NF-κB p65 and TCF-4 interactions are associated with LPS-stimulated IL-6 secretion of macrophages.

Proinflammatory cytokine plays a central role in host defense and acute inflammatory responses. Both positive and negative correlations of NF-κB and Wnt/ß-catenin pathways have been reported depending on cell types in response to inflammatory stimuli for IL-6 cytokine production. Macrophages are vital to the regulation of immune responses and the development of inflammation, but the crosstalk between two pathways has not been elucidated so far in macrophages. We observed a positive cross-regulation between the NF-κB and Wnt/ß-catenin pathways for IL-6 production in human macrophages. To verify the functional validity of this interaction, LY294002 or PNU74654, representative blockers of each pathway, were treated. IL-6 secretion was reduced to the basal level by both inhibitor treatments, even when stimulated by LPS. We also found that NF-κB p65 migrated to the nucleus and interacted with the transcription factor TCF-4 in macrophages upon LPS stimulation.

Lithium chloride inhibits the migration and invasion of osteosarcoma cells by blocking nuclear translocation of phospho-Erk.

Lithium chloride (LiCl) is an important mood-stabilizing therapeutic agent for bipolar disorders, which has also been shown to inhibit cancer cell metastasis. Investigations of LiCl-induced signaling have focused mainly on extracellular signal regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3 (GSK-3). However, little is known about the differences in cellular activities resulting from specific signaling via each of these pathways. In this study, we investigated the difference in responses between the Wnt/ß-catenin and ERK pathways by LiCl or epidermal growth factor (EGF) treatment of osteosarcoma cells. In particular, we analyzed the mechanisms responsible for differences in cell mobility and cell proliferation when pERK or ß-catenin is activated. In osteosarcoma cells treated with LiCl or EGF, active ß-catenin and p-ERK protein levels were significantly increased compared to those in the control group. However, in wound healing and transwell invasion assays, U2OS and SaOS2 cell migration was significantly reduced by LiCl treatment but increased by EGF treatment. In addition, the proliferation of U2OS cells was reduced by LiCl treatment but increased by EGF treatment. Using immunofluorescence microscopy, we observed nuclear accumulation of phosphorylated ERK (pERK) with EGF treatment, but pERK was restricted to the perinuclear area with LiCl treatment. These results were confirmed using immunoblot assays after subcellular fractionation. Together, these data suggest that LiCl interferes with the translocation of pERK from the cytoplasm to the nucleus.

Macrophage-preferable delivery of the leucine-rich repeat domain of NLRX1 ameliorates lethal sepsis by regulating NF-κB and inflammasome signaling activation.

Sepsis is an acute systemic inflammatory disease triggered by bacterial infection leading organ dysfunctions that macrophages are responsible for major triggering of systemic inflammation. Treatment options are limited to antibiotics and drugs to manage the symptoms of sepsis, but there are currently no molecular-targeted therapies. Here, we identified a novel macrophage-preferable delivery peptide, C10, which we conjugated to truncated domains of NLRX1 (leucine-rich repeat region (LRR), and nucleotide binding domain (NBD)) to obtain C10-LRR and C10-NBD. Leucine rich amino acid of C10 enables macrophage preferable moieties that efficiently deliver a cargo protein into macrophages in vitro and in vivo. C10-LRR but not C10-NBD significantly improved survival in an LPS-mediated lethal endotoxemia sepsis model. C10-LRR efficiently inhibited IL-6 production in peritoneal macrophages via prevention of IκB degradation and p65 phosphorylation. In addition, C10-LRR negatively regulated IL-1ß production by preventing caspase-1 activation with a sustained mitochondrial MAVS level. Finally, co-treatment with anti-TNFα antibody and C10-LRR had a synergistic effect in an LPS-induced sepsis model. Collectively, these findings indicate that C10-LRR could be an effective therapeutic agent to treat systemic inflammation in sepsis by regulating both NF-κB and inflammasome signaling activation.

Characteristics of Asian Mosquito Saliva Allergens for Specific Diagnoses and Effective Therapies.

An allergic reaction to mosquitoes can result in severe or abnormal local or systemic reactions such as anaphylaxis, angioedema, and general urticarial or wheezing. The aim of this review is to provide information on mosquito saliva allergens that can support the production of highly specific recombinant saliva allergens. In particular, candidate allergens of mosquitoes that are well suited to the ecology of mosquitoes that occur mainly in East Asia will be identified and introduced. By doing so, the diagnosis and treatment of patients with severe sensitivity to mosquito allergy will be improved by predicting the characteristics of East Asian mosquito allergy, presenting the future direction of production of recombinant allergens, and understanding the difference between East and West.

WDR76 is a RAS binding protein that functions as a tumor suppressor via RAS degradation.

Stability regulation of RAS that can affect its activity, in addition to the oncogenic mutations, occurs in human cancer. However, the mechanisms for stability regulation of RAS involved in their activity and its roles in tumorigenesis are poorly explored. Here, we identify WD40-repeat protein 76 (WDR76) as one of the HRAS binding proteins using proteomic analyses of hepatocellular carcinomas (HCC) tissue. WDR76 plays a role as an E3 linker protein and mediates the polyubiquitination-dependent degradation of RAS. WDR76-mediated RAS destabilization results in the inhibition of proliferation, transformation, and invasion of liver cancer cells. WDR76-/- mice are more susceptible to diethylnitrosamine-induced liver carcinogenesis. Liver-specific WDR76 induction destabilizes Ras and markedly reduces tumorigenesis in HRasG12V mouse livers. The clinical relevance of RAS regulation by WDR76 is indicated by the inverse correlation of their expressions in HCC tissues. Our study demonstrates that WDR76 functions as a tumor suppressor via RAS degradation.

ß-Catenin Accumulation Is Associated With Increased Expression of Nanog Protein and Predicts Maintenance of MSC Self-Renewal.

Human mesenchymal stem cells (hMSCs) are self-renewing cells with the ability to differentiate into organized, functional network of cells. Recent studies have revealed that activation of the Wnt/ß-catenin pathway by a glycogen synthase kinase (GSK)-3-specific pharmacological inhibitor, Bio, results in the maintenance of self-renewal in both mouse and human ES cells. The molecular mechanism behind the maintenance of hMSCs by these factors, however, is not fully understood. We found that rEGF enhances the level of ß-catenin, a component of the Wnt/ß-catenin signaling pathway. Furthermore, it was found that ß-catenin upregulates Nanog. EGF activates the ß-catenin pathway via the Ras protein and also increased the Nanog protein and gene expression levels 2 h after rEGF treatment. These results suggest that adding EGF can enhance ß-catenin and Nanog expression in MSCs and facilitate EGF-mediated maintenance of MSC self-renewal. EGF was shown to augment MSC proliferation while preserving early progenitors within MSC population and thus did not induce differentiation. Thus, EGF not only can be used to expand MSC in vitro but also be utilized to autologous transplantation of MSCs in vivo.

Sur8/Shoc2 promotes cell motility and metastasis through activation of Ras-PI3K signaling.

Sur8 (also known as Shoc2) is a Ras-Raf scaffold protein that modulates signaling through extracellular signal-regulated kinase (ERK) pathway. Although Sur8 has been shown to be a scaffold protein of the Ras-ERK pathway, its interaction with other signaling pathways and its involvement in tumor malignancy has not been reported. We identified that Sur8 interacts with the p110α subunit of phosphatidylinositol 3-kinase (PI3K), as well as with Ras and Raf, and these interactions are increased in an epidermal growth factor (EGF)- and oncogenic Ras-dependent manner. Sur8 regulates cell migration and invasion via activation of Rac and matrix metalloproteinases (MMPs). Interestingly, using inhibitors of MEK and PI3K we found Sur8 mediates these cellular behaviors predominantly through PI3K pathway. We further found that human metastatic melanoma tissues had higher Sur8 content followed by activations of Akt, ERK, and Rac. Lentivirus-mediated Sur8-knockdown attenuated metastatic potential of highly invasive B16-F10 melanoma cells indicating the role of Sur8 in melanoma metastasis. This is the first report to identify the role of scaffold protein Sur8 in regulating cell motility, invasion, and metastasis through activation of both ERK and PI3K pathways.

A novel isoform of met receptor tyrosine kinase blocks hepatocyte growth factor/Met signaling and stimulates skeletal muscle cell differentiation.

Hepatocyte growth factor (HGF) and its receptor, Met, regulate skeletal muscle differentiation. In the present study, we identified a novel alternatively spliced isoform of Met lacking exon 13 (designated Δ13Met), which is expressed mainly in human skeletal muscle. Alternative splicing yielded a truncated Met having extracellular domain only, suggesting an inhibitory role. Indeed, Δ13Met expression led to a decrease in HGF-induced tyrosine phosphorylation of Met and ERK phosphorylation, as well as cell proliferation and migration via sequestration of HGF. Interestingly, in human primary myoblasts undergoing differentiation, Δ13Met mRNA and protein levels were rapidly increased, concomitantly with a decrease in wild type Met mRNA and protein. Inhibition of Δ13Met with siRNA led to a decreased differentiation, whereas its overexpression potentiated differentiation of human primary myoblasts. Furthermore, in notexin-induced mouse injury model, exogenous Δ13Met expression enhanced regeneration of skeletal muscle, further confirming a stimulatory role of the isoform in muscle cell differentiation. In summary, we identified a novel alternatively spliced inhibitory isoform of Met that stimulates muscle cell differentiation, which confers a new means to control muscle differentiation and/or regeneration.

Comparative analysis of mesenchymal stem cell surface marker expression for human dental mesenchymal stem cells.

AIM: Human dental mesenchymal stem cells (hDMSCs) have been isolated from extracted human teeth and proven to have different proliferation and differentiation abilities among the subtypes. Despite increasing interest in the clinical use of hDMSCs, a well-defined specific marker has been absent for these stem cells. In this study, a comparative analysis with known mesenchymal stem cell surface markers such as STRO-1, CD90, CD146, CD34 and TfR (CD71) was performed. MATERIALS & METHODS: Four subtypes of the hDMSCs were obtained and cultured. The hDMSCs were processed by flow cytometric analysis, fluorescence immunocytostaining for in vitro study and in situ immunohistochemical staining for in vivo study. RESULTS & CONCLUSION: The previously known positive and negative MSC markers, such as STRO-1, CD90, CD146 and CD34 showed comparative expression profiles of hDMSC subtypes. TfR was highly positive in hDMSCs compared with the control cells; therefore, TfR was suggested as a new marker for hDMSCs in this study.

Silkworm 30K protein inhibits ecdysone-induced apoptosis by blocking the binding of ultraspiracle to ecdysone receptor-B1 in cultured Bm5 cells.

This study investigates the mechanism through which increased 30K protein inhibits ecdysone-induced apoptosis in the Bm5 silkworm ovarian cell line. Treatment of Bm5 cells with 20-hydroxyecdysone (20E) after transfection with the pIZT/V5-His control vector triggered apoptosis, but 20E treatment did not trigger apoptosis in Bm5 cells transfected with the pIZT/30K/V5-His vector. To confirm its inhibitory effect on apoptosis, 30K protein was first purified from Escherichia coli transformed with a 30K expression vector and used to generate specific antibodies in mice. Anti-30K antiserum was used to confirm synthesis of the 30K protein in pIZT/30K/V5-His-transfected Bm5 cells and to detect 30K protein binding to the ecdysone receptor-B1 (EcR-B1). Anti-30K antiserum was used to immunoprecipitate protein complexes containing 30K from Bm5 cells transfected with pIZT/30K/V5-His vector and treated with 20E. We observed that 30K proteins bound primarily to the EcR-B1 and not to ultraspiracle (USP). Reciprocal immunoprecipitation of EcR-B1-containing complexes from Bm5 cells transfected with control pIZT/V5-His vector and treated with 20E showed that EcR-B1 bound to USP in the absence of 30K but did not bind to USP in pIZT/30K/V5-His-transfected Bm5 cells. These results demonstrate that 30K proteins block USP binding to EcR-B1 through formation of a 30K/EcR-B1 complex, resulting in inhibition of 20E-induced Bm5 cell apoptosis.

Increase of 30K protein in identified motoneurons by hemolymph results in inhibition of programmed cell death in silkworm, Bombyx mori (Lepidoptera, Bombycidae).

This study demonstrates that a 30K protein was gradually synthesized in primary-cultured motoneurons from the accessory planta retractor (APR) of the 6th abdominal ganglion (APR6) in silkworm ventral ganglia through stimulation of hemolymph. An increase in 30K protein synthesis resulted in an inhibition of programmed cell death (PCD) of APR6 motoneurons. The 30K protein was gradually synthesized from the 30Kc6 gene of identified APR6s in day-6 4th instars to day-9 5th instar larvae, but synthesis of the 30K protein ceased in isolated APR6s of day-1 pupa, which normally begin to undergo PCD. When pupal APR6s were treated with larval hemolymph, however, the 30K protein was synthesized suggesting the existence of an anti-PCD factor in the larval hemolymph. An increase of 30K protein within the APR6s was confirmed by antiserum made against the recombinant 30K protein that originated from the APR 30Kc6 gene. Larval APR6, in which PCD was induced with 20-hydroxyecdysone (20E) added to the primary culture, exhibited some PCD characteristics of shrinkage of cell bodies, axonal fragmentation and loss of mitochondrial function. These results provide new insights on the survival or PCD of insect motoneurons through stimulation of hemolymph.

Platelet-rich fibrin is a Bioscaffold and reservoir of growth factors for tissue regeneration.

The platelet-rich fibrin (PRF) is known as a rich source of autologous cytokines and growth factors and universally used for tissue regeneration in current clinical medicine. However, the microstructure of PRF has not been fully investigated nor have been studied the key molecules that differ PRF from platelet-rich plasma. We fabricated PRF under Choukroun's protocol and produced its extract (PRFe) by freezing at -80°C. The conventional histological, immunohistological staining, and scanning electron microscopy images showed the microstructure of PRF, appearing as two zones, the zone of platelets and the zone of fibrin, which resembled a mesh containing blood cells. The PRFe increased proliferation, migration, and promoted differentiation of the human alveolar bone marrow stem cells (hABMSCs) at 0.5% concentration in vitro. From the results of proteome array, matrix metalloproteinase 9 (MMP9) and Serpin E1 were detected especially in PRFe but not in concentrated platelet-rich plasma. Simultaneous elevation of MMP9, CD44, and transforming growth factor ß-1 receptor was shown at 0.5% PRFe treatment to the hABMSC in immunoblot. Mineralization assay showed that MMP9 directly regulated mineralization differentiation of hABMSC. Transplantation of the fresh PRF into the mouse calvarias enhanced regeneration of the critical-sized defect. Our results strongly support the new characteristics of PRF as a bioscaffold and reservoir of growth factors for tissue regeneration.

Efficacy of periodontal stem cell transplantation in the treatment of advanced periodontitis.

Periodontitis is the most common cause for tooth loss in adults and advanced types affect 10-15% of adults worldwide. The attempts to save tooth and regenerate the periodontal apparatus including cementum, periodontal ligament, and alveolar bone reach to the dental tissue-derived stem cell therapy. Although there have been several periodontitis models suggested, the apical involvement of tooth root is especially challenging to be regenerated and dental stem cell therapy for the state has never been investigated. Three kinds of dental tissue-derived adult stem cells (aDSCs) were obtained from the extracted immature molars of beagle dogs (n = 8), and ex vivo expanded periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and periapical follicular stem cells (PAFSCs) were transplanted into the apical involvement defect. As for the lack of cementum-specific markers, anti-human cementum protein 1 (rhCEMP1) antibody was fabricated and the aDSCs and the regenerated tissues were immunostained with anti-CEMP1 antibody. Autologous PDLSCs showed the best regenerating capacity of periodontal ligament, alveolar bone, and cementum as well as peripheral nerve and blood vessel, which were evaluated by conventional and immune histology, 3D micro-CT, and clinical index. The rhCEMP1 was expressed strongest in PDLSCs and in the regenerated periodontal ligament space. We suggest here the PDLSCs as the most favorable candidate for the clinical application among the three dental stem cells and can be used for treatment of advanced periodontitis where tooth removal was indicated in the clinical cases.

Comparative study of LHX8 expression between odontoma and dental tissue-derived stem cells.

BACKGROUND: LHX8 (LIM-homeobox gene 8) is known as an important regulating factor in tooth morphogenesis. Odontoma is a mixed odontogenic tumor where epithelium and mesenchyme differentiated together, resulting in anomalous tooth structures. In this study, gene and protein expressions of LHX8 were analyzed in human odontoma-derived mesenchymal cells (HODC) compared to adult dental mesenchymal stem cells (aDSC), as well as morphological and histological characteristics of odontoma were analyzed. METHODS: aDSCs were isolated from normal teeth, and HODCs were isolated from surgically removed odontoma mass. Morphological and histological evaluations were performed to compare between compound odontomas and normal premolars. RT-PCR and real-time PCR were performed to identify LHX8 mRNA expression in the HODCs and aDSCs. LHX8 protein expression levels were observed by immunoblotting and immunofluorescent staining. RESULTS: The compound odontoma was composed of multiple tooth-like structures, which contained disorganized but recognizable enamel matrix, dentin, pulp, and cementum. LHX8 mRNA and LHX8 protein expressions were all higher in HODCs compared to those in aDSCs examined by RT-PCR, immunoblot, and immunofluorescent staining. Especially, real-time PCR showed 2.77-fold higher LHX8 expression in HODCs than in normal periodontal ligament stem cells (PDLSCs), while alveolar bone marrow stem cells (ABMSCs) expressed 0.12-fold LHX8 than PDLSCs. CONCLUSIONS: Based on these observations, LHX8 might play an important role in odontoma formation. This is the first report regarding the comparison of LHX8 expression between HODC and normal aDSCs and its overexpression in human samples. The specific mechanism of LHX8 in odontoma morphogenesis awaits further study.

Effect of platelet-rich plasma on dental stem cells derived from human impacted third molars.

AIM: Platelet-rich plasma (PRP) is fabricated from autologous blood and extensively used to promote soft and hard tissue healing. In the dental field, autologous PRP is widely used combined with dental implant installation and bone graft. This study will evaluate the biologic effect of PRP on the proliferation and the differentiation of human dental stem cells, and find the key cytokines inducing these effects to estimate the clinical feasibility of PRP for dental tissue engineering. MATERIALS & METHODS: Venous blood was obtained from four individuals and each PRP was fabricated. The human dental stem cells were obtained from the periodontal ligament (PDL) and dental pulp of the surgically extracted human third molars and expanded in vitro. Immunocytochemical staining and flow cytometry with STRO-1 and CD146 confirmed existence of mesenchymal stem cells in the PDL and dental pulp. The effect of PRP on the proliferation of PDL stem cells (PDLSCs) and dental pulp stem cells (DPSCs) was assessed by colony-forming ability measurement, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and bromodeoxyuridine incorporation assay. Alkaline phosphatase activity and calcium deposit were measured to evaluate the mineralization effect of PRP PDLSCs and DPSCs. Alizarin red S staining was used to detect mineral nodules. Odontogenic and osteogenic gene expressions were evaluated in the PRP-treated PDLSCs and DPSCs by real-time quantitative PCR. A protein array was performed to detect the key cytokines that have an important role in the tissue regenerative effect of PRP. RESULTS: Flow cytometry cell sorting showed that the cells from human PDL and dental pulp contained mesenchymal stem cell populations. Colony-forming ability and cellular proliferation of the dental stem cells were increased at 0.5 and 1% PRP concentration but decreased at 5% concentration. Long-term treatment with 1% PRP enhanced proliferation of the human dental stem cells PDLSCs and DPSCs by 120 h and showed the most significant enhancement at 96 h. PRP also promoted mineralization differentiation of the two kinds of dental stem cells as shown by measurement of alkaline phosphatase activity and calcium deposit under mineralization conditioned media. Increased formation of mineral nodules stained with alizarin red was observed in both PDLSCs and DPSCs after treatment with 1% PRP. Real-time quantitative PCR showed higher odontogenic and osteogenic gene expressions in PRP-treated PDLSCs and DPSCs. RANTES/CCL5 and ICAM-1 were the two key cytokines that were detected in human cytokine array with PRP. CONCLUSION: The appropriate concentration of the PRP treatment enhanced proliferation and mineralization differentiation of human dental stem cells. RANTES/CCL5 and ICAM-1 might play an important role in PRP-induced tissue regeneration but further study is needed to investigate the whole mechanism.

L1 cell adhesion molecule is a novel therapeutic target in intrahepatic cholangiocarcinoma.

PURPOSE: Intrahepatic cholangiocarcinoma (ICC), a highly malignant hepatobiliary cancer, has a poor prognosis and is refractory to conventional therapies. The aim of this study is to discover a novel molecular target for the treatment of ICC. EXPERIMENTAL DESIGN: To discover novel cancer-associated membrane antigens expressed in ICC cells, we generated monoclonal antibodies (mAb) by immunizing mice with intact ICC cell lines and screened for those that bind to the plasma membrane of ICC cells but not to normal cells. The mAb A10-A3 was selected and its target antigen was identified as the L1 cell adhesion molecule. Expression of L1 in ICC was evaluated by immunohistochemical analysis of tumor samples from 42 ICC patients. The functional significance of L1 expression in the tumor progression of ICC was investigated by L1 suppression, L1 overexpression, and antibody treatment. RESULTS: L1 was not expressed in normal hepatocytes and intrahepatic bile duct epithelium but highly expressed in 40.5% of ICC patients, remarkably at the invasive front of the tumors. Suppression of L1 with short hairpin RNA significantly decreased proliferation, migration, and invasion of ICC cells in vitro. Consistently, L1 overexpression in ICC cells enhanced proliferation, migration, invasion, and apoptosis resistance. In addition, L1 short hairpin RNA or anti-L1 mAb significantly reduced the tumor growth in nude mice bearing ICC xenograft. CONCLUSIONS: We identified that L1 is expressed in ICC. L1 plays an important role in the tumor progression of ICC by enhancing cell proliferation, migration, invasion, and survival. L1 may represent a novel therapeutic target for ICC.

Dental stem cell therapy with calcium hydroxide in dental pulp capping.

Calcium hydroxide has been extensively and steadily used for direct pulp capping in modern clinical dentistry. As it was known to have potential to induce hard tissue repair, this chemical has been applied to the exposed dental pulp and the hard tissue is expected to be regenerated above the pulp. During the reparative process of exposed pulp, primary odontoblasts that were lost as a result of extensive damage are replaced with newly differentiated odontoblast-like cells. This process is known to follow the sequential steps of proliferation, migration, and differentiation of progenitor cells. This research will examine the relationship between calcium hydroxide and the recruitment, proliferation, and mineralization of postnatal dental stem cells, obtained from an immature dental tissue of beagle dogs. Immunocytochemical staining and reverse transcriptase-polymerase chain reaction were used to identify the putative stem cell markers. Immunoblot analysis, wound healing assay, cell migration assay, and alizarin red staining were used to evaluate proliferation, migration, and mineralization capacity of the calcium hydroxide-treated stem cells. As an in vivo study, a combination of calcium hydroxide and autologous dental pulp stem cells (DPSCs) was applied for the treatment of intentionally created tooth defects on the premolars and the molars in beagle dogs to observe dentin regeneration. Ex vivo expanded DPSCs and periodontal ligament stem cells expressed STRO-1 and CD146, the mesenchymal stem cell markers. It was evident that calcium hydroxide increased recruitment, migration, proliferation, and mineralization of the DPSCs and periodontal ligament stem cells. Such results are valuable for future availability of DPSCs, which are recently focused as the stem cell reservoir for regeneration of dentin upon tooth injury, as well as for elucidation of the role of calcium hydroxide in pulp capping therapy.

H-Ras is degraded by Wnt/beta-catenin signaling via beta-TrCP-mediated polyubiquitylation.

Ras is an important proto-protein that is regulated primarily by GDP/GTP exchange. Here, we report a novel regulatory mechanism whereby turnover of both endogenous and overexpressed H-Ras protein is controlled by beta-TrCP-mediated ubiquitylation, proteasomal degradation and the Wnt/beta-catenin signaling pathway. The interaction of H-Ras with the WD40 domain of beta-TrCP targeted H-Ras for polyubiquitylation and degradation. This process was stimulated by Axin or adenomatous polyposis coli (Apc), and was inhibited by Wnt3a. Ras-mediated cellular transformation was also inhibited by the expression of beta-TrCP and/or Axin. In vivo regulation of Ras stability by Wnt/beta-catenin signaling was determined via measurements of the status of Ras in the intestines of mice stimulated with recombinant Wnt3a by intravenous tail vein injection. The regulation of Ras stability by Wnt/beta-catenin signaling provides a mechanical basis for crosstalk between the Wnt/beta-catenin and the Ras-ERK pathways involved in transformation.

Akt is involved in the inhibition of cell proliferation by EGF.

Axin is a negative regulator of the Wnt/beta-catenin pathway and is involved in the regulation of axis formation and proliferation. Involvement of Axin in the regulation of other signaling pathways is poorly understood. In this study, we investigated the involvement of Akt in growth regulation by Axin in L929 fibroblasts stimulated by EGF. Akt activity was increased by EGF treatment and Ras activation, respectively. Both the EGF- and Ras-induced Akt activations were abolished by Axin induction, as revealed by both Western blot and immunocytochemical analyses. The proliferation and Akt activation induced by EGF were decreased by Axin induction, and the effects of EGF were abolished by treatment of an Akt-specific inhibitor. Therefore, Axin inhibits EGF-induced proliferation of L929 fibroblasts by blocking Akt activation.

Axin inhibits extracellular signal-regulated kinase pathway by Ras degradation via beta-catenin.

Interactions between the Wnt/beta-catenin and the extracellular signal-regulated kinase (ERK) pathways have been posited, but the molecular mechanisms and cooperative roles of such interaction in carcinogenesis are poorly understood. In the present study, the Raf-1, MEK, and ERK activities were concomitantly decreased in fibroblasts, which inhibit morphological transformation and proliferation by Axin induction. The inhibition of the components of the ERK pathway by Axin occurred in cells retaining wild-type beta-catenin, including primary hepatocytes, but not in cells retaining non-degradable mutant beta-catenin. Axin inhibits cellular proliferation and ERK pathway activation induced by either epidermal growth factor or Ras, indicating a role of Axin in the regulation of growth induced by ERK pathway activation. ERK pathway regulation by Axin occurs at least partly via reduction of the protein level of Ras. Both wild-type and mutant Ras proteins are subjected to regulation by Axin, which occurs in cells retaining wild-type but not mutant beta-catenin gene. The role of beta-catenin in the regulation of the Ras-ERK pathway was further confirmed by Ras reduction and subsequent inhibitions of the ERK pathway components by knock down of mutated form of beta-catenin. The Ras regulation by Axin was blocked by treatment of leupeptin, an inhibitor of the lysosomal protein degradation machinery. Overall, Axin inhibits proliferation of cells at least partly by reduction of Ras protein level via beta-catenin. This study provides evidences for the role of the Ras-ERK pathway in carcinogenesis caused by mutations of the Wnt/beta-catenin pathway components.