Role for notch signaling in salivary acinar cell growth and differentiation

The Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. The Notch signaling is essential for Drosophila salivary gland development but its role in mammalian salivary gland remains unclear. The human salivary epithelial cell line, HSG, was studied to determine the role of Notch signaling in salivary epithelial cell differentiation. HSG expressed Notch 1 to 4, and the Notch ligands Jagged 1 and 2 and Delta 1. Treatment of HSG cells with inhibitors of γ‐secretase, which is required for Notch cleavage and activation, blocked vimentin and cystatin S expression, an indicator of HSG differentiation. HSG differentiation was also associated with Notch downstream signal Hes‐1 expression, and Hes‐1 expression was inhibited by γ‐secretase inhibitors. siRNA corresponding to Notch 1 to 4 was used to show that silencing of all four Notch receptors was required to inhibit HSG differentiation. Normal human submandibular gland expressed Notch 1 to 4, Jagged 1 and 2, and Delta 1, with nuclear localization indicating Notch signaling in vivo. Hes‐1 was also expressed in the human tissue, with staining predominantly in the ductal cells. In salivary tissue from rats undergoing and recovering from ductal obstruction, we found that Notch receptors and ligands were expressed in the nucleus of the regenerating epithelial cells. Taken together, these data suggest that Notch signaling is critical for normal salivary gland cell growth and differentiation. Developmental Dynamics 238:724–731, 2009. © 2009 Wiley‐Liss, Inc.     

The Notch pathway mediates expansion of a progenitor pool and neuronal differentiation in adult neural progenitor cells after stroke

Molecular mechanisms by which stroke increases neurogenesis have not been fully investigated. Using neural progenitor cells isolated from the subventricular zone (SVZ) of the adult rat subjected to focal cerebral ischemia, we investigated the Notch pathway in regulating proliferation and differentiation of adult neural progenitor cells after stroke. During proliferation of neural progenitor cells, ischemic neural progenitor cells exhibited substantially increased levels of Notch, Notch intracellular domain (NICD), and hairy enhancer of split (Hes) 1, which was associated with a significant increase of proliferating cells. Blockage of the Notch pathway by short interfering ribonucleic acid (siRNA) against Notch or a γ secretase inhibitor significantly reduced Notch, NICD and Hes1 expression and cell proliferation induced by stroke. During differentiation of neural progenitor cells, Notch and Hes1 expression was downregulated in ischemic neural progenitor cells, which was coincident with a significant increase of neuronal population. Inhibition of the Notch pathway with a γ secretase inhibitor further substantially increased neurons, but did not alter astrocyte population in ischemic neural progenitor cells. These data suggest that the Notch signaling pathway mediates adult SVZ neural progenitor cell proliferation and differentiation after stroke.     

Overexpression of miR-34c inhibits high glucose-induced apoptosis in podocytes by targeting Notch signaling pathways

Recent findings have shown that microRNAs play critical roles in the pathogenesis of diabetic nephropathy. miR-34c has been found to inhibit fibrosis and the epithelial-mesenchymal transition of kidney cells. However, the role of miR-34c in diabetic nephropathy has not been well studied. The current study was designed to investigate the role and potential underlying mechanism of miR-34c in regulating diabetic nephropathy. After treating podocytes with high glucose (HG) in vitro, we found that miR-34c was downregulated and that overexpression of miR-34c inhibited HG-induced podocyte apoptosis. The direct interaction between miR-34c and the 3’-untranslated region (UTR) of Notch1 and Jagged1 was validated by dual-luciferase reporter assay. Moreover, Notch1 and Jagged1 as putative targets of miR-34c were downregulated by miR-34c overexpression in HG-treated podocytes. Overexpression of miR-34c inhibited HG-induced Notch signaling pathway activation, as indicated by decreased expression of the Notch intracellular domain (NICD) and downstream genes including Hes1 and Hey1. Furthermore, miR-34c overexpression increased the expression of the anti-apoptotic gene Bcl-2, and decreased the expression of the pro-apoptotic protein Bax and cleaved Caspase-3. Additionally, the phosphorylation of p53 was also downregulated by miR-34c overexpression. Taken together, our findings suggest that miR-34c overexpression inhibits the Notch signaling pathway by targeting Notch1 and Jaggged1 in HG-treated podocytes, representing a novel and potential therapeutic target for the treatment of diabetic nephropathy.     

IKKα aggravates renal fibrogenesis by positively regulating the Wnt/β-catenin pathway

AKI (acute kidney injury) with maladaptive repair plays exacerbated role in renal fibrosis characterized by tubulointerstitial fibrosis. Previously, we reported that IKKα contributed to kidney regeneration and inhibited inflammation. Here, we first identified the role and mechanism of IKKα on TGF-β1-induced fibrosis in human tubular epithelial cells and fibrotic kidneys. IKKα was up-regulated in kidney tubular epithelium in unilateral ureteral obstruction (UUO) and unilateral ischemic reperfusion injury (UIRI) mice. Immunohistochemical staining showed that IKKα was positively correlated with the extent of kidney fibrosis in tissue biopsies from chronic kidney disease (CKD) patients. Compared with wild-type controls, Ksp-IKKα^−/− mice exhibited inactivated Wnt/β-catenin pathway, decreased serum creatinine and interstitial fibrosis in the kidney after IRI. In TGF-β1-stimulated human tubular epithelial cells, IKKα overexpression enhanced β-catenin nuclear translocation. Blocking IKKα by siRNA specifically suppressed β-catenin activation and downstream profibrotic genes such as fibronectin and α-smooth muscle actin (α-SMA). Taken together, our study demonstrated that IKKα aggravated renal fibrogenesis by activating Wnt/β-catenin signalling pathway, providing a new target for the treatment of kidney fibrosis.     

Notch信号通路蛋白在糖尿病肾病患者肾组织中的表达及意义

目的观察糖尿病肾病肾组织中Notch信号通路的表达情况,探讨其与糖尿病肾病肾脏损害的关系。方法收集10例手术切除的远离肿瘤的瘤旁肾组织及34例糖尿病肾病肾穿刺组织,免疫组化检测Jagged1、Notch1、NICD1和Hes1蛋白表达情况。结果 Jagged1、Notch1、NICD1和Hes1蛋白在糖尿病肾病肾组织中高表达,并与24小时尿蛋白成正相关,而与肾小球滤过率成负相关。结论 Notch信号通路在糖尿病肾病肾组织中激活,与糖尿病肾病肾脏损伤有关。     

Calsenilin Contributes to Neuronal Cell Death in Ischemic Stroke

Calsenilin is a calcium sensor protein that interacts with presenilin and increases calcium‐triggered neuronal apoptosis, and γ‐secretase activity. Notch is a cell surface receptor that regulates cell‐fate decisions and synaptic plasticity in brain. The aim of the present study was to characterize the role of calsenilin as a regulator of the γ‐secretase cleavage of Notch in ischemic stroke. Here, we determined the modulation of expression level and cellular distribution of calsenilin in neurons subjected to ischemic‐like conditions. The levels of calsenilin and presenilin were increased in primary neurons after oxygen and glucose deprivation. Furthermore, calsenilin was found to enhance the γ‐secretase cleavage of Notch and to contribute to cell death under ischemia‐like conditions. The inhibition of γ‐secretase activity and a presenilin deficiency were both found to protect against calsenilin‐mediated ischemic neuronal death. The expression of calsenilin was found to be increased in brain following experimental ischemic stroke. These findings establish a specific molecular mechanism by which the induction of calsenilin enhances Notch activation in ischemic stroke, and identify calsenilin as an upstream of the γ‐secretase cleavage of Notch.     

Notch in the kidney: development and disease

Notch signalling is a highly conserved cell-cell communication mechanism that regulates development, tissue homeostasis, and repair. Within the kidney, Notch has an important function in orchestrating kidney development. Recent studies indicate that Notch plays a key role in establishing proximal epithelial fate during nephron segmentation as well as the differentiation of principal cells in the renal collecting system. Notch signalling is markedly reduced in the adult kidney; however, increased Notch signalling has been noted in both acute and chronic kidney injury. Increased glomerular epithelial Notch signalling has been associated with albuminuria and glomerulosclerosis, while tubular epithelial Notch activation caused fibrosis development most likely inducing an improper epithelial repair pathway. Recent studies thereby indicate that Notch is a key regulator of kidney development, repair, and injury.     

Angiotensin II increases connective tissue growth factor in the kidney

Connective tissue growth factor (CTGF) has been described as a novel fibrotic mediator. CTGF is overexpressed in several kidney diseases and is induced by different factors involved in renal injury. Angiotensin II (AngII) participates in the pathogenesis of kidney damage, contributing to fibrosis; however, whether AngII regulates CTGF in the kidney has not been explored. Systemic infusion of AngII into normal rats for 3 days increased renal CTGF mRNA and protein levels. At day 7, AngII-infused rats presented overexpression of CTGF in glomeruli, tubuli, and renal arteries, as well as tubular injury and elevated fibronectin deposition. Only treatment with an AT(1) receptor antagonist, but not an AT(2), diminished CTGF and fibronectin overexpression and ameliorated tubular damage. In rats with immune complex nephritis, renal overexpression of CTGF was diminished by the ACE inhibitor quinapril, correlated with a diminution in fibrosis. In cultured renal cells (mesangial and tubular epithelial cells) AngII, via AT(1), increased CTGF mRNA and protein production, and a CTGF antisense oligonucleotide decreased AngII-induced fibronectin synthesis. Our data show that AngII regulates CTGF in the kidney and cultured in mesangial and tubular cells. This novel finding suggests that CTGF could be a mediator of the profibrogenic effects of AngII in the kidney.     

Notch signaling: distinct ligands induce specific signals during lymphocyte development and maturation

Notch signaling is a highly conserved pathway involved in cell fate choice during development with Delta and Jagged constituting the two evolutionary conserved families of Notch ligands. These ligands are transmembrane proteins with conserved biochemical structure that share their receptors and signal through a common mechanism. Upon ligand binding Notch receptors are proteoliticaly cleaved, the intracellular domain of Notch (NICD) is released and translocated to the nucleus, where it activates target genes. In mammals, four receptors and five ligands have been described. Delta-1, Delta-3 and Delta-4 are homologues to Drosophila Delta and Jagged-1 and Jagged-2 to Drosophila Serrate. Despite strong domain homology, there is growing evidence that signals transmitted through Delta or Jagged ligands can differentially affect the target cell. At least during embryonic development, Notch receptors and Notch ligands functions cannot be compensated by other members. Knock-out mice for Notch-1, Notch-2, Delta-1 and Jagged-1 are embryonic lethal . Similarly, mice heterozygous for Delta-4 inactivation also die before birth . Invalidation of Jagged-2 results in defaults in thymus morphology and gammadelta development . Altogether, these data suggest that each Notch member can exert unique specific effects. In this review, we will thus focus on recent data about differential effects of Notch ligands on T cell development and differentiation. In light of recent biochemical and molecular advances on Notch-signaling pathway, we will examine how specific effects can be mediated by a given ligand.     

Epithelial–mesenchymal transition of renal tubules: Divergent processes of repairing in acute or chronic injury?

Epithelial–mesenchymal transition (EMT) of tubular epithelial cells (TECs) is commonly considered as the major mechanism leading to renal fibrosis in chronic kidney diseases (CKD) injury. We raise the hypothesis that EMT in adult kidney may be an event of “atavistic” phenotypic transition, which mimics but reverses the genetic and cellular processes of development of renal tubules. Transformed TECs may be regarded as induced mesenchymal stem-like cells, representing a cellular self-adaptation when in acute or chronic injury. The reasons are as follows: (1) Embryonic gene WT1 and Pax2, which govern tubule development, have been found to re-express during tubular EMT when facing injury. (2) The common factors that induce EMT in vitro, like IL-1, angiotension II and hypoxia could also promote WT1 and/or Pax2 re-expression. (3) Expression of WT1 and Pax2 are found to be associated with progenitor cells. (4) Beside embryonic gene WT1 and Pax2, we also found that some stem cell markers like CD133 were expressed during EMT process. (5) The process of EMT is not only take place in chronic kidney injury (CKD), but also in acute kidney injury (AKI) as well. (6) The phenotype transition of TECs and genetic event during AKI are entirely consistent with what happened in CKD, but the outcome is completely different. Thus, we thought tubular injury of CKD and AKI may share a common initiative repair mechanism: tubular EMT, that is TECs are transformed into induced mesenchymal stem-like cells, and then interpret the injurious signal differently in acute versus chronic conditions, so as to possess a divergent fates, tubular regeneration or fibrosis formation, depending on a different microenvironment or the duration of the injury. In this sense, tubular EMT could be purposefully orientated into a constructively pathway that repair kidney injury via tubular regeneration, matrix remodeling and tissue structure and function restoration.     

Kinetics of chemokines and their receptors in mercuric chloride-induced tubulointerstitial lesions in brown Norway rats

We investigated the kinetics of chemokines and their receptors in mercuric chloride-treated brown Norway (BN) rats from the viewpoint of its relation to the development of tubulointerstitial fibrosis with mononuclear cell infiltration. BN rats were injected subcutaneously with 1 mg/kg b.w. of mercuric chloride one or three times. The kidney was examined histopathologically and the kinetics of chemokines and their receptors in the kidney were also examined using immunohistochemistry and RT-PCR. As a result, mercuric chloride induced tubular injury and subsequent tubulointerstitial fibrosis accompanied with mononuclear cell infiltration. Macrophages were the most predominant population of infiltrating cells and lymphocytes were the next. In the lesions, the expression of MCP-1 mRNA was most prominently elevated, and those of RANTES and IP-10 mRNAs also increased, and their proteins were localized in tubular epithelium. As to their receptors, the levels of CCR1, CCR2, and CXCR3 mRNAs showed significant and prominent elevations, CCR5 mRNA also increased moderately, and their receptor protein-expressing cells also increased. The present findings suggest that MCP-1, RANTES, and IP-10 may participate in the pathogenesis of mercuric chloride-induced tubulointerstitial fibrosis with mononuclear cell infiltration, via CCR2, CCR1 or CCR5, and CXCR3, respectively.     

ADAM10 mediates ectopic proximal tubule development and renal fibrosis through Notch signalling

Disturbed intrauterine development increases the risk of renal disease. Various studies have reported that Notch signalling plays a significant role in kidney development and kidney diseases. A disintegrin and metalloproteinase domain 10 (ADAM10), an upstream protease of the Notch pathway, is also reportedly involved in renal fibrosis. However, how ADAM10 interacts with the Notch pathway and causes renal fibrosis is not fully understood. In this study, using a prenatal chlorpyrifos (CPF) exposure mouse model, we investigated the role of the ADAM10/Notch axis in kidney development and fibrosis. We found that prenatal CPF-exposure mice presented overexpression of Adam10, Notch1 and Notch2, and led to premature depletion of Six2⁺ nephron progenitors and ectopic formation of proximal tubules (PTs) in the embryonic kidney. These abnormal phenotypic changes persisted in mature kidneys due to the continuous activation of ADAM10/Notch and showed aggravated renal fibrosis in adults. Finally, both ADAM10 and NOTCH2 expression were positively correlated with the degree of renal interstitial fibrosis in IgA nephropathy patients, and increased ADAM10 expression was negatively correlated with decreased kidney function evaluated by serum creatinine, cystatin C, and estimated glomerular filtration rate. Regression analysis also indicated that ADAM10 expression was an independent risk factor for fibrosis in IgAN. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.     

Regulation of Notch signaling during T- and B-cell development by O-fucose glycans

Summary:  Notch signaling is required for the development of all T cells and marginal zone (MZ) B cells. Specific roles in T- and B-cell differentiation have been identified for different Notch receptors, the canonical Delta-like (Dll) and Jagged (Jag) Notch ligands, and downstream effectors of Notch signaling. Notch receptors and ligands are post-translationally modified by the addition of glycans to extracellular domain epidermal growth factor-like (EGF) repeats. The O -fucose glycans of Notch cell-autonomously modulate Notch–ligand interactions and the strength of Notch signaling. These glycans are initiated by protein O -fucosyltransferase 1 (Pofut1), and elongated by the transfer of N -acetylglucosamine (GlcNAc) to the fucose by β1,3GlcNAc-transferases termed lunatic, manic, or radical fringe. This review discusses T- and B-cell development from progenitors deficient in O -fucose glycans. The combined data show that Lfng and Mfng regulate T-cell development by enhancing the interactions of Notch1 in T-cell progenitors with Dll4 on thymic epithelial cells. In the spleen, Lfng and Mfng cooperate to modify Notch2 in MZ B progenitors, enhancing their interaction with Dll1 on endothelial cells and regulating MZ B-cell production. Removal of O -fucose affects Notch signaling in myelopoiesis and lymphopoiesis, and the O -fucose glycan in the Notch1 ligand-binding domain is required for optimal T-cell development.     

Novel signalling mechanisms and targets in renal ischaemia and reperfusion injury

Acute kidney injury (AKI) induced by ischaemia and reperfusion (I/R) injury is a common and severe clinical problem. Vascular dysfunction, immune system activation and tubular epithelial cell injury contribute to functional and structural deterioration. The search for novel therapeutic interventions for I/R‐induced AKI is a dynamic area of experimental research. Pharmacological targeting of injury mediators and corresponding intracellular signalling in endothelial cells, inflammatory cells and the injured tubular epithelium could provide new opportunities yet may also pose great translational challenge. Here, we focus on signalling mediators, their receptors and intracellular signalling pathways which bear potential to abrogate cellular processes involved in the pathogenesis of I/R‐induced AKI. Sphingosine 1 phosphate (S1P) and its respective receptors, cytochrome P450 (CYP450)‐dependent vasoactive eicosanoids, NF‐κB‐ and protein kinase‐C (PKC)‐related pathways are representatives of such ‘druggable’ pleiotropic targets. For example, pharmacological agents targeting S1P and PKC isoforms are already in clinical use for treatment for autoimmune diseases and were previously subject of clinical trials in kidney transplantation where I/R‐induced AKI occurs as a common complication. We summarize recent in vitro and in vivo experimental studies using pharmacological and genomic targeting and highlight some of the challenges to clinical application of these advances.     

Targeting Notch Signaling and Autophagy Increases Cytotoxicity in Glioblastoma Neurospheres

Glioblastomas are highly aggressive tumors that contain treatment resistant stem‐like cells. Therapies targeting developmental pathways such as Notch eliminate many neoplastic glioma cells, including those with stem cell features, but their efficacy can be limited by various mechanisms. One potential avenue for chemotherapeutic resistance is the induction of autophagy, but little is known how it might modulate the response to Notch inhibitors. We used the γ‐secretase inhibitor MRK003 to block Notch pathway activity in glioblastoma neurospheres and assessed its effects on autophagy. A dramatic, several fold increase of LC3B‐II/LC3B‐I autophagy marker was noted on western blots, along with the emergence of punctate LC3B immunostaining in cultured cells. By combining the late stage autophagy inhibitor chloroquine (CQ) with MRK003, a significant induction in apoptosis and reduction in growth was noted as compared to Notch inhibition alone. A similar beneficial effect on inhibition of cloogenicity in soft agar was seen using the combination treatment. These results demonstrated that pharmacological Notch blockade can induce protective autophagy in glioma neurospheres, resulting in chemoresistance, which can be abrogated by combination treatment with autophagy inhibitors.