Sacubitril/valsartan inhibits the proliferation of vascular smooth muscle cells through notch signaling and ERK1/2 pathway.

AIMS: To explore the role and mechanism of Notch signaling and ERK1/2 pathway in the inhibitory effect of sacubitril/valsartan on the proliferation of vascular smooth muscle cells (VSMCs). MAIN METHODS: Human aortic vascular smooth muscle cells (HA-VSMCs) were cultured in vitro. The proliferating VSMCs were divided into three groups as control group, Ang II group and Ang II + sacubitril/valsartan group. Cell proliferation and migration were detected by CCK8 and scratch test respectively. The mRNA and protein expression of PCNA, MMP-9, Notch1 and Jagged-1 were detected by qRT-PCR and Western blot respectively. The p-ERK1/2 expression was detected by Western blot. KEY FINDINGS: Compared with the control group, proliferation and migration of VSMCs and the expression of PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 was increased in Ang II group. Sacubitril/valsartan significantly reduced the proliferation and migration. Additionally, pretreatment with sacubitril/valsartan reduced the PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 expression.

Jagged1 contained in MSC-derived small extracellular vesicles promotes squamous differentiation of cervical cancer by activating NOTCH pathway.

PURPOSE: Cervical cancer is the fourth most common cancer in women and poses a major threat to women's health, urgently requiring new treatment methods. METHODS: This study first successfully extracted and identified small extracellular vesicles secreted by human umbilical cord-derived mesenchymal stem cells. We studied the effects of MSC-sEV on the squamous differentiation levels of cervical cancer CaSki cells in vitro, and explored the effects of MSC-sEV on the NOTCH pathway, the growth, proliferation, migration abilities and squamous differentiation levels of cervical cancer cells. The roles of MSC-sEV were also verified in human keratinocyte HaCaT cells. RESULTS: The results showed that Jagged1 protein on MSC-sEV can bind to NOTCH1 on cervical cancer cells, activate NOTCH signaling, and promote squamous differentiation levels in CaSki cells, thus inhibiting the growth, proliferation and migration abilities of CaSki cells. MSC-sEV can also activate the NOTCH pathway in HaCaT cells, but promote the viability of HaCaT cells. CONCLUSION: MSC-sEV can activate the NOTCH pathway to promote squamous differentiation of CaSki cells and inhibit the growth proliferation and migration abilities of CaSki cells which may be a new mechanism for cervical cancer treatment.

Asiaticoside conveys an antifibrotic effect by inhibiting activation of hepatic stellate cells via the Jagged-1/Notch-1 pathway.

The aim of this study was to investigate the underlying protective mechanisms of asiaticoside (AS) against liver fibrosis (LF) both in vivo and in vitro. A rat model with carbon tetrachloride (CCl4)-induced liver fibrosis is employed to verify the effect and mechanism of AS on the process of liver fibrosis in vivo experiment. Hematoxylin/eosin and sirius red staining was conducted to assess the severity of liver injury and fibrosis. Further, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), glutamyl transferase (GGT), and total bilirubin (TBil) were measured. In addition, LX2 cells were cultured for vitro experiment to investigate the influence of AS on hepatic stellate cells (HSCs). Overproduction of α-smooth muscle actin and type I collagen is characteristic of LF and HSCs, as determined by immunohistochemical and Western blot analyses. The expression levels of molecules associated with the Notch signaling pathway (i.e., Notch-1, Jagged-1, and Delta-like-4) were assessed by Western blot analysis. The results revealed that AS attenuated LF, as defined by reduced deposition of collagen, expression of α-smooth muscle actin and collagen type 1, and expression of biochemical parameters (alanine aminotransferase, aspartate aminotransferase, and hydroxyproline). Notably, AS suppressed the expression levels of Notch-1, Jagged-1, and Delta-like-4 in activated HSCs and LF. Collectively, these results demonstrate that AS prevented the progression of LF by modulating the Notch signaling pathway, indicating that AS has potential therapeutic effects against LF.

[Hesperidin Regulates Jagged1/Notch1 Pathway to Promote Macrophage Polarization and Alleviate Lung Injury in Mice with Bronchiolitis].

Objective To explore the effect and mechanism of hesperidin in treating the lung injury in the mouse model of respiratory syncytial virus (RSV)-induced bronchiolitis. Methods A mouse model of RSV-induced bronchiolitis was established,and 60 BALB/c mice were assigned into a control group,a model group,a low-dose hesperidin (18 mg/kg) group,a high-dose hesperidin (36 mg/kg) group,and a high-dose hesperidin (36 mg/kg)+Jagged1(1 mg/kg) group by random number table method,with 12 mice in each group. Corresponding doses of drugs were administrated for intervention,and the control group and model group were administrated with the same amount of saline.The bronchoalveolar lavage fluid (BALF) samples were collected and alveolar macrophages were isolated.ELISA was employed to detect the levels of interleukin (IL)-4,IL-6,tumor necrosis factor-α (TNF-α),and IL-10 in BALF,and flow cytometry to detect the M1/M2 polarization of macrophages.qRT-PCR and Western blotting were respectively conducted to detect the mRNA and protein levels of inducible nitric oxide synthase (iNOS),arginase 1 (Arg-1),Jagged1,and Notch1 in the lung tissue. Results Compared with the control group,the modeling of RSV-induced bronchiolitis elevated the IL-4,IL-6,and TNF-α levels,increased the proportion of M1-type macrophages and the lung inflammation and mucus secretion scores,and up-regulated the mRNA and protein levels of iNOS,Jagged1,and Notch1 in BALF (all P<0.001).Meanwhile,the modeling lowered the IL-10 level,decreased the proportion of M2-type macrophages,and down-regulated the mRNA and protein levels of Arg-1 (all P<0.001).Compared with the model group,low- and high-dose hesperidin lowered the IL-4,IL-6,TNF-α levels,decreased the proportion of M1-type macrophages and the lung inflammation and mucus secretion scores,and down-regulated the mRNA and protein levels of iNOS,Jagged1,and Notch1 in BALF (all P<0.05).Moreover,hesperidin elevated the IL-10 level,increased the proportion of M2-type macrophages,and up-regulated the mRNA and protein levels of Arg-1 (all P<0.001).Using recombinant Jagged1 protein to activate Notch1 signaling pathway can significantly attenuate the promotion of high-dose hesperidin on M2 macrophage polarization and amelioration of lung inflammation damage (all P<0.01). Conclusion Hesperidin may alleviate the lung inflammation damage in mice with RSV-induced bronchiolitis by inhibiting the Jagged1/Notch1 signaling pathway and promoting the M2-type polarization of macrophages.

Ginsenoside Rg1 Nanoparticles Induce Demethylation of H3K27me3 in VEGF-A and Jagged 1 Promoter Regions to Activate Angiogenesis After Ischemic Stroke.

Background: Compared with traditional drugs, nanomaterial drugs have the benefits of improving the solubility, bioavailability, and absorption rate of insoluble drugs. Nanoporous complexes can increase the efficiency with which drugs can penetrate the blood-brain barrier and reach target organs. Ginsenoside Rg1 is an effective drug that promotes angiogenesis. Ginsenoside Rg1 composite nanoparticles were employed to induce the expression of several key epigenetic enzymes and then activate the VEGF and Notch pathways after the onset of ischemic brain lesions. Methods: We constructed nanoparticles to fully encapsulate the therapeutic drug (ginsenoside Rg1), which can be transferred into brain tissue via the receptor-mediated transfer of drug-encapsulated nanoparticles. Evaluation of the therapeutic effect of ginsenoside Rg1 complex nanovesicles (CNV) was performed by in vitro and in vivo experiments. Real-time polymerase chain reaction (RT- PCR), Western blot, immunohistochemistry staining (IHC), and Co-immunoprecipitation (co-IP) were employed to screen for epigenetic enzymes with an up-regulated expression post ginsenoside Rg1-CNV intervention. RNA sequencing, shRNA knockdown, and chromatin Immunoprecipitation (ChIP) sequencing were performed to detect the target genes of ginsenoside Rg1-CNV that regulate angiogenesis. Then, bioinformatic analysis was performed to investigate the mechanism of action of epigenetic modifying enzymes in regulating target genes. Results: The average of the synthesized ginsenoside Rg1-CNV was 203.78±6.83 nm, the polydispersion index was 0.135±0.007, and the Zeta potential was 23.13±1.65 mV. Through in vivo and in vitro experiments, we found that it promotes the proliferation, migration, and tubular formation of brain microvascular endothelial cells (BMECs). Meanwhile, the intervention of ginsenoside Rg1-CNV promoted the demethylation of H3K27me3 within the promoter region of VEGF-A and Jagged1 genes and reduced the H3K27me3 modification within this region. Conclusion: The ginsenoside Rg1 nanoparticles may be an available blood-brain barrier penetrating agent for ischemic stroke.

LINC00173 regulates polycystic ovarian syndrome progression by promoting apoptosis and repressing proliferation in ovarian granulosa cells via the microRNA-124-3p (miR-124-3p)/jagged canonical Notch ligand 1 (JAG1) pathway.

As an endocrine and metabolic disorder, polycystic ovarian syndrome (PCOS) is common in females at childbearing age. Our work was intended to uncover the underlying role of LINC00173 and its potential regulatory mechanism in PCOS based on two cell lines (PCOS granulosa cells and KGN cells) and an in vivo model established from Sprague Dawley rats. It was revealed that LINC00173 and JAG1 expressions were upregulated, while miR-124-3p was poorly expressed in PCOS patients and PCOS rats. Functional assays showed that LINC00173 overexpression repressed proliferation and stimulated apoptosis in granulosa cells and KGN cells, while LINC00173 downregulation exhibited the opposite effects. Besides, it was verified that LINC00173 upregulated JAG1 expression in KGN cells via competitively binding to miR-124-3p. Similarly, miR-124-3p abundance was inversely related to LINC00173 and JAG1 level in PCOS. Subsequently, rescue assays elucidated that miR-124-3p upregulation or downregulation eliminated the effects on KGN cell proliferation and apoptosis mediated by LINC00173 overexpression or knockdown. In addition, it was found that the JAG1 level in KGN cells was adversely modulated by miR-124-3p and positively modulated by LINC00173. Moreover, it was further demonstrated that the reduced cell vitality and increased apoptosis of KGN cells induced by overexpressing LINC00173 could be relieved by JAG1 deletion. These findings suggested that LINC00173 could be a latent regulating factor for PCOS progression via modulating the miR-124-3p/JAG1 cascade.

Endothelial PERK-ATF4-JAG1 axis activated by T-ALL remodels bone marrow vascular niche.

The endoplasmic reticulum unfolded protein response (UPR) is a conserved adaptive signaling in ER homeostasis and has emerged as critical in highly proliferating cells and potential treatment target for acute T-cell lymphoblastic leukemia (T-ALL). Methods: in this study, we assessed the transcriptomic and phenotypic alterations in UPR response of the bone marrow endothelial cells (ECs) in mice engrafted with T-ALL and in bone marrow specimens from patients who have T-ALL. We used PERK inhibitor and generated endothelial specific PERK knockout mice to study the impact of PERK on leukemia progression and hematopoiesis. We performed chromatin immunoprecipitation (ChIP) to study the mechanistic regulation of JAG1 by ATF4. We characterized small extracellular vesicles (SEV) from leukemia-developing mice and studied the effect of SEVs on EC function. Results: we found that T-ALL development induced a robust activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dominant UPR in the bone marrow endothelial vascular niche. The activation of PERK-eIF2a-ATF4 axis remodels the vascular niche, upregulates angiogenic factors including VEGFα and ATF4-regulated JAG1, and suppresses the expression of SCF and CXCL12, which are important to HSC maintenance and regeneration. Further, targeting endothelial PERK significantly improved T-ALL outcome. EC-specific deletion of PERK abolished the aberrant JAG1 up-regulation, improved HSC maintenance, promoted leukemia apoptosis, and improved overall survival. Finally, we showed that small extracellular vesicles are critical mediators of endothelial PERK-eIF2a-ATF4 activation and JAG1 up-regulation in leukemia. Corroborating animal model studies, activation of PERK-ATF4-JAG1 is prominent in human T-ALL bone marrow and T-ALL xenografts. Conclusion: our studies thus revealed for the first time that the leukemia-initiated PERK-ATF4-JAG1 axis plays a critical role in the remodeling of the bone marrow vascular niche and that targeting vascular niche UPR is a potential therapeutic opportunity in T-ALL.

Propofol alleviates neuropathic pain in chronic constriction injury rat models via the microRNA-140-3p/Jagged-1 peptide/Notch signaling pathway.

Chronic constriction injury (CCI) of the sciatic nerve was used to establish neuropathic pain (NP) models in rats. CCI rats were then treated with propofol (Pro) and their paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured. In addition, the expression patterns of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-10 were detected. CCI rats treated with propofol were further injected with antagomiR-140-3p to verify the role of miR-140-3p in propofol's analgesic actions. In addition to confirming the relationship between miR-140-3p and JAG1, the expression patterns of JAG1 itself were detected. Propofol-treated CCI rats were also injected with Ad-JAG1 (adenovirus-packaged JAG1 overexpression vector and Ad-NC) to test the role of JAG1 in propofol's analgesic mechanism of action. Finally, the levels of JAG1 and Notch pathway-related proteins were detected RESULTS: Propofol was found to alleviate NP, including thermal hyperalgesia and mechanical pain threshold. Propofol could also ameliorate neuroinflammation by up-regulating the expression of IL-10 and inhibiting the release of TNF-α and IL-1ß. Mechanically, propofol enhanced the amount of miR-140-3p in CCI rats via the regulation of JAG1. Down-regulation of miR-140-3p, or up-regulation of JAG1, could reduce the protective effect of propofol against NP. Propofol inhibited the activation of Notch signaling via miR-140-3p/JAG1 to realize its analgesic effect CONCLUSION: Our findings indicated that propofol inhibits inflammatory responses and the Notch signaling pathway via miR-140-3p/JAG1 to alleviate NP. These data provide evidence to support a potential clinical therapy for NP.

Curcumin attenuates hypoxia/reoxygenation­induced cardiomyocyte injury by downregulating Notch signaling.

Recovery of the blood supply is the most effective treatment against ischemic heart disease; however, it is also a major cause of myocardial ischemia/reperfusion injury in clinical therapy. Curcumin has been reported to possess beneficial effects against hypoxia/reoxygenation (H/R)­induced cardiomyocyte injury by regulating cell proliferation, apoptosis and antioxidant enzyme activity. The aim of the present study was to investigate the molecular mechanisms underlying the effects of curcumin on H/R­injured cardiomyocytes. H9C2 cardiomyocytes were pretreated with curcumin, and then cultured under H/R conditions. The viability of H9C2 cells was measured using a Cell Counting kit­8 assay, and the levels of intracellular lactate dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD) were measured to assess cell injury. Levels of reactive oxygen species (ROS) and apoptosis were evaluated by flow cytometry. The expression levels of Notch intracellular domain (NICD) and numerous downstream genes were analyzed via reverse transcription­quantitative polymerase chain reaction and western blotting. The results revealed that curcumin protected H9C2 cells against H/R­induced injury, reversing the H/R­induced increases in LDH and MDA levels, and decreases in SOD levels. ROS levels in H/R­induced cells were also significantly downregulated by curcumin treatment (P<0.01), and the apoptotic rate was significantly decreased from 15.13% in the H/R group to 7.7% in the H/R + curcumin group (P<0.01). The expression levels of NICD, hairy and enhancer of split (Hes)­1, Hes­5 and hairy/enhancer­of­split related with YRPW motif protein 1 (Hey­1) were significantly decreased in H/R­treated cells following curcumin treatment. Treatment with Jagged1 attenuated the effects of curcumin on cell viability, ROS levels and apoptosis; the Notch pathway was also reactivated. The present study indicated that there was a role for the Notch pathway in the protective effects of curcumin against H/R­induced cardiomyocyte injury, suggesting that downregulation of the Notch pathway may alleviate H/R­induced injury in H9C2 cells.

Jagged1 promotes mineralization in human bone-derived cells.

OBJECTIVES: The present study aimed to investigate the expression of Notch signaling components during osteogenic differentiation in vitro and bone healing in vivo. In addition, the influence of Notch signaling on osteogenic differentiation of human bone-derived cells was examined. METHODS: Gene expression profiling of osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro (GSE80614) and bone healing period of murine tibial fracture in vivo (GSE99388) was downloaded from Gene Expression Omnibus database. The expression of Notch signaling components was obtained from bioinformatic tools. Human bone-derived cells were isolated from alveolar and iliac bone. Cells were seeded on Jagged1 immobilized surface. Osteogenic marker gene expression and mineralization were examined using real-time polymerase chain reaction and alizarin red s staining, respectively. RESULTS: From bioinformatic analysis of gene expression profiling, various Notch signaling components were differentially expressed during osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro and bone healing period of murine tibial fracture in vivo. The common genes differentially regulated of these two datasets were Hes1, Aph1a, Nsctn, Furin, Adam17, Hey1, Pcsk5, Nedd4, Jag1, Heyl, Notch3, Dlk1, and Hey2. For an in vitro analysis, the mineral deposition markedly increased after seeding human bone-derived cells on Jagged1 immobilized surface, correspondingly with the increase of ALP mRNA expression. Jagged1 treatment downregulated TWIST2 mRNA expression in both human alveolar and iliac bone-derived cells. CONCLUSION: Notch signaling is regulated during osteogenic differentiation and bone healing. In addition, the activation of Notch signaling promotes osteogenic differentiation in human alveolar and iliac bone-derived cells. Therefore, Notch signaling manipulation could be a useful approach for enhancing bone regeneration.

A non-canonical JAGGED1 signal to JAK2 mediates osteoblast commitment in cranial neural crest cells.

During craniofacial development, cranial neural crest (CNC) cells migrate into the developing face and form bone through intramembranous ossification. Loss of JAGGED1 (JAG1) signaling in the CNC cells is associated with maxillary hypoplasia or maxillary bone deficiency (MBD) in mice and recapitulates the MBD seen in humans with Alagille syndrome. JAGGED1, a membrane-bound NOTCH ligand, is required for normal craniofacial development, and Jagged1 mutations in humans are known to cause Alagille Syndrome, which is associated with cardiac, biliary, and bone phenotypes and these children experience increased bony fractures. Previously, we demonstrated deficient maxillary osteogenesis in Wnt1-cre;Jagged1f/f (Jag1CKO) mice by conditional deletion of Jagged1 in maxillary CNC cells. In this study, we investigated the JAG1 signaling pathways in a CNC cell line. Treatment with JAG1 induced osteoblast differentiation and maturation markers, Runx2 and Ocn, respectively, Alkaline Phosphatase (ALP) production, as well as classic NOTCH1 targets, Hes1 and Hey1. While JAG1-induced Hes1 and Hey1 expression levels were predictably decreased after DAPT (NOTCH inhibitor) treatment, JAG1-induced Runx2 and Ocn levels were surprisingly constant in the presence of DAPT, indicating that JAG1 effects in the CNC cells are independent of the canonical NOTCH pathway. JAG1 treatment of CNC cells increased Janus Kinase 2 (JAK2) phosphorylation, which was refractory to DAPT treatment, highlighting the importance of the non-canonical NOTCH pathway during CNC cells osteoblast commitment. Pharmacologic inhibition of JAK2 phosphorylation, with and without DAPT treatment, upon JAG1 induction reduced ALP production and, Runx2 and Ocn gene expression. Collectively, these data suggest that JAK2 is an essential component downstream of a non-canonical JAG1-NOTCH1 pathway through which JAG1 stimulates expression of osteoblast-specific gene targets in CNC cells that contribute to osteoblast differentiation and bone mineralization.

Notch activation promotes osteoblast mineralization by inhibition of apoptosis.

Notch activator Jagged1 (JAG1) plays a critical role in the regulation of osteoblast differentiation and bone metabolism. In this study, JAG1-induced osteoblast proliferation, differentiation, and mineralization has been analyzed in primary osteoblasts for up to 7 days. Alkaline phosphatase and Alizarin red staining showed an enhanced osteoblast maturation and mineralization in JAG1 treated cells, as well as higher mRNA levels of late osteoblast differentiation markers. In contrast, Notch inhibitor DAPT and deletion of Runx2 totally blocked JAG1 effects on osteoblast mineralization. Flow cytometry data further showed a significantly higher cell proliferation in early stages of culture at day 3, and lower levels of osteoblast apoptosis in late stages of culture at day 7. More importantly, activation of anti-apoptotic factor BCL-2 was enhanced, while pro-apoptotic factor Caspase3 was reduced in JAG1 treated osteoblasts. Therefore, we conclude that cell mineralization is enhanced via anti-apoptotic actions of Notch signaling within the osteoblast cells.

Indirect immobilized Jagged1 suppresses cell cycle progression and induces odonto/osteogenic differentiation in human dental pulp cells.

Notch signaling regulates diverse biological processes in dental pulp tissue. The present study investigated the response of human dental pulp cells (hDPs) to the indirect immobilized Notch ligand Jagged1 in vitro. The indirect immobilized Jagged1 effectively activated Notch signaling in hDPs as confirmed by the upregulation of HES1 and HEY1 expression. Differential gene expression profiling using an RNA sequencing technique revealed that the indirect immobilized Jagged1 upregulated genes were mainly involved in extracellular matrix organization, disease, and signal transduction. Downregulated genes predominantly participated in the cell cycle, DNA replication, and DNA repair. Indirect immobilized Jagged1 significantly reduced cell proliferation, colony forming unit ability, and the number of cells in S phase. Jagged1 treated hDPs exhibited significantly higher ALP enzymatic activity, osteogenic marker gene expression, and mineralization compared with control. Pretreatment with a γ-secretase inhibitor attenuated the Jagged1-induced ALP activity and mineral deposition. NOTCH2 shRNA reduced the Jagged1-induced osteogenic marker gene expression, ALP enzymatic activity, and mineral deposition. In conclusion, indirect immobilized Jagged1 suppresses cell cycle progression and induces the odonto/osteogenic differentiation of hDPs via the canonical Notch signaling pathway.

Notch signaling is significantly suppressed in basal cell carcinomas and activation induces basal cell carcinoma cell apoptosis.

A subset of basal cell carcinomas (BCCs) are directly derived from hair follicles (HFs). In some respects, HFs can be defined as 'ordered' skin appendage growths, while BCCs can be regarded as 'disordered' skin appendage growths. The aim of the present study was to examine HFs and BCCs to define the expression of common and unique signaling pathways in each skin appendage. Human nodular BCCs, along with HFs and non­follicular skin epithelium from normal individuals, were examined using microarrays, qPCR, and immunohistochemistry. Subsequently, BCC cells and root sheath keratinocyte cells from HFs were cultured and treated with Notch signaling peptide Jagged1 (JAG1). Gene expression, protein levels, and cell apoptosis susceptibility were assessed using qPCR, immunoblotting, and flow cytometry, respectively. Specific molecular mechanisms were found to be involved in the process of cell self­renewal in the HFs and BCCs, including Notch and Hedgehog signaling pathways. However, several key Notch signaling factors showed significant differential expression in BCCs compared with HFs. Stimulating Notch signaling with JAG1 induced apoptosis of BCC cells by increasing Fas ligand expression and downstream caspase-8 activation. The present study showed that Notch signaling pathway activity is suppressed in BCCs, and is highly expressed in HFs. Elements of the Notch pathway could, therefore, represent targets for the treatment of BCCs and potentially in hair follicle engineering.

PlexinD1 Is a Novel Transcriptional Target and Effector of Notch Signaling in Cancer Cells.

The secreted semaphorin Sema3E controls cell migration and invasiveness in cancer cells. Sema3E-receptor, PlexinD1, is frequently upregulated in melanoma, breast, colon, ovarian and prostate cancers; however, the mechanisms underlying PlexinD1 upregulation and the downstream events elicited in tumor cells are still unclear. Here we show that the canonical RBPjk-dependent Notch signaling cascade controls PlexinD1 expression in primary endothelial and cancer cells. Transcriptional activation was studied by quantitative PCR and promoter activity reporter assays. We found that Notch ligands and constitutively activated intracellular forms of Notch receptors upregulated PlexinD1 expression; conversely RNAi-based knock-down, or pharmacological inhibition of Notch signaling by gamma-secretase inhibitors, downregulated PlexinD1 levels. Notably, both Notch1 and Notch3 expression positively correlates with PlexinD1 levels in prostate cancer, as well as in other tumor types. In prostate cancer cells, Sema3E-PlexinD1 axis was previously reported to regulate migration; however, implicated mechanisms were not elucidated. Here we show that in these cells PlexinD1 activity induces the expression of the transcription factor Slug, downregulates E-cadherin levels and enhances cell migration. Moreover, our mechanistic data identify PlexinD1 as a pivotal mediator of this signaling axis downstream of Notch in prostate cancer cells. In fact, on one hand, PlexinD1 is required to mediate cell migration and E-cadherin regulation elicited by Notch. On the other hand, PlexinD1 upregulation is sufficient to induce prostate cancer cell migration and metastatic potential in mice, leading to functional rescue in the absence of Notch. In sum, our work identifies PlexinD1 as a novel transcriptional target induced by Notch signaling, and reveals its role promoting prostate cancer cell migration and downregulating E-cadherin levels in Slug-dependent manner. Collectively, these findings suggest that Notch-PlexinD1 signaling axis may be targeted to impair prostate cancer cell invasiveness and metastasis.

Effect of Jagged-1 and Dll-1 on osteogenic differentiation by stem cells from human exfoliated deciduous teeth.

OBJECTIVE: The aim of the present study was to determine the influence of Notch ligands, Jagged-1 and Dll-1, on osteogenic differentiation by stem cells from human exfoliated deciduous teeth. DESIGN: Notch ligands were immobilized on tissue culture surface using an indirect affinity immobilization technique. Cells from the remaining of dental pulp tissues from human deciduous teeth were isolated and characterized using flow cytometry and differentiation assay. Alkaline phosphatase (ALP) enzymatic activity, osteogenic marker gene expression, and mineralization were determined using ALP assay, real-time polymerase chain reaction, and alizarin red staining, respectively. RESULTS: The isolated cells exhibited CD44, CD90, and CD105 expression but lack of CD45 expression. Further, these cells were able to differentiate toward osteogenic lineage. The upregulation of HES-1 and HEY-1 was observed in those cells on Dll-1 and Jagged-1 coated surface. The significant increase of ALP activity and mineralization was noted in those cells seeded on Jagged-1 surface and these results were attenuated when cells were pretreated with gamma secretase inhibitor. The significant upregulation of ALP and collagen type I gene expression was also observed in those cells seeded on Jagged-1 surface. The inconsistent Dll-1 induced osteogenic differentiation was found and high Dll-1 immobilized dose (50 nM) slightly enhanced alkaline phosphatase enzymatic activity. However, the statistical significant difference was not obtained as compared to the hFc control. CONCLUSION: The surface immobilization of Notch ligands, Jagged-1 and Dll-1, likely to enhance osteogenic differentiation of SHEDs. However, Jagged-1 had more ability in enhancing osteogenic differentiation than Dll-1 in our model.

Jagged1 inhibits osteoprotegerin expression by human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Notch signaling regulates bone homeostasis. The present study investigated the effect of Jagged1 on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human periodontal ligament stromal (hPDL) cells. MATERIAL AND METHODS: hPDL cells were seeded on to indirect immobilized Jagged1 surfaces. OPG expression was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Lentiviral small hairpin RNA particles against NOTCH2 were employed to inhibit NOTCH2 expression. Osteoclast formation was evaluated using RAW264.7 cells. An influence of exogenous OPG on osteogenic differentiation was determined by real-time polymerase chain reaction and Alizarin Red S staining. RESULTS: Jagged1 significantly enhanced HES1 and HEY1mRNA expression in a dose-dependent manner. Furthermore, OPG mRNA and protein levels dramatically decreased upon exposing hPDL cells to Jagged1. However, RANKL mRNA levels were not significantly different. There was also no difference in M-CSF and MCP-1mRNA expression. A γ-secretase inhibitor and cycloheximide treatment rescued Jagged1-attenuated OPG expression. Furthermore, shNOTCH2 overexpressing hPDL cells did not exhibit a decrease in OPG expression upon exposure to Jagged1, implying the involvement of NOTCH2 in the regulatory mechanism. Culturing RAW264.7 cells with conditioned medium from Jagged1-treated hPDL cells enhanced osteoclast formation compared with those cultured with conditioned medium of the control group. Lastly, OPG treatment did not influence osteogenic differentiation by hPDL cells. CONCLUSION: These results suggest that Jagged1 activates Notch signaling in hPDL cells, leading to decreased OPG expression. This may imply an indirect role of Jagged1 on the regulation of osteoclast differentiation via hPDL cells.