Alpha‐fetoprotein accelerates the progression of hepatocellular carcinoma by promoting Bcl‐2 gene expression through an RA‐RAR signalling pathway

Previous studies have found that alpha‐fetoprotein (AFP) can promote the proliferation of hepatoma cells and accelerate the progression of hepatocellular carcinoma (HCC). However, the exact mechanism of action remains unclear. Recent bioinformatics studies have predicted the possible interaction between AFP and retinoic acid receptors (RARs). Thus, the purpose of this study was to investigate the molecular mechanism through which AFP promotes tumour cell proliferation by interfering with the RA‐RAR signal pathway. Our data indicated that AFP could significantly promote the proliferation and weaken ATRA‐induced apoptosis of hepatoma cells. Besides, cytoplasmic AFP interacts with RAR, disrupting its entrance into the nucleus, which in turn affects the expression of the Bcl‐2 gene. In addition, knockdown of AFP in HepG2 cells was synchronously associated with an incremental increase of RAR binding to DNA, as well as down‐regulation of Bcl‐2; the opposite effect was observed in AFP gene‐transfected HLE cells. Moreover, a similar effect of AFP was detected in tumour tissues with high serum AFP, but not in adjacent non‐cancerous liver tissues, or HCC tissues with low serum AFP levels. These results indicate that AFP acts as signalling molecule and prevents RAR from entering into the nucleus by interacting with RAR, thereby promoting the expression of Bcl‐2. Our data reveal a novel mechanism through which AFP regulates Bcl‐2 expression and further suggest that AFP may be used as a novel target for treating HCC.     

Sex‐related differential susceptibility to ponatinib cardiotoxicity and differential modulation of the Notch1 signalling pathway in a murine model

Ponatinib (PON), a tyrosine kinase inhibitor approved in chronic myeloid leukaemia, has proven cardiovascular toxicity. We assessed mechanisms of sex‐related PON‐induced cardiotoxicity and identified rescue strategies in a murine model. PON+scrambled siRNA‐treated male mice had a higher number of TUNEL‐positive cells (%TdT+6.12 ± 0.17), higher percentage of SA‐β‐gal‐positive senescent cardiac area (%SA‐β‐gal 1.41 ± 0.59) and a lower reactivity degree (RD) for the survival marker Bmi1 [Abs (OD) 5000 ± 703] compared to female (%TdT+3.75 ± 0.35; %SA‐β‐gal 0.77 ± 0.02; Bmi1 [Abs (OD) 8567 ± 2173]. Proteomics analysis of cardiac tissue showed downstream activation of cell death in PON+siRNA scrambled compared to vehicle or PON+siRNA‐Notch1‐treated male mice. Upstream analysis showed beta‐oestradiol activation, and downstream analysis showed activation of cell survival and inhibition of cell death in PON+scrambled siRNA compared to vehicle or PON+siRNA‐Notch1‐treated female mice. PON+scrambled siRNA‐treated mice also had a downregulation of cardiac actin—more marked in males—and vessel density—more marked in females. Female hearts showed greater cardiac fibrosis than their male counterparts at baseline, with no significant change after PON treatment. PON+siRNA‐scrambled mice had less fibrosis than vehicle or PON+siRNA‐Notch1‐treated mice. The left ventricular systolic dysfunction showed by PON+scrambled siRNA‐treated mice (male %EF 28 ± 9; female %EF 36 ± 7) was reversed in both PON+siRNA‐Notch1‐treated male (%EF 53 ± 9) and female mice (%EF 52 ± 8). We report sex‐related differential susceptibility and Notch1 modulation in PON‐induced cardiotoxicity. This can help to identify biomarkers and potential mechanisms underlying sex‐related differences in PON‐induced cardiotoxicity.     

Androgen receptor suppresses vasculogenic mimicry in hepatocellular carcinoma via circRNA7/miRNA7‐5p/VE‐cadherin/Notch4 signalling

Androgen receptor (AR) can suppress hepatocellular carcinoma (HCC) invasion and metastasis at an advanced stage. Vasculogenic mimicry (VM), a new vascularization pattern by which tumour tissues nourish themselves, is correlated with tumour progression and metastasis. Here, we investigated the effect of AR on the formation of VM and its mechanism in HCC. The results suggested that AR could down‐regulate circular RNA (circRNA) 7, up‐regulate micro RNA (miRNA) 7‐5p, and suppress the formation of VM in HCC Small hairpin circR7 (ShcircR7) could reverse the impact on VM and expression of VE‐cadherin and Notch4 increased by small interfering AR (shAR) in HCC, while inhibition of miR‐7‐5p blocked the formation of VM and expression of VE‐cadherin and Notch4 decreased by AR overexpression (oeAR) in HCC. Mechanism dissection demonstrated that AR could directly target the circR7 host gene promoter to suppress circR7, and miR‐7‐5p might directly target the VE‐cadherin and Notch4 3′UTR to suppress their expression in HCC. In addition, knockdown of Notch4 and/or VE‐cadherin revealed that shVE‐cadherin or shNotch4 alone could partially reverse the formation of HCC VM, while shVE‐cadherin and shNotch4 together could completely suppress the formation of HCC VM. Those results indicate that AR could suppress the formation of HCC VM by down‐regulating circRNA7/miRNA7‐5p/VE‐Cadherin/Notch4 signals in HCC, which will help in the design of novel therapies against HCC.     

Notch signalling in healthy and diseased vasculature

Notch signalling is an evolutionarily highly conserved signalling mechanism governing differentiation and regulating homeostasis in many tissues. In this review, we discuss recent advances in our understanding of the roles that Notch signalling plays in the vasculature. We describe how Notch signalling regulates different steps during the genesis and remodelling of blood vessels (vasculogenesis and angiogenesis), including critical roles in assigning arterial and venous identities to the emerging blood vessels and regulation of their branching. We then proceed to discuss how experimental perturbation of Notch signalling in the vasculature later in development affects vascular homeostasis. In this review, we also describe how dysregulated Notch signalling, as a consequence of direct mutations of genes in the Notch pathway or aberrant Notch signalling output, contributes to various types of vascular disease, including CADASIL, Snedden syndrome and pulmonary arterial hypertension. Finally, we point out some of the current knowledge gaps and identify remaining challenges in understanding the role of Notch in the vasculature, which need to be addressed to pave the way for Notch-based therapies to cure or ameliorate vascular disease.     

USP15 promotes the apoptosis of degenerative nucleus pulposus cells by suppressing the PI3K/AKT signalling pathway

ObjectivesDegenerative disc disease is characterized by an enhanced breakdown of its existing nucleus pulposus (NP) matrix due to the dysregulation of matrix enzymes and factors. Ubiquitin‐specific protease 15 (USP15) is reported to be abnormal in certain human diseases. However, its role in NP degeneration remains unclear. Therefore, we aimed to explore the function of USP15 in degenerative NP cell specimens.MethodsWe induced gene silencing and overexpression of USP15 in degenerative NP cells using RNA interference (RNAi) and a lentiviral vector, respectively. qRT‐PCR and Western blotting were used to determine gene and protein expression levels. Cell apoptosis was analysed via flow cytometry. Protein interaction was examined by performing a co‐immunoprecipitation assay. Furthermore, the PI3K inhibitor LY294002 and agonist IGF‐1 were used to investigate the link between USP15 and AKT in NP degeneration.ResultsWe found that USP15 was up‐regulated in degenerative NP cells and that its overexpression accelerated the process of apoptosis. Moreover, USP15 expression levels negatively correlated with AKT phosphorylation in degenerative NP cells. Furthermore, targeting and silencing USP15 with miR‐338‐3p and studying its interaction with FK506‐binding protein 5 (FKBP5) revealed enhancement of FKBP5 ubiquitination, indicating that USP15 is a component of the FKBP5/AKT signalling pathway in degenerative NP cells.ConclusionsOur results show that USP15 exacerbates NP degradation by deubiquitinating and stabilizing FKBP5. This in turn results in the suppression of AKT phosphorylation in degenerative NP cells. Therefore, our study provides insights into the understanding of USP15 function as a potential molecule in the network of NP degeneration.     

Notch3 expression in capillary pericytes predicts worse graft outcome in human renal grafts with antibody‐mediated rejection

Microvasculature consisting of endothelial cells and pericytes is the main site of injury during antibody‐mediated rejection (ABMR) of renal grafts. Little is known about the mechanisms of activation of pericytes in this pathology. We have found recently that activation of Notch3, a mediator of vascular smooth muscle cell proliferation and dedifferentiation, promotes renal inflammation and fibrosis and aggravates progression of renal disease. Therefore, we studied the pericyte expression of Notch3 in 49 non‐selected renal graft biopsies (32 for clinical cause, 17 for graft surveillance). We analysed its relationship with patients’ clinical and morphological data, and compared with the expression of partial endothelial mesenchymal transition (pEndMT) markers, known to reflect endothelial activation during ABMR. Notch3 was de novo expressed in pericytes of grafts with ABMR, and was significantly correlated with the microcirculation inflammation scores of peritubular capillaritis and glomerulitis and with the expression of pEndMT markers. Notch3 expression was also associated with graft dysfunction and proteinuria at the time of biopsy and in the long term. Multivariate analysis confirmed pericyte expression of Notch3 as an independent risk factor predicting graft loss. These data suggest that Notch3 is activated in the pericytes of renal grafts with ABMR and is associated with poor graft outcome.     

Metformin attenuates trauma‐induced heterotopic ossification via inhibition of Bone Morphogenetic Protein signalling

AMP‐activated protein kinase (AMPK) is an intracellular sensor of energy homoeostasis that is activated under energy stress and suppressed in energy surplus. AMPK activation leads to inhibition of anabolic processes that consume ATP. Osteogenic differentiation is a process that highly demands ATP during which AMPK is inhibited. The bone morphogenetic proteins (BMPs) signalling pathway plays an essential role in osteogenic differentiation. The present study examines the inhibitory effect of metformin on BMP signalling, osteogenic differentiation and trauma‐induced heterotopic ossification. Our results showed that metformin inhibited Smad1/5 phosphorylation induced by BMP6 in osteoblast MC3T3‐E1 cells, concurrent with up‐regulation of Smad6, and this effect was attenuated by knockdown of Smad6. Furthermore, we found that metformin suppressed ALP activity and mineralization of the cells, an event that was attenuated by the dominant negative mutant of AMPK and mimicked by its constitutively active mutant. Finally, administration of metformin prevented the trauma‐induced heterotopic ossification in mice. In conjuncture, AMPK activity and Smad6 and Smurf1 expression were enhanced by metformin treatment in the muscle of injured area, concurrently with the reduction of ALK2. Collectively, our study suggests that metformin prevents heterotopic ossification via activation of AMPK and subsequent up‐regulation of Smad6. Therefore, metformin could be a potential therapeutic drug for heterotopic ossification induced by traumatic injury.     

Management of intrauterine adhesions using human amniotic mesenchymal stromal cells to promote endometrial regeneration and repair through Notch signalling

Intrauterine adhesions (IUAs) severely hamper women's reproductive functions. Human amniotic mesenchymal stromal cell (hAMSC) transplantation is effective in treating IUAs. Here, we examined the function of Notch signalling in IUA treatment with hAMSC transplantation. Forty-five Sprague-Dawley female rats were randomly divided into the sham operation, IUA, IUA + E2, IUA + hAMSCs and IUA + hAMSCs + E2 groups. After IUA induction in the rats, hAMSCs promoted endometrial regeneration and repair via differentiation into endometrial epithelial cells. In all groups, the expression of key proteins in Notch signalling was detected in the uterus by immunohistochemistry. The results indicated Notch signalling activation in the hAMSCs and hAMSCs + E2 groups. We could also induce hAMSC differentiation to generate endometrial epithelial cells in vitro. Furthermore, the inhibition of Notch signalling using the AdR-dnNotch1 vector suppressed hAMSC differentiation (assessed by epithelial and mesenchymal marker levels), whereas its activation using the AdR-Jagged1 vector increased differentiation. The above findings indicate Notch signalling mediates the differentiation of hAMSCs into endometrial epithelial cells, thus promoting endometrial regeneration and repair; Notch signalling could have an important function in IUA treatment.     

Unravelling the evolution of neural stem cells in arthropods: Notch signalling in neural stem cell development in the crustacean Daphnia magna

The genetic regulatory networks controlling major developmental processes seem to be conserved in bilaterians regardless of an independent or a common origin of the structures. This has been explained by the employment of a genetic toolkit that was repeatedly used during bilaterian evolution to build the various forms and body plans. However, it is not clear how genetic networks were incorporated into the formation of novel structures and how homologous genes can regulate the disparate morphological processes. Here we address this question by analysing the role of Notch signalling, which is part of the bilaterian toolkit, in neural stem cell evolution in arthropods. Within arthropods neural stem cells have evolved in the last common ancestor of insects and crustaceans (Tetraconata). We analyse here for the first time the role of Notch signalling in a crustacean, the branchiopod Daphnia magna, and show that it is required in neural stem cells for regulating the time of neural precursor production and for binary cell fate decisions in the ventral neuroectoderm. The function of Notch signalling has diverged in the ventral neuroectoderm of insects and crustaceans accompanied by changes in the morphogenetic processes. In the crustacean, Notch controlled mechanisms of neuroblast regulation have evolved that are surprisingly similar to vertebrates and thus present a remarkable case of parallel evolution. These new data on a representative of crustaceans complete the arthropod data set on Notch signalling in the nervous system and allow for reconstructing how the Notch signalling pathway has been co-opted from pre-existing structures to the development of the evolving neural stem cells in the Tetraconata ancestor.     

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.     

PTH‐induced EndMT via miR‐29a‐5p/GSAP/Notch1 pathway contributed to valvular calcification in rats with CKD

BackgroundEndothelial‐to‐mesenchymal transition (EndMT) is a common pathophysiology in valvular calcification (VC) among non‐chronic kidney disease (CKD) patients. However, few studies were investigated in CKD‐induced VC. Parathyroid hormone (PTH) was considered to be an important component of EndMT in CKD‐induced cardiovascular diseases. Therefore, determining whether PTH could induce valvular EndMT and elucidating corresponding mechanism involved further study.MethodsPerforming a 5/6 nephrectomy with a high phosphorus diet was done to construct VC models in rats with CKD. miRNA sequencing was used to ascertain changes in microRNA in human umbilical vein endothelial cells (HUVECs) intervened by PTH. VC was observed by Von Kossa staining and scanning electron microscope.ResultsPTH induced valvular EndMT in VC. Global microRNA expression profiling of HUVECs was examined in PTH versus the control in vitro, in which miR‐29a‐5p was most notably decreased and was resumed by PTHrP(7‐34) (PTH‐receptor1 inhibitor). Overexpression of miR‐29a‐5p could inhibit PTH‐induced EndMT in vitro and valvular EndMT in vivo. The dual‐luciferase assay verified that γ‐secretase‐activating protein (GASP) served as the target of miR‐29a‐5p. miR‐29a‐5p‐mimics, si‐GSAP and DAPT (γ‐secretase inhibitor) inhibited PTH‐induced γ‐secretase activation, thus blocking Notch1 pathway activation to inhibit EndMT in vitro. Moreover, Notch1 pathway activation was observed in VC. Blocking Notch1 pathway activation via AAV‐miR‐29a and DAPT inhibited valvular EndMT. In addition, blocking Notch1 pathway activation was also shown to alleviate VC.ConclusionPTH activates valvular EndMT via miR‐29a‐5p/GSAP/Notch1 pathway, which can contribute to VC in CKD rats.     

Oestradiol promotes the intrahepatic bile duct development of C57BL/6CrSlc mice during embryonic period via Notch signalling pathway

Oestradiol (E2) is a critical factor for multiple systems' development during the embryonic period. Here, we aimed to investigate the effects of oestradiol on intrahepatic bile duct development, which may allow a better understanding of congenital bile duct dysplasia. DLK⁺ hepatoblasts were extracted from the C57BL/6CrSlc foetal mice and randomly divided into control group, oestradiol groups (1, 10, 100 nM) and oestradiol (10 nM) + DAPT (inhibitor of Notch signalling; 40 µM) group for in vitro experiments. For in vivo analysis, pregnant mice were divided into control group, oestradiol (intraperitoneal injection of 0.6 mg/kg/day) ± DAPT (subcutaneous injection of 10 mg/kg/day) groups and tamoxifen (gavage administration of 0.4 mg/kg/day) group. The results showed that oestradiol promoted hepatoblast differentiation into cholangiocytes and intrahepatic bile duct development during the embryonic period. Tamoxifen, an antioestrogenic drug, inhibited the above processes. Moreover, oestradiol promoted the expression of Notch signalling pathway-associated proteins and genes both in vitro and in vivo. Notably, DAPT addition inhibited the oestradiol-mediated effects. In conclusion, oestradiol can promote hepatoblast differentiation into cholangiocytes and intrahepatic bile duct development of C57BL/6CrSlc mice during embryonic period via the Notch signalling pathway.     

SHED aggregate exosomes shuttled miR‐26a promote angiogenesis in pulp regeneration via TGF‐β/SMAD2/3 signalling

ObjectivesPulp regeneration brings big challenges for clinicians, and vascularization is considered as its determining factor. We previously accomplished pulp regeneration with autologous stem cells from deciduous teeth (SHED) aggregates implantation in teenager patients, however, the underlying mechanism needs to be clarified for regenerating pulp in adults. Serving as an important effector of mesenchymal stem cells (MSCs), exosomes have been reported to promote angiogenesis and tissue regeneration effectively. Here, we aimed to investigate the role of SHED aggregate‐derived exosomes (SA‐Exo) in the angiogenesis of pulp regeneration.Materials and MethodsWe extracted exosomes from SHED aggregates and utilized them in the pulp regeneration animal model. The pro‐angiogenetic effects of SA‐Exo on SHED and human umbilical vein endothelial cells (HUVECs) were evaluated. The related mechanisms were further investigated.ResultsWe firstly found that SA‐Exo significantly improved pulp tissue regeneration and angiogenesis in vivo. Next, we found that SA‐Exo promoted SHED endothelial differentiation and enhanced the angiogenic ability of HUVECs, as indicated by the in vitro tube formation assay. Mechanistically, miR‐26a, which is enriched in SA‐Exo, improved angiogenesis both in SHED and HUVECs via regulating TGF‐β/SMAD2/3 signalling.ConclusionsIn summary, these data reveal that SA‐Exo shuttled miR‐26a promotes angiogenesis via TGF‐β/SMAD2/3 signalling contributing to SHED aggregate‐based pulp tissue regeneration. These novel insights into SA‐Exo may facilitate the development of new strategies for pulp regeneration.     

Larotrectinib induces autophagic cell death through AMPK/mTOR signalling in colon cancer

Larotrectinib (Lar) is a highly selective and potent small‐molecule inhibitor used in patients with tropomyosin receptor kinase (TRK) fusion‐positive cancers, including colon cancer. However, the underlying molecular mechanisms specifically in patients with colon cancer have not yet been explored. Our data showed that Lar significantly suppressed proliferation and migration of colon cancer cells. In addition, Lar suppressed the epithelial–mesenchymal transition (EMT) process, as evidenced by elevation in E‐cadherin (E‐cad), and downregulation of vimentin and matrix metalloproteinase (MMP) 2/9 expression. Furthermore, Lar was found to activate autophagic flux, in which Lar increased the ratio between LC3II/LC3I and decreased the expression of p62 in colon cancer cells. More importantly, Lar also increased AMPK phosphorylation and suppressed mTOR phosphorylation in colon cancer cells. However, when we silenced AMPK in colon cancer cells, Lar‐induced accumulation of autolysomes as well as Lar‐induced suppression of the EMT process were significantly diminished. An in vivo assay also confirmed that tumour volume and weight decreased in Lar‐treated mice than in control mice. Taken together, this study suggests that Lar significantly suppresses colon cancer proliferation and migration by activating AMPK/mTOR‐mediated autophagic cell death.