Hes1 mRNA Expression Research Articles

Inhibition of gamma-secretase activity impedes uterine serous carcinoma growth in a human xenograft model

ObjectiveUterine serous carcinoma (USC) represents an aggressive subtype of endometrial cancer. We sought to understand Notch pathway activity in USC and determine if pathway inhibition has anti-tumor activity. MethodsPatient USC tissue blocks were obtained and used to correlate clinical outcomes with Notch1 expression. Three established USC cell lines were treated with gamma-secretase inhibitor (GSI) in vitro. Mice harboring cell line derived or patient derived USC xenografts (PDXs) were treated with vehicle, GSI, paclitaxel and carboplatin (P/C), or combination GSI and P/C. Levels of cleaved Notch1 protein and Hes1 mRNA were determined in GSI treated samples. Statistical analysis was performed using the Wilcoxon rank sum and Kaplan–Meier methods. ResultsHigh nuclear Notch1 protein expression was observed in 58% of USC samples with no correlation with overall survival. GSI induced dose-dependent reductions in cell number and decreased levels of cleaved Notch1 protein and Hes1 mRNA in vitro. Treatment of mice with GSI led to decreased Hes1 mRNA expression in USC xenografts. In addition, GSI impeded tumor growth of cell line xenografts as well as UT1 USC PDXs. When GSI and P/C were combined, synergistic anti-tumor activity was observed in UT1 xenografts. ConclusionsNotch1 is expressed in a large subset of USC. GSI-mediated Notch pathway inhibition led to both reduced cell numbers in vitro and decreased tumor growth of USC some xenograft models. When combined with conventional chemotherapy, GSI augmented anti-tumor activity in one USC PDX line suggesting that targeting of the Notch signaling pathway is a potential therapeutic strategy for future investigation.

Prognostic implications of mutations in NOTCH1 and FBXW7 in childhood T-all treated according to the NOPHO ALL-1992 and ALL-2000 protocols

In children, T-cell acute lymphoblastic leukemia (T-ALL) has inferior prognosis compared with B-cell precursor ALL. In order to improve survival, individualized treatment strategies and thus risk stratification algorithms are warranted, ideally already at the time of diagnosis. We analyzed the frequency and prognostic implication of mutations in NOTCH1 and FBXW7 in 79 cases of Swedish childhood T-ALL treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL-1992 and ALL-2000 protocols. In a subgroup of patients, we also investigated the functional relevance of NOTCH1 mutations measured as expression of the HES1, MYB, and MYC genes. Forty-seven of the cases (59%) displayed mutations in NOTCH1 and/or FBXW7. There was no difference in overall (P = 0.14) or event-free survival (EFS) (P = 0.10) in patients with T-ALL with mutation(s) in NOTCH1/FBXW7 compared with patients with T-ALL without mutations in any of these genes. T-ALL carrying NOTCH1 mutations had increased HES1 and MYB mRNA expression (HES1 9.2 ± 1.9 (mean ± SEM), MYB 8.7 ± 0.8 (mean ± SEM)) compared to T-ALL with wild-type NOTCH1 (HES1 1.8 ± 0.7, MYB 5.1 ± 1.2, P = 0.02 and 0.008, respectively). In cases of T-ALL with high HES1 expression, improved overall (P = 0.02) and EFS (P = 0.028) was seen. Increased NOTCH activity, reflected by increased HES1 expression, is associated with improved outcome in pediatric T-ALL, but its role as a diagnostic tool or a therapeutic target in future clinical treatment protocols remains to be elucidated.

The Jagged-2/Notch-1/Hes-1 Pathway Is Involved in Intestinal Epithelium Regeneration after Intestinal Ischemia-Reperfusion Injury

BackgroundNotch signaling plays a critical role in the maintenance of intestinal crypt epithelial cell proliferation. The aim of this study was to investigate the role of Notch signaling in the proliferation and regeneration of intestinal epithelium after intestinal ischemia reperfusion (I/R) injury.MethodsMale Sprague-Dawley rats were subjected to sham operation or I/R by occlusion of the superior mesenteric artery (SMA) for 20 min. Intestinal tissue samples were collected at 0, 1, 2, 4, and 6 h after reperfusion. Proliferation of the intestinal epithelium was evaluated by immunohistochemical staining of proliferating nuclear antigen (PCNA). The mRNA and protein expression levels of Notch signaling components were examined using Real-time PCR and Western blot analyses. Immunofluorescence was also performed to detect the expression and location of Jagged-2, cleaved Notch-1, and Hes-1 in the intestine. Finally, the γ-secretase inhibitor DAPT and the siRNA for Jagged-2 and Hes-1 were applied to investigate the functional role of Notch signaling in the proliferation of intestinal epithelial cells in an in vitro IEC-6 culture system.ResultsI/R injury caused increased intestinal crypt epithelial cell proliferation and increased mRNA and protein expression of Jagged-2, Notch-1, and Hes-1. The immunofluorescence results further confirmed increased protein expression of Jagged-2, cleaved Notch-1, and Hes-1 in the intestinal crypts. The inhibition of Notch signaling with DAPT and the suppression of Jagged-2 and Hes-1 expression using siRNA both significantly inhibited the proliferation of IEC-6 cells.ConclusionThe Jagged-2/Notch-1/Hes-1 signaling pathway is involved in intestinal epithelium regeneration early after I/R injury by increasing crypt epithelial cell proliferation.

Hes-1-targeting siRNA inhibits the maturation of murine myeloid-derived dendritic cells

Abstract Activation of Notch by Jagged-1 may plays a pivotal role in maturation of dendritic cells (DCs), but the mechanism has not been completely defined. In the present study, Hes-1 (Hairy/enhancer-of-split)-targeting siRNA was used to confirm a role of Jagged-1-Notch signaling pathway activation in maturation of murine bone marrow-derived DCs and to search for a target that plays a critical role. The results showed that compared with the control, lipopolysaccharide or Zymosan A groups, Jagged-1 (a soluble Jagged 1/Fc chimera protein) effectively increased expression of Hes-1 and Deltex-1 mRNA, which could be reversed by DAPT (2, 4-diamino-5-phenylthiazole), a specific inhibitor of the Notch signaling pathway. Hes-1-targeting siRNA could successfully down-regulate the endogenous Hes-1 expression in the DCs. Concurrently, a significant down-regulation of CD40, CD80, CD86 and MHC-II expressions on the surface of the DCs was found with the reduction of IL-12 yielded by the DCs. Our results demonstrate that Hes-1-targeting siRNA can inhibit the maturation of the DCs induced by Jagged-1, indicating Hes-1 may be an important target of Notch signaling mediating the maturation of DCs.

HES1, a target of Notch signaling, is elevated in canine osteosarcoma, but reduced in the most aggressive tumors

BackgroundHairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Notch signaling and HES1 expression have been linked to growth and survival in a variety of human cancer types and have been associated with increased metastasis and invasiveness in human osteosarcoma cell lines. Osteosarcoma (OSA) is an aggressive cancer demonstrating both high metastatic rate and chemotherapeutic resistance. The current study examined expression of Notch signaling mediators in primary canine OSA tumors and canine and human osteosarcoma cell lines to assess their role in OSA development and progression.ResultsReverse transcriptase - quantitative PCR (RT-qPCR) was utilized to quantify HES1, HEY1, NOTCH1 and NOTCH2 gene expression in matched tumor and normal metaphyseal bone samples taken from dogs treated for appendicular OSA at the Colorado State University Veterinary Teaching Hospital. Gene expression was also assessed in tumors from dogs with a disease free interval (DFI) of <100 days compared to those with a DFI > 300 days following treatment with surgical amputation followed by standard chemotherapy. Immunohistochemistry was performed to confirm expression of HES1. Data from RT-qPCR and immunohistochemical (IHC) experiments were analyzed using REST2009 software and survival analysis based on IHC expression employed the Kaplan-Meier method and log rank analysis. Unbiased clustered images were generated from gene array analysis data for Notch/HES1 associated genes.Gene array analysis of Notch/HES1 associated genes suggested alterations in the Notch signaling pathway may contribute to the development of canine OSA. HES1 mRNA expression was elevated in tumor samples relative to normal bone, but decreased in tumor samples from dogs with a DFI < 100 days relative to those with a DFI > 300 days. NOTCH2 and HEY1 mRNA expression was also elevated in tumors relative to normal bone, but was not differentially expressed between the DFI tumor groups. Survival analysis confirmed an association between decreased HES1 immunosignal and shorter DFI.ConclusionsOur findings suggest that activation of Notch signaling occurs and may contribute to the development of canine OSA. However, association of low HES1 expression and shorter DFI suggests that mechanisms that do not alter HES1 expression may drive the most aggressive tumors.

AB0115 BFGF increases HES1 and HEY1 expressions in murine chondrocytes in vitro

BackgroundDuring osteoarthritis (OA), articular chondrocytes undergo cellular changes, such as proliferation and dedifferenciation, reminiscent of growth plate (GP) terminal différentiation. Among the factors and the signaling molecules which control this...

Sa1731 Neurotensin-HIF-1α-MicroRNA 210 Axis Orchestrates Hypoxia, Colonic Inflammation and Intestinal Angiogenesis

G A A b st ra ct s or constructs overexpressing HA-tagged 14-3-3σ plasmids. 14-3-3σ, Rictor (an mTORC2 protein), MUC2, cleaved Notch1, p-Akt(S473), Akt1 and Akt2 protein expression was determined by Western blotting. Enterocyte differentiation was induced by treatment of HT29 cells with sodium butyrate (NaBT) and assayed by measuring intestinal alkaline phosphatase (IAP) activity, an enterocyte differentiation marker. The expression of the goblet cell differentiation marker MUC2 and the Notch1 target gene HES1 was also determined by RT-PCR. The interaction between Akt andmTORC2was determined by immunoprecipitation followed by Western blot analysis. RESULTS. Knockdown of 14-3-3 σ did not alter basal or NaBT-induced of IAP activity. However, knockdown of 14-3-3 σ significantly increased MUC2 expression associated with the inhibition of Notch1 signaling as shown by decreased cleaved Notch1 protein and decreased HES1 mRNA expression. These results suggested that 14-3-3σ plays an important role in the regulation of goblet cell differentiation without impacting enterocyte differentiation. In addition, overexpression of 14-3-3σ decreased Akt (Ser473) phosphorylation and knockdown of either Akt1 or Akt2 decreased MUC2 expression. Moreover, overexpression of 14-3-3σ decreased the interaction between Akt and mTORC2. These results suggest that 14-3-3σmay regulate Akt activity through the inhibition of mTORC2-mediated Akt (Ser473) phosphorylation. CONCLUSIONS. Our results suggest a role for 14-3-3σ in goblet cell differentiation through the alteration of TSC2, Notch and Akt signaling pathways. Importantly, our data demonstrate that 14-3-3 σ regulation of Akt phosphorylation is mediated through the altered interaction between mTORC2 and Akt.

The Jagged-1/Notch-1/Hes-1 Pathway Is Involved in Intestinal Adaptation in a Massive Small Bowel Resection Rat Model

Notch signaling is required for the maintenance of intestinal epithelial proliferation. Dysfunction of this signaling pathway is associated with the loss of proliferated crypt epithelial cells. The aim of this study was to investigate the role of Notch signaling in small bowel resection (SBR)-associated crypt epithelial cell proliferation. Male Sprague-Dawley rats were subjected to sham operation (bowel transection and reanastomosis) or 70% mid-SBR. Intestinal tissue samples were collected at 0.5, 1, 6, 12, 24, 72, and 168 h after operation. The expression of Notch pathway mRNAs and proteins was analyzed using RT-PCR and Western blot. The expression of the Notch pathway proteins Jagged-1, NICD and Hes-1 was also determined through immunohistochemical staining using day 3 postoperative intestinal tissues. The degree of crypt epithelial cell proliferation was evaluated using the immunohistochemical staining of proliferating cell nuclear antigen (PCNA). Furthermore, IEC-6 cells were used to examine the function of the Jagged-1 signaling system. SBR led to increased crypt epithelial cell proliferation and increased expression of Jagged-1 and Hes-1 mRNA and protein along with cleaved Notch-1. Immunohistochemical staining showed that Jagged-1, cleaved Notch-1 and Hes-1 colocalized in the same proliferated crypt epithelial cell population. Recombinant Jagged-1 significantly stimulated the proliferation of IEC-6 cells. Transient upregulation of Jagged-2 expression was found 1 h after SBR, and it was accompanied by cleaved Notch-1 and Hes-1 upregulation. The Jagged-1/Notch-1/Hes-1 signaling pathway is involved in intestinal adaptation through increasing crypt epithelial cell proliferation.

Effects of matrine on oval cell-mediated liver regeneration and expression of RBP-Jκ and HES1

In the present study a rat model of oval cell‑mediated liver regeneration was constructed to examine the molecular mechanisms of matrine in oval cell‑mediated liver regeneration and the effects of matrine on hepatic function and the expression of OV6 protein and recombination signal sequence‑binding proteinJκ (RBP‑Jκ) and HES1 mRNA. A total of 48Sprague Dawley rats were equally and randomly assigned to two groups. The model group underwent oval cell‑mediated liver regeneration, whereas the matrine group underwent oval cell‑mediated liver regeneration and received oral gavage of matrine. Expression of OV6 protein was tested by immunohistochemistry and RBP‑Jκ and HES1 mRNA expression was determined by reverse‑transcription polymerase chain reaction. Recovery of hepatic function was faster in the matrine group compared with the model (P<0.05). OV6 protein, RBP‑Jκ and HES1 mRNA expression levels were lower in the matrine than the model group (P<0.05). Matrine promotes oval cell‑mediated liver regeneration through downregulation of the RBP‑Jκ‑HES1 signaling pathway.

Notch Signaling Is Involved in Neurogenic Commitment of Human Periodontal Ligament-Derived Mesenchymal Stem Cells

Notch signaling plays critical roles in stem cells by regulating cell fate determination and differentiation. The aim of this study was to evaluate the participation of Notch signaling in neurogenic commitment of human periodontal ligament-derived mesenchymal stem cells (hPDLSCs) and to examine the ability to control differentiation of these cells using modified surfaces containing affinity immobilized Notch ligands. Neurogenic induction of hPDLSCs was performed via neurosphere formation. Cells were aggregated and form spheres as early 1 day in culture. In addition, the induced cells exhibited increased mRNA and protein expression of neuronal markers that is, β3-tubulin and neurofilament. During neuronal differentiation, a significant increase of Hes1 and Hey1 mRNA expression was noted. Using pharmacological inhibition (γ-secretase inhibitor) or genetic manipulation (overexpression of dominant negative mastermind-like transcription co-activators), neurosphere formation was attenuated and a marked decrease in neurogenic mRNA expression was observed. To confirm the role of Notch signaling in neuronal differentiation of hPDLSCs, the Notch ligand, Jagged-1, is bound to the surface using an affinity immobilization technique. The hPDLSC cultured on a Jagged-1-modified surface had increased expression of Notch signaling target genes, Hes-1 and Hey-1, confirming the activity and potency of surface-bound Jagged-1. Further, hPDLSC on surface-bound Jagged-1 under serum-free conditions showed multiple long and thin neurite-like extensions, and an increase in the expression of neurogenic mRNA markers was observed. Pretreatment of the cells with γ-secretase inhibitor, DAPT, before seeding on the Jagged-1-modified surface blocked development of the neurite-like morphology. Together, the results in this study suggest the involvement of Notch signaling in neurogenic commitment of hPDLSCs.

MicroRNA9 regulates neural stem cell differentiation by controlling Hes1 expression dynamics in the developing brain

Earlier studies show that Hes1 expression is oscillatory in neural stem cells but sustained and high in the roof plate and the floor plate, and that such different dynamics of Hes1 expression (oscillatory versus sustained) regulate different proliferation and differentiation characteristics of these cells (active in neural stem cells but rather dormant in roof/floor plate cells). The mechanism of how different dynamics of Hes1 expression is controlled remains to be determined. Here, we found that the seed sequence of microRNA-9 (miR-9) is complementary to the 3'-UTR sequence of Hes1 mRNA. MiR-9 is highly expressed in the ventricular zone of the developing brain, which contains neural stem cells, but it is not expressed in the roof plate or the floor plate. Over-expression of miR-9 negatively regulates the Hes1 protein expression by interacting with the 3'-UTR of Hes1 mRNA, thereby inducing cell cycle exit and neuronal differentiation. Conversely, knockdown of miR-9 inhibits neuronal differentiation. Furthermore, knockdown of miR-9 inhibits the oscillatory expression of Hes1 mRNA in neural stem cells. These results indicate that miR-9 regulates the proliferation and differentiation of neural stem cells by controlling the dynamics of Hes1 expression in the developing brain.

Abstract 1195: Erk activity promotes Notch-dependent HES1 expression in pancreatic cancer cells

Abstract The oncogene Ras is well-recognized for its involvement in tumorigenesis in part through activation of downstream pathways including the PI3K/Akt and Mek/Erk pathways. Recent studies have suggested that the ability of Ras to transform cells also depends on cooperation with the Notch signalling pathway. However, the molecular and cellular mechanisms involved in this cooperation remain largely unknown. The aim of this study was to evaluate whether specific activation of the Ras/Raf/Mek/Erk pathway impacts Notch-dependent signalling. METHODS. Experiments were done using the pancreatic cancer cells MIA PaCa-2 and BxPC-3. Both cell lines express endogenous levels of the activated Notch1 (NIC). To inhibit Mek/Erk activity, cells were treated with the Mek inhibitor U0126 (10uM). Activation of the Mek/Erk pathway was achieved by treatment with phorbol 12-myristate 13-acetate (PMA, 100nM). The hes1-luciferase reporter gene was used to evaluate Notch-dependent transcriptional activity. The gamma-secretase inhibitor DAPT (25µM) was used to inhibit Notch activation. RESULTS. 1- Treatment of pancreatic cancer cells with U0126 reduced the activity of the hes1-luciferase reporter gene to the same extent than treatment with the gamma-secretase inhibitor DAPT. 2- Combined treatment (U0126 + DAPT) did not result in an additive inhibition of hes1-luciferase activity suggesting a Notch-dependent impact of the Mek inhibitor on hes1 transcription. 3- Increased HES1 mRNA expression levels were observed following treatment with PMA, an effect prevented by addition of U0126. 4- Massive activation of the Notch receptors by treatment with EGTA promoted HES1 protein expression. 5- Interestingly, strong stimulation of Erk activity by PMA promoted the EGTA-induced HES1 protein expression. 6- Conversely, inhibition of Erk activity completely prevented both the EGTA-induced and EGTA+PMA-induced HES1 protein expression. CONCLUSION. Taken together, our results suggest that Erk activity promotes Notch-dependent transcriptional activity and consequently promotes the expression of Notch target genes such as HES1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1195. doi:1538-7445.AM2012-1195

Effect of trastuzumab on Notch-1 signaling pathway in breast cancer SK-BR3 cells

To investigate the effects and mechanisms of trastuzumab on Notch-1 pathway in breast cancer cells, recognizing the significance of Notch-1 signaling pathway in trastuzumab resistance. Immunocytochemistry staining and Western blotting were employed to justify the expression of Notch-1 protein in HER2-overexpressing SK-BR3 cells and HER2-non-overexpressing breast cancer MDA-MB-231 cells. Western blotting and reverse transcription PCR (RT-PCR) were used to detect the activated Notch-1 and Notch-1 target gene HES-1 mRNA expression after SK-BR3 cells were treated with trastuzumab. Double immunofluorescence staining and co-immunoprecipitation were used to analyze the relationship of Notch-1 and HER2 proteins. The level of Notch-1 nuclear localization and activated Notch-1 proteins in HER2-overexpressing cells were significantly lower than in HER2-non-overexpressing cells (P<0.01), and the expressions of activated Notch-1 and HES-1 mRNA were obviously increased after trastuzumab treatment (P<0.05), but HER2 expression did not change significantly for trastuzumab treating (P>0.05). Moreover, Notch-1 was discovered to co-localize and interact with HER2 in SK-BR3 cells. Overexpression of HER2 decreased Notch-1 activity by the formation of a HER2-Notch1 complex, and trastuzumab can restore the activity of Notch-1 signaling pathway, which could be associated with cell resistance to trastuzumab.

The Notch-1 signaling pathway is required for a functional colonic barrier in WT mice

Gut lympho-epithelial interactions occur in the epithelial layer and in the sub-epithelial space. We have recently shown that the presence of LPLs promotes mucosal barrier function in the RAG1−/− transfer model of colitis putatively due to the Notch-1 signaling pathway. This finding was corroborated in vitro as epithelial barrier function correlated with the activation of the Notch-1 signaling pathway in vitro. Moreover, the activation of Notch-1 extends throughout the epithelium in CD mucosa compared to normal or UC mucosa. To determine whether the Notch pathway is mandatory for barrier function in vivo. A specific Notch-1 or a scrambled siRNA sequence was freshly complexed with Lipofectamine 2000. Anesthetized WT mice received 2 intra-rectal injections of complexed siRNA (5 nmol in 20 μl of solution/mouse) 4 days apart. The injected mice were sacrificed one day after the last injection. Colonic tissues were either mounted into Ussing chambers to assess permeability or snap-frozen and subjected to Real-Time PCR for Notch-1, Hes-1, CDX-2, Villin, Occludin, Claudin-5, β-catenin, and E-cadherin. Notch-1 mRNA expression was decreased in the Notch-1 siRNA treated WT mice (p=0.001). Hes-1, CDX-2, β-catenin and E-cadherin mRNA expression was decreased in the Notch-1 siRNA treated WT mice (p=0.01, p=0.009, p<0.0001 and p=0.0004). These findings correlated with a decrease in villin, occludin, and claudin-5 mRNA expression (p=0.0002, p=0.0002 and p=0.002). While no differences in resistance were seen between the groups, there was a significant decrease in flux in mice that received the Notch-1 siRNA (10925 ± 3389 ng of Dextran-FITC/ml/min/cm2 vs. 2912 ± 1054 ng, p=0.01). Local knock down of Notch-1 impaired expression of the genes related to Notch-1 and to barrier function affecting epithelial integrity. Thus, the Notch-1 signaling pathway is critical for the epithelium in order to achieve differentiation and functional barrier integrity.

Up-regulation of cochlear Hes1 expression in response to noise exposure.

Hes1, a hairy and enhancer of split homolog, negatively regulates inner ear hair cell differentiation. The main objective of this study was to investigate the status of the Hes1 gene in the noise-damaged cochlea in relation to the degree of noise-induced hearing loss (NIHL). Adult albino guinea pigs were exposed to white-band noise (115 dB sound pressure level). Noise exposure for either 1 or 3 hours induced significant elevations of threshold in auditory brainstem response (ABR) compared with unexposed controls. Succinate dehydrogenase staining showed that white-band noise exposure caused significant outer hair cell losses. In addition, we found significant up-regulations of cochlear Hes1 mRNA and protein expressions following acoustic trauma, and Hes1 mRNA expression was positively correlated with NIHL. These findings suggest that up-regulation of Hes1 expression in response to noise exposure may be one of the underlying mechanisms of NIHL.

Engagement of activated Notch signalling in collagen II-specific T helper type 1 (Th1)- and Th17-type expansion involving Notch3 and Delta-like1

Our previous study demonstrated that T helper (Th) cells from patients with rheumatoid arthritis (RA) display an altered expression profile of Notch receptors and enhanced activation of Notch signalling. The aim of this study was to investigate the role of distinct Notch receptors and ligands in the activation and differentiation of collagen II (CII)-reactive Th cells upon antigen-specific restimulation. Spleen mononuclear cells (SMNCs) from CII-immunized DBA/1J mice were restimulated by culturing with CII. CII-specific proliferation and differentiation of T cells were determined by tritiated thymidine ((3) [H]-TdR) incorporation and flow cytometric analysis, respectively. The mRNA expression of Notch receptors and Hes1 was assessed by real-time polymerase chain reaction (PCR). There was a clear increase in the percentage of Th1 cells and Th17 cells after CII restimulation. No significant difference was observed in the percentage of regulation T cells (T(reg) ) in SMNCs with or without CII restimulation. CII restimulation induced up-regulated transcript levels of Hes1 in CII-reactive CD4(+) T cells. The mRNA level of Notch3 was also up-regulated significantly, while the levels of the other three Notch receptors were not increased. Inhibition of Notch signalling by N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and Notch3 antibody decreased the collagen-specific T cell proliferation and attenuated Th1- and Th17-type responses, while treatment with Notch ligand Delta-like 1 promoted such a response. The present study demonstrates that Notch signalling is engaged in CII-specific Th1- and Th17-type expansion in which Notch3 and Delta-like1 were involved. Selective inhibition of Notch signalling mediated by Notch3 or Delta-like1 may offer a new strategy for the treatment of RA.

Decreased colony formation of high proliferative potential colony-forming cells and granulocyte-macrophage colony-forming units and increased Hes-1 expression in bone marrow mononuclear cells from patients with psoriasis

Psoriasis is a chronic inflammatory disease of the skin. The dysfunctional immunity experienced by patients with psoriasis is believed to influence the bone marrow haematopoietic cells and their surrounding microenvironment. Phagocytes derived from the bone marrow of patients with active psoriasis exhibit enhanced monocytopoietic activity and hyperplasia in vitro. However, direct evidence supporting the hypothesis that bone marrow is involved in the pathogenesis of psoriasis has yet to be established. To investigate the involvement of bone marrow in the pathogenesis of psoriasis. Bone marrow mononuclear cells (BMMNCs) were isolated from patients with psoriasis and healthy individuals. The high proliferative potential colony-forming cells (HPP-CFCs), granulocyte-macrophage colony-forming units (CFU-GM) and erythroid colony-forming units (CFU-E) were cultured in the presence of defined cytokines, and the effects of secreted factors from psoriatic peripheral blood mononuclear cells (PBMCs) on colony formation of normal haematopoietic cells were analysed. Furthermore, the telomere activity of psoriatic and normal BMMNCs was determined using the polymerase chain reaction (PCR)-based telomeric repeat amplification protocol, while the expression of human telomerase reverse transcriptase (hTERT) and HES1 mRNA was detected by reverse transcription-PCR assay. The numbers of HPP-CFCs and CFU-GM, but not CFU-E, were significantly reduced in cultured haematopoietic cells from patients with psoriasis. The culture supernatant of PBMCs from patients with psoriasis was found to inhibit the colony formation capacity of HPP-CFCs, CFU-GM and CFU-E of normal haematopoietic cells. We also detected low levels of telomerase activity and hTERT gene expression in psoriatic and control BMMNCs that was statistically similar between the two groups. In contrast, the HES1 gene expression appeared to be significantly elevated in psoriatic BMMNCs (P < 0.05). Together, our results indicate the involvement of bone marrow in the immunopathogenesis of psoriasis, and suggest a mechanism mediated by certain inflammatory or haematopoietic cytokines present in the bone marrow microenvironment. Elevated expression levels of HES1 mRNA suggest a potential role for the Notch signalling pathway in this process.

Osteogenic oxysterol, 20(S)-hydroxycholesterol, induces notch target gene expression in bone marrow stromal cells

We previously reported that specific oxysterols stimulate osteogenic differentiation of pluripotent bone marrow stromal cells (MSCs) through activation of hedgehog (Hh) signaling and may serve as potential future therapies for intervention in osteopenia and osteoporosis. In this study we report that the osteogenic oxysterol 20(S)-hydroxycholesterol (20S) induces the expression of genes associated with Notch signaling. Using M2-10B4 (M2) MSCs, we found that 20S significantly induced HES-1, HEY-1, and HEY-2 mRNA expression compared with untreated cells, with maximal induction after 48 hours, whereas the nonosteogenic oxysterols did not. Similar observations were made when M2 cells were treated with sonic hedgehog (Shh), and the specific Hh pathway inhibitor cyclopamine blocked 20S-induced Notch target gene expression. 20S did not induce Notch target genes in Smo−/− mouse embryonic fibroblasts, further confirming the role of Hh signaling in 20S-induced expression of Notch target genes. Despite the inability of liver X-receptor (LXR) synthetic ligand TO901317 to induce Notch target genes in M2 cells, LXR knockdown studies using siRNA showed inhibition of 20S-induced HEY-1 but not HES-1 expression, suggesting the partial role of LXR signaling in MSC responses to 20S. Moreover, 20S-induced Notch target gene expression was independent of canonical Notch signaling because neither 20S nor Shh induced CBF1 luciferase reporter activity or NICD protein accumulation in the nucleus, which are hallmarks of canonical Notch signaling activation. Finally, HES-1 and HEY-1 siRNA transfection significantly inhibited 20S-induced osteogenic genes, suggesting that the pro-osteogenic effects of 20S are regulated in part by HES-1 and HEY-1. © 2010 American Society for Bone and Mineral Research

Expression of the RNA‐binding protein Musashi1 in adult liver stem‐like cells

Musashi1 is an RNA-binding protein that regulates the Notch signaling pathway in stem cells. Our previous study revealed that Musashi1 is expressed in early hepatocytes during liver development in the mouse. However, whether this unique protein is expressed with Notch signaling markers in adult liver stem-like cells remains unknown. Established hepatic stem-like cells (HSLC), which were derived from adult Sprague-Dawley rats, were used for experiments in vitro. HSLC were differentiated into mature cells in terms of producing albumin when co-cultured with epidermal growth factor (EGF). The mRNA expression of Musashi1, Notch family (Notch1 and Notch2), Jagged1 and Hes1 was examined in HSLC before and after cell differentiation using polymerase chain reaction-based techniques. Protein expression of Musashi1 was examined in the HSLC and normal mature hepatocytes by immunofluorescence staining. The mRNA expression of Musashi1, Notch1, Jagged1 and Hes1 was detected in the original HSLC before culturing with EGF but not in primary cultured mature hepatocytes. The mRNA expression of Musashi1 and Hes1 was found to be downregulated in differentiated HSLC that produce albumin. Protein expression of Musashi1 was detectable in the original HSLC but not in both differentiated HSLC and mature hepatocytes. These findings demonstrate that the RNA-binding protein Musashi1 is expressed with Notch signaling markers in adult liver stem-like cells.

Conditional deletion of Stat3 promotes neurogenesis and inhibits astrogliogenesis in neural stem cells

Although signal transducer and activator of transcription 3 (Stat3) plays crucial roles in the determination of neural stem cell (NSC) fate, Stat3 has multiple roles in NSC function. Moreover, Stat3 plays important roles in neuronal survival and tumorigenesis. To investigate the overall effects of Stat3 on NSC fate, NSC were isolated from Stat3 flox/flox mouse embryos (E14–15d), in which both Stat3 alleles are flanked by LoxP sites. Isolated NSC was inoculated with an adenovirus vector expressing Cre recombinase (Ad.nCre) or a control adenovirus vector expressing β-galactosidase (Ad.nLz). Three days later, quantitative real-time PCR (qPCR) analysis revealed that treatment with Ad.nCre eliminated stat3 mRNA expression in NSC. Promoter assay confirmed that overexpression of nCre inhibited transactivation of acute responsive element (APRE) and blocked Stat3 function in NSC. Moreover, Western blot analysis and immunocytochemical analysis revealed that elimination of Stat3 in NSC promoted neurogenesis and inhibited astrogliogenesis. In addition, we investigated the effects of Stat3 elimination in NSC on the mRNA expression of Notch family members and bHLH factors. Consequently, qPCR analysis showed that elimination of Stat3 in NSC promoted neurogenesis and inhibited astrogliogenesis through down-regulation of notch1, notch2 and hes5, but not hes1 mRNA expression.