The Landscape of microRNAs in βCell: Between Phenotype Maintenance and Protection

Diabetes mellitus is a group of heterogeneous metabolic disorders characterized by chronic hyperglycaemia mainly due to pancreatic β cell death and/or dysfunction, caused by several types of stress such as glucotoxicity, lipotoxicity and inflammation. Different patho-physiological mechanisms driving β cell response to these stresses are tightly regulated by microRNAs (miRNAs), a class of negative regulators of gene expression, involved in pathogenic mechanisms occurring in diabetes and in its complications. In this review, we aim to shed light on the most important miRNAs regulating the maintenance and the robustness of β cell identity, as well as on those miRNAs involved in the pathogenesis of the two main forms of diabetes mellitus, i.e., type 1 and type 2 diabetes. Additionally, we acknowledge that the understanding of miRNAs-regulated molecular mechanisms is fundamental in order to develop specific and effective strategies based on miRNAs as therapeutic targets, employing innovative molecules.     

Skin Resident γδ T Cell Function and Regulation in Wound Repair

The skin is a critical barrier that protects against damage and infection. Within the epidermis and dermis reside γδ T cells that play a variety of key roles in wound healing and tissue homeostasis. Skin-resident γδ T cells require T cell receptor (TCR) ligation, costimulation, and cytokine reception to mediate keratinocyte activity and inflammatory responses at the wound site for proper wound repair. While both epidermal and dermal γδ T cells regulate inflammatory responses in wound healing, the timing and factors produced are distinct. In the absence of growth factors, cytokines, and chemokines produced by γδ T cells, wound repair is negatively impacted. This disruption in γδ T cell function is apparent in metabolic diseases such as obesity and type 2 diabetes. This review provides the current state of knowledge on skin γδ T cell activation, regulation, and function in skin homeostasis and repair in mice and humans. As we uncover more about the complex roles played by γδ T cells in wound healing, novel targets can be discovered for future clinical therapies.     

CHIR99021 Augmented the Function of Late Endothelial Progenitor Cells by Preventing Replicative Senescence

Endothelial progenitor cells (EPCs) are specialized cells in circulating blood, well known for their ability to form new vascular structures. Aging and various ailments such as diabetes, atherosclerosis and cardiovascular disease make EPCs vulnerable to decreasing in number, which affects their migration, proliferation and angiogenesis. Myocardial ischemia is also linked to a reduced number of EPCs and their endothelial functional role, which hinders proper blood circulation to the myocardium. The current study shows that an aminopyrimidine derivative compound (CHIR99021) induces the inhibition of GSK-3β in cultured late EPCs. GSK-3β inhibition subsequently inhibits mTOR by blocking the phosphorylation of TSC2 and lysosomal localization of mTOR. Furthermore, suppression of GSK-3β activity considerably increased lysosomal activation and autophagy. The activation of lysosomes and autophagy by GSK-3β inhibition not only prevented replicative senescence of the late EPCs but also directed their migration, proliferation and angiogenesis. To conclude, our results demonstrate that lysosome activation and autophagy play a crucial role in blocking the replicative senescence of EPCs and in increasing their endothelial function. Thus, the findings provide an insight towards the treatment of ischemia-associated cardiovascular diseases based on the role of late EPCs.     

Effect of Hypoxia on Pulmonary Endothelial Cells from Bleomycin-Induced Pulmonary Fibrosis Model Mice

Pulmonary fibrosis is a progressive and fatal disorder characterized by dysregulated repair after recurrent injury. Destruction of the lung architecture with excess extracellular matrix deposition induces respiratory failure with hypoxia and progressive dyspnea. The impact of hypoxia on pulmonary endothelial cells during pulmonary fibrogenesis is unclear. Using a magnetic-activated cell sorting system, pulmonary endothelial cells were isolated from a mouse model of pulmonary fibrosis induced by intratracheally administered bleomycin. When endothelial cells were exposed to hypoxic conditions, a hypoxia-inducible factor (HIF)-2α protein was detected in CD31- and α-smooth muscle actin (SMA)-positive cells. Levels of plasminogen activator inhibitor 1, von Willebrand factor, and matrix metalloproteinase 12 were increased in endothelial cells isolated from bleomycin-treated mice exposed to hypoxic conditions. When endothelial cells were cultured under hypoxic conditions, levels of fibrotic mediators, transforming growth factor-β and connective tissue growth factor, were elevated only in endothelial cells from bleomycin-treated and not from saline-treated lungs. The increased expression of α-SMA and mesenchymal markers and collagen production in bleomycin- or hypoxia-stimulated endothelial cells were further elevated in endothelial cells from bleomycin-treated mouse lungs cultured under hypoxic conditions. Exposure to hypoxia damaged endothelial cells and enhanced fibrogenesis-related damage in bleomycin-treated pulmonary endothelial cells.     

Context Dependent Sulf1/Sulf2 Functional Divergence in Endothelial Cell Activity

Signalling activities are tightly regulated to control cellular responses. Heparan sulfate proteoglycans (HSPGs) at the cell membrane and extracellular matrix regulate ligand availability and interaction with a range of key receptors. SULF1 and SULF2 enzymes modify HSPG sulfation by removing 6-O sulfates to regulate cell signalling but are considered functionally identical. Our in vitro mRNA and protein analyses of two diverse human endothelial cell lines, however, highlight their markedly distinct regulatory roles of maintaining specific HSPG sulfation patterns through feedback regulation of HS 6-O transferase (HS6ST) activities and highly divergent roles in vascular endothelial growth factor (VEGF) and Transforming growth factor β (TGFβ) cell signalling activities. Unlike Sulf2, Sulf1 over-expression in dermal microvascular HMec1 cells promotes TGFβ and VEGF cell signalling by simultaneously upregulating HS6ST1 activity. In contrast, Sulf1 over-expression in venous ea926 cells has the opposite effect as it attenuates both TGFβ and VEGF signalling while Sulf2 over-expression maintains the control phenotype. Exposure of these cells to VEGF-A, TGFβ1, and their inhibitors further highlights their endothelial cell type-specific responses and integral growth factor interactions to regulate cell signalling and selective feedback regulation of HSPG sulfation that additionally exploits alternative Sulf2 RNA-splicing to regulate net VEGF-A and TGFβ cell signalling activities.     

Cerebral Pericytes and Endothelial Cells Communicate through Inflammasome-Dependent Signals

By upregulation of cell adhesion molecules and secretion of proinflammatory cytokines, cells of the neurovascular unit, including pericytes and endothelial cells, actively participate in neuroinflammatory reactions. As previously shown, both cell types can activate inflammasomes, cerebral endothelial cells (CECs) through the canonical pathway, while pericytes only through the noncanonical pathway. Using complex in vitro models, we demonstrate here that the noncanonical inflammasome pathway can be induced in CECs as well, leading to a further increase in the secretion of active interleukin-1β over that observed in response to activation of the canonical pathway. In parallel, a more pronounced disruption of tight junctions takes place. We also show that CECs respond to inflammatory stimuli coming from both the apical/blood and the basolateral/brain directions. As a result, CECs can detect factors secreted by pericytes in which the noncanonical inflammasome pathway is activated and respond with inflammatory activation and impairment of the barrier properties. In addition, upon sensing inflammatory signals, CECs release inflammatory factors toward both the blood and the brain sides. Consequently, CECs activate pericytes by upregulating their expression of NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), an inflammasome-forming pattern recognition receptor. In conclusion, cerebral pericytes and endothelial cells mutually activate each other in inflammation.     

Crosstalk between β2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether β-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of β-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a β-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a β2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the β2-AR stimulation. We conclude that the crosstalk between the β2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy.     

Synthesis of Reactive Sulfur Species in Cultured Vascular Endothelial Cells after Exposure to TGF-β1: Induction of Cystathionine γ-Lyase and Cystathionine β-Synthase Expression Mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 Pathways

Transforming growth factor-β₁ (TGF-β₁) occurs at high levels at damage sites of vascular endothelial cell layers and regulates the functions of vascular endothelial cells. Reactive sulfur species (RSS), such as cysteine persulfide, glutathione persulfide, and hydrogen persulfide, are cytoprotective factors against electrophiles such as reactive oxygen species and heavy metals. Previously, we reported that sodium trisulfide, a sulfane sulfur donor, promotes vascular endothelial cell proliferation. The objective of the present study was to clarify the regulation and significance of RSS synthesis in vascular endothelial cells after exposure to TGF-β₁. Bovine aortic endothelial cells in a culture system were treated with TGF-β₁ to assess the expression of intracellular RSS, the effect of RSS on cell proliferation in the presence of TGF-β₁, induction of RSS-producing enzymes by TGF-β₁, and intracellular signal pathways that mediate this induction. The results suggest that TGF-β₁ increased intracellular RSS levels to modulate its inhibitory effect on proliferation. The increased production of RSS, probably high-molecular-mass RSS, was due to the induction of cystathionine γ-lyase and cystathionine β-synthase, which are RSS-producing enzymes, and the induction was mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 pathways in vascular endothelial cells. TGF-β₁ regulates vascular endothelial cell functions such as proliferation and fibrinolytic activity; intracellular high-molecular-mass RSS, which are increased by TGF-β₁, may modulate the regulation activity in vascular endothelial cells.     

Conventional and Unconventional Mechanisms by which Exocytosis Proteins Oversee β-cell Function and Protection

Type 2 diabetes (T2D) is one of the prominent causes of morbidity and mortality in the United States and beyond, reaching global pandemic proportions. One hallmark of T2D is dysfunctional glucose-stimulated insulin secretion from the pancreatic β-cell. Insulin is secreted via the recruitment of insulin secretory granules to the plasma membrane, where the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and SNARE regulators work together to dock the secretory granules and release insulin into the circulation. SNARE proteins and their regulators include the Syntaxins, SNAPs, Sec1/Munc18, VAMPs, and double C2-domain proteins. Recent studies using genomics, proteomics, and biochemical approaches have linked deficiencies of exocytosis proteins with the onset and progression of T2D. Promising results are also emerging wherein restoration or enhancement of certain exocytosis proteins to β-cells improves whole-body glucose homeostasis, enhances β-cell function, and surprisingly, protection of β-cell mass. Intriguingly, overexpression and knockout studies have revealed novel functions of certain exocytosis proteins, like Syntaxin 4, suggesting that exocytosis proteins can impact a variety of pathways, including inflammatory signaling and aging. In this review, we present the conventional and unconventional functions of β-cell exocytosis proteins in normal physiology and T2D and describe how these insights might improve clinical care for T2D.     

Treadmill Exercise Reduces Neuroinflammation,Glial Cell Activation and Improves Synaptic Transmission in the Prefrontal Cortex in 3 × Tg-AD Mice

Physical exercise improves memory and cognition in physiological aging and Alzheimer’s disease (AD), but the mechanisms remain poorly understood. Here, we test the hypothesis that Aβ oligomer accumulation, neuroinflammation, and glial cell activation may lead to disruption of synaptic transmission in the prefrontal cortex of 3 × Tg-AD Mice, resulting in impairment of learning and memory. On the other hand, treadmill exercise could prevent the pathogenesis and exert neuroprotective effects. Here, we used immunohistochemistry, western blotting, enzyme-linked immunosorbent assay, and slice electrophysiology to analyze the levels of GSK3β, Aβ oligomers (Aβ dimers and trimers), pro-inflammatory cytokines (IL-1β, IL-6, and TNFα), the phosphorylation of CRMP2 at Thr514, and synaptic currents in pyramidal neurons in the prefrontal cortex. We show that 12-week treadmill exercise beginning in three-month-old mice led to the inhibition of GSK3β kinase activity, decreases in the levels of Aβ oligomers, pro-inflammatory cytokines (IL-1β, IL-6, and TNFα), and the phosphorylation of CRMP2 at Thr514, reduction of microglial and astrocyte activation, and improvement of excitatory and inhibitory synaptic transmission of pyramidal neurons in the prefrontal cortex of 3 × Tg-AD Mice. Thus, treadmill exercise reduces neuroinflammation, glial cell activation and improves synaptic transmission in the prefrontal cortex in 3 × Tg-AD mice, possibly related to the inhibition of GSK3β kinase activity.     

Coordinated Regulation of Mesenchymal Stem Cell Migration by Various Chemotactic Stimuli

Endogenous bone marrow-derived mesenchymal stem cells are mobilized to peripheral blood and injured tissues in response to changes in the expression of various growth factors and cytokines in the injured tissues, including substance P (SP), transforming growth factor-beta (TGF-β), and stromal cell-derived factor-1 (SDF-1). SP, TGF-β, and SDF-1 are all known to induce the migration of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it is not yet clear how these stimuli influence or interact with each other during BM-MSC mobilization. This study used mouse bone marrow-derived mesenchymal stem cell-like ST2 cells and human BM-MSCs to evaluate whether SP, TGF-β, and SDF-1 mutually regulate their respective effects on the mobilization of BM-MSCs. SP pretreatment of ST2 and BM-MSCs impaired their response to TGF-β while the introduction of SP receptor antagonist restored the mobilization of ST2 and BM-MSCs in response to TGF-β. TGF-β pretreatment did not affect the migration of ST2 and BM-MSCs in response to SP, but downregulated their migration in response to SDF-1. SP pretreatment modulated the activation of TGF-β noncanonical pathways in ST2 cells and BM-MSCs, but not canonical pathways. These results suggest that the migration of mesenchymal stem cells is regulated by complex functional interactions between SP, TGF-β, and SDF-1. Thus, understanding the complex functional interactions of these chemotactic stimuli would contribute to ensuring the development of safe and effective combination treatments for the mobilization of BM-MSCs.     

Cytoplasmic p53β Isoforms Are Associated with Worse Disease-Free Survival in Breast Cancer

TP53 mutations are associated with tumour progression, resistance to therapy and poor prognosis. However, in breast cancer, TP53′s overall mutation frequency is lower than expected (~25%), suggesting that other mechanisms may be responsible for the disruption of this critical tumour suppressor. p53 isoforms are known to enhance or disrupt p53 pathway activity in cell- and context-specific manners. Our previous study revealed that p53 isoform mRNA expression correlates with clinicopathological features and survival in breast cancer and may account for the dysregulation of the p53 pathway in the absence of TP53 mutations. Hence, in this study, the protein expression of p53 isoforms, transactivation domain p53 (TAp53), p53β, Δ40p53, Δ133p53 and Δ160p53 was analysed using immunohistochemistry in a cohort of invasive ductal carcinomas (n = 108). p53 isoforms presented distinct cellular localisation, with some isoforms being expressed in tumour cells and others in infiltrating immune cells. Moreover, high levels of p53β, most likely to be N-terminally truncated β variants, were significantly associated with worse disease-free survival, especially in tumours with wild-type TP53. To the best of our knowledge, this is the first study that analysed the endogenous protein levels of p53 isoforms in a breast cancer cohort. Our findings suggest that p53β may be a useful prognostic marker.     

The Role of β-Arrestins in Regulating Stem Cell Phenotypes in Normal and Tumorigenic Cells

β-Arrestins (ARRBs) are ubiquitously expressed scaffold proteins that mediate inactivation of G-protein-coupled receptor signaling, and in certain circumstances, G-protein independent pathways. Intriguingly, the two known ARRBs, β-arrestin1 (ARRB1) and β-Arrestin2 (ARRB2), seem to have opposing functions in regulating signaling cascades in several models in health and disease. Recent evidence suggests that ARRBs are implicated in regulating stem cell maintenance; however, their role, although crucial, is complex, and there is no universal model for ARRB-mediated regulation of stem cell characteristics. For the first time, this review compiles information on the function of ARRBs in stem cell biology and will discuss the role of ARRBs in regulating cell signaling pathways implicated in stem cell maintenance in normal and malignant stem cell populations. Although promising targets for cancer therapy, the ubiquitous nature of ARRBs and the plethora of functions in normal cell biology brings challenges for treatment selectivity. However, recent studies show promising evidence for specifically targeting ARRBs in myeloproliferative neoplasms.     

Mycophenolate Antagonizes IFN-γ-Induced Catagen-Like Changes via β-Catenin Activation in Human Dermal Papilla Cells and Hair Follicles

Recently, various immunosuppressant drugs have been shown to induce hair growth in normal hair as well as in alopecia areata and androgenic alopecia; however, the responsible mechanism has not yet been fully elucidated. In this study, we investigate the influence of mycophenolate (MPA), an immunosuppressant, on the proliferation of human dermal papilla cells (hDPCs) and on the growth of human hair follicles following catagen induction with interferon (IFN)-γ. IFN-γ was found to reduce β-catenin, an activator of hair follicle growth, and activate glycogen synthase kinase (GSK)-3β, and enhance expression of the Wnt inhibitor DKK-1 and catagen inducer transforming growth factor (TGF)-β2. IFN-γ inhibited expression of ALP and other dermal papillar cells (DPCs) markers such as Axin2, IGF-1, and FGF 7 and 10. MPA increased β-catenin in IFN-γ-treated hDPCs leading to its nuclear accumulation via inhibition of GSK3β and reduction of DKK-1. Furthermore, MPA significantly increased expression of ALP and other DPC marker genes but inhibited expression of TGF-β2. Therefore, we demonstrate for the first time that IFN-γ induces catagen-like changes in hDPCs and in hair follicles via inhibition of Wnt/β-catenin signaling, and that MPA stabilizes β-catenin by inhibiting GSK3β leading to increased β-catenin target gene and DP signature gene expression, which may, in part, counteract IFN-γ-induced catagen in hDPCs.     

Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid β-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal β-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid β-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid β-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.     

Hsp90 Maintains the Stability and Function of the Tau Phosphorylating Kinase GSK3β

Hyperphosphorylation of tau leading to aggregated tau and tangle formation is a common pathological feature of tauopathies, including Alzheimer’s disease. Abnormal phosphorylation of tau by kinases, in particular GSK3β, has been proposed as a pathogenic mechanism in these diseases. In this study we demonstrate that the heat shock protein 90 (Hsp90) maintains the stability and function of the GSK3β. By using both rat primary cortical neurons and COS-7 cells, we show that Hsp90 inhibitors lead to a reduction of the protein level of GSK3β, and that this effect is associated with both a decrease in tau phosphorylation at putative GSK3β sites and an induction in heat shock protein 70 (Hsp70) levels. We further show that Hsp90 associates with the GSK3β regulating its stability and function and preventing its degradation by the proteasome.     

β3-Adrenoreceptors as ROS Balancer in Hematopoietic Stem Cell Transplantation

In the last decades, the therapeutic potential of hematopoietic stem cell transplantation (HSCT) has acquired a primary role in the management of a broad spectrum of diseases including cancer, hematologic conditions, immune system dysregulations, and inborn errors of metabolism. The different types of HSCT, autologous and allogeneic, include risks of severe complications including acute and chronic graft-versus-host disease (GvHD) complications, hepatic veno-occlusive disease, lung injury, and infections. Despite being a dangerous procedure, it improved patient survival. Hence, its use was extended to treat autoimmune diseases, metabolic disorders, malignant infantile disorders, and hereditary skeletal dysplasia. HSCT is performed to restore or treat various congenital conditions in which immunologic functions are compromised, for instance, by chemo- and radiotherapy, and involves the administration of hematopoietic stem cells (HSCs) in patients with depleted or dysfunctional bone marrow (BM). Since HSCs biology is tightly regulated by oxidative stress (OS), the control of reactive oxygen species (ROS) levels is important to maintain their self-renewal capacity. In quiescent HSCs, low ROS levels are essential for stemness maintenance; however, physiological ROS levels promote HSC proliferation and differentiation. High ROS levels are mainly involved in short-term repopulation, whereas low ROS levels are associated with long-term repopulating ability. In this review, we aim summarize the current state of knowledge about the role of β3-adrenoreceptors (β3-ARs) in regulating HSCs redox homeostasis. β3-ARs play a major role in regulating stromal cell differentiation, and the antagonist SR59230A promotes differentiation of different progenitor cells in hematopoietic tumors, suggesting that β3-ARs agonism and antagonism could be exploited for clinical benefit.