-3β and induced its ubiquitination and subsequent proteasomal degradation, and hence upregulated β-catenin in Col2a1-Smurf2 transgenic chondrocytes ex vivo. It is therefore likely that Smurf2-mediated upregulation of β-catenin through induction of proteasomal degradation of GSK-β in chondrocytes may activate articular chondrocyte maturation and associated alteration of gene expression, the early events of OA.     

Lithium chloride decreases proliferation and migration of C6 glioma cells harboring isocitrate dehydrogenase 2 mutant via GSK-3β

The gene encoding isocitrate dehydrogenase (IDH) is somatically mutated predominantly in secondary glioblastoma multiforme. Mutations of IDH1 and IDH2 lead to simultaneous loss and gain of activities in the production of α-ketoglutarate and 2-hydroxyglutarate, respectively. Lithium chloride was recently proved efficient in inhibiting glioma cell migration. The mechanism of lithium chloride on C6 glioma cells harboring IDH2 mutation has not been studied. Here, we found lithium chloride induced inhibitive effects on cell proliferation of both C6 glioma cells with and without IDH2 mutation, although IDH2 mutation increased the stability of HIF-1α. GSK-3β could be phosphorylated at Ser9 and its activity was inhibited when C6 glioma cells were treated by lithium chloride. The degree of phosphorylation in IDH2^R172G treatment group was lower than that as compared to the control and IDH2 treatment groups. At the same time, the accumulation of β-catenin in C6 cell nucleus was decreased. Moreover, although the β-catenin and HIF-1α increased the secretion of metalloproteinase-2,-9 in C6 glioma cells harboring IDH2 mutation, the migration potential of lithium chloride-treated C6 glioma cells harboring the IDH2 and its mutant was uniform. These results indicated lithium chloride could decrease the proliferation and migration potential of C6 glioma cells harboring IDH2 mutation.     

Induction of aromaticl-amino acid decarboxylase mRNA by interleukin-1β and prostaglandin E2 in PC12 cells

Aromatic 1-amino acid decarboxylase (AADC) is involved in the synthesis of the putative neurotransmitters dopamine (DA), norepinephrine (NA) and 5-hydroxytryptamine (5-HT). We report here that the gene expression of AADC can be regulated by interleukin (IL) 1- and prostaglandin (PG) E₂ in PC12 cells. The cells were treated with different doses of IL 1- and PGE₂ for 3 days. Slot blot hybridization was performed to detect AADC mRNA and Western immunoblot to detect AADC protein. The cDNA probe for rat AADC was generated by the PCR method. IL 1- and PGE₂ produced a dose- and time-dependent up-regulation in AADC mRNA levels (up to 200% of the control values) which was followed by a stable increase in AADC protein. The data further support the suggestion that AADC is a regulated enzyme and that the regulation occurs at the level of gene expression. Because IL-1 is synthesized, and acts locally, within the brain to influence neuronal and glial functions, it has been proposed to be a mediator with both beneficial and detrimental responses to inflammation and injury. The regulation of AADC by IL-1 may indicate a possible involvement for AADC in neuronal injury and recovery. Since IL-1 promotes PGE₂ formation, its effects may be occurring by increasing level of PGE₂.Abbreviations AADC aromatic 1-amino acid decarboxylase - IL-1 interleukin 1 - PGE₂ prostaglandin E₂ - GITC guanidinium isothiocyanate - DEPC diethyl pyrocarbonate - MOPS 3-(4-morpholino)propanesulfonic acid - SSPE 0.18M NaCl, 0.001M sodium phosphate, and 0.001M EDTA Special issue dedicated to Dr. Bernard W. Agranoff.     

Altered distribution of the nuclear receptor rarβ accompanies proliferation and differentiation changes caused by retinoic acid in Caco-2 cells

Summary All epithelial cells require retinoic acid for growth, maintenance, and differentiation. Although the epithelial cells that line the gastrointestinal tract are exposed to extreme retinoid concentration fluctuations in luminal fluid, whether proliferation and differentiation in these cells are significantly affected is not known. We have investigated this question using Caco-2 cells as a model because, although they are derived from a colon adenocarcinoma, they differentiate spontaneously in a manner similar to enterocytes in the small intestine. We found that retinoic acid caused maximum inhibition of cell growth and ornithine decarboxylase activity during the proliferative period. Retinoic acid increased brush border enzyme activities only in differentiating cells but stimulated transglutaminase activity in cells at all stages. In untreated proliferating cells, we found an early peak of transglutaminase activity that has not been reported before. Retinoic acid in intestinal cells acts through its nuclear receptor, RARβ. The nuclear distribution of this receptor has not been demonstrated. In this study, we show that RARβ responds to increasing concentrations of retinoic acid with a shift to the nuclear membrane in undifferentiated cells and progressive aggregation, diffusion, and loss in differentiated cells. We conclude that retinoic acid can inhibit proliferation and stimulate differentiation in Caco-2 cells depending on concentration and cell stage, and that these effects are accompanied by changes in distribution, as well as by the loss of RARβ.     

Role of endogenous TGF-β family in myogenic differentiation of C2C12 cells

The present study evaluated endogenous activities and the role of BMP and transforming growth factor-β (TGF-β), representative members of the TGF-β family, during myotube differentiation in C2C12 cells. Smad phosphorylation at the C-terminal serines was monitored, since TGF-β family members signal via the phosphorylation of Smads in a ligand-dependent manner. Expression of phosphorylated Smad1/5/8, which is an indicator of BMP activity, was higher before differentiation, and rapidly decreased after differentiation stimulation. Differentiation-related changes were consistent with those in the expression of Ids, well-known BMP-responsive genes. Treatment with inhibitors of BMP type I receptors or noggin in C2C12 myoblasts down-regulated the expression of myogenic regulatory factors, such as Myf5 and MyoD, leading to impaired myotube formation. Addition of BMP-2 during the myoblast phase also inhibited myotube differentiation through the down-regulation of Myf5 and MyoD. In contrast to endogenous BMP activity, the phosphorylation of Smad2, a TGF-β-responsive Smad, was higher 8-16 days after differentiation stimulation. A-83-01, an inhibitor of TGF-β type I receptor, increased the expression of Myf5 and MyoD, and enhanced myotube formation. The present results reveal that endogenous activities of the TGF-β family are changed during myogenesis in a pathway-specific manner, and that the activities are required for myogenesis.     

Inhibition of gecko GSK-3β promotes elongation of neurites and oligodendrocyte processes but decreases the proliferation of blastemal cells

GSK-3β signaling is involved in regulation of both neuronal and glial cell functions, and interference of the signaling affects central nervous system (CNS) development and regeneration. Thus, GSK-3β was proposed to be an important therapeutic target for promoting functional recovery of adult CNS injuries. To further clarify the regulatory function of the kinase on the CNS regeneration, we characterized gecko GSK-3β and determined the effects of GSK-3β inactivation on the neuronal and glial cell lines, as well as on the gecko tail (including spinal cord) regeneration. Gecko GSK-3β shares 91.7-96.7% identity with those of other vertebrates, and presented higher expression abundance in brain and spinal cord. The kinase strongly colocalized with the oligodendrocytes while less colocalized with neurons in the spinal cord. Phosphorylated GSK-3β (pGSK-3β) levels decreased gradually during the normally regenerating spinal cord ranging from L13 to the 6th caudal vertebra. Lithium injection increased the pGSK-3β levels of the corresponding spinal cord segments, and in vitro experiments on neurons and oligodendrocyte cell line revealed that the elevation of pGSK-3β promoted elongation of neurites and oligodendrocyte processes. In the normally regenerate tails, pGSK-3β kept stable in 2 weeks, whereas decreased at 4 weeks. Injection of lithium led to the elevation of pGSK-3β levels time-dependently, however destructed the regeneration of the tail including spinal cord. Bromodeoxyuridine (BrdU) staining demonstrated that inactivation of GSK-3β decreased the proliferation of blastemal cells. Our results suggested that species-specific regulation of GSK-3β was indispensable for the complete regeneration of CNS.     

Substance P inhibits high urea-induced apoptosis through the AKT/GSK-3β pathway in human corneal epithelial cells

To investigate the effect of substance P (SP) on human corneal epithelial cells (HCECs) that have been stressed by a high urea environment and to determine the relationship between SP and the protein kinase B (AKT)/glycogen synthase kinase-3β (GSK-3β) signaling pathway. An in vitro model of chronic renal failure (CRF)-related dry eye was used to study HCECs that were treated with high urea concentrations. Cell proliferation was assayed using a cell counting kit-8 test. Besides, cell apoptosis was evaluated by flow cytometry. Furthermore, the effects of SP and the AKT inhibitor perifosine on the urea-treated HCECs were examined using immunofluorescence, quantitative real time polymerase chain reaction (qRT-PCR), and Western blot analysis. SP markedly reduced the number of apoptotic HCECs and decreased the cleaved caspase-3 expression levels while contributing to increased cellular proliferation (P < 0.05). The Western blot analysis and qRT-PCR experiments revealed that SP significantly increased the expression of p-AKT and p-GSK-3β (P < 0.05); additionally, these increases were attenuated after the perifosine inhibition of the AKT signaling pathway (P < 0.05). These in vitro experiments demonstrated that SP may protect against the apoptotic damage of HCECs caused by the high urea condition. The underlying mechanism may be related to the activation of the AKT/GSK-3β signaling pathway.     

TMPyP4-regulated cell proliferation and apoptosis through the Wnt/β-catenin signaling pathway in SW480 cells

Background: The aim of this study was to investigate the potential effects of the 5, 10, 15, 20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4) on the proliferation and apoptosis of SW480 cells and the underlying mechanisms by which TMPyP4 exerted its actions. Methods: After treated with different doses of TMPyP4, cell viability was determined by MTT method, the apoptosis was observed by flow cytometry (FCM) and the expression of Wnt, GSK-3β, β-catenin and cyclinD1 was measured by RT-PCR and Western blot analysis. Results: The analysis revealed that TMPyP4 potently suppressed cell viability and induced the apoptosis of SW480 cells in a dose-dependent manner. In addition, the downregulation of Wnt, β-catenin and cyclinD1 expression levels was detected in TMPyP4-treated SW480 cells. However, followed by the block of Wnt signaling pathway using siRNA methods, the effects of TMPyP4 on proliferation and apoptosis of SW480 cells were significantly reduced. Conclusion: It indicates that the TMPyP4-inhibited proliferation and -induced apoptosis in SW480 cells was accompanied by the suppression of Wnt/β-catenin signaling pathway. Therefore, TMPyP4 may represent a potential therapeutic method for the treatment of colon carcinoma.