Peripheral 5-HT/HTR6 axis is responsible for obesity-associated hypertension.

Hypertension is one of the major life-threatening complications of obesity. Recently adipose multipotent mesenchymal stromal cells (MSCs) were implicated to the pathogenesis of obesity-associated hypertension. These cells amplify noradrenaline-induced vascular cell contraction via cAMP-mediated signaling pathway. In this study we tested the ability of several cAMP-mediated hormones to affect the adrenergic sensitivity of MSCs and their associated contractility. Despite that adipose MSCs express a plethora of receptors capable of cAMP signaling activation, only 5-HT was able to elevate α1A-adrenoceptor-induced Ca2+ signaling in MSCs. Furthermore, 5-HT markedly enhanced noradrenaline-induced MSCs contractility. Using HTR isoform-specific antagonists followed by CRISPRi-mediated knockdown, we identified that the observed 5-HT effect on MSCs was mediated by the HTR6 isoform. This receptor was previously associated exclusively with 5-HT central nervous system activity. Discovered effect of HTR6 on MSCs contractility points to it as a potential therapeutic target for the prevention and treatment of obesity-associated hypertension.

Pharmacological and Genetic Suppression of VDAC1 Alleviates the Development of Mitochondrial Dysfunction in Endothelial and Fibroblast Cell Cultures upon Hyperglycemic Conditions.

Prolonged hyperglycemia related to diabetes and its complications leads to multiple cellular disorders, the central one being the dysfunction of mitochondria. Voltage-dependent anion channels (VDAC) of the outer mitochondrial membrane control the metabolic, ionic, and energy cross-talk between mitochondria and the rest of the cell and serve as the master regulators of mitochondrial functions. Here, we have investigated the effect of pharmacological suppression of VDAC1 by the newly developed inhibitor of its oligomerization, VBIT-4, in the primary culture of mouse lung endotheliocytes and downregulated expression of VDAC1 in human skin fibroblasts on the progression of mitochondrial dysfunction upon hyperglycemic stress. The cells were grown in high-glucose media (30 mM) for 36 h. In response to hyperglycemia, the mRNA level of VDAC1 increased in endotheliocytes and decreased in human skin fibroblasts. Hyperglycemia induced overproduction of mitochondrial ROS, an increase in the susceptibility of the organelles to mitochondrial permeability transition (MPT) pore opening and a drop in mitochondrial membrane potential, which was accompanied by a decrease in cell viability in both cultures. Treatment of endotheliocytes with 5 µM VBIT-4 abolished the hyperglycemia-induced increase in susceptibility to spontaneous opening of the MPT pore and ROS generation in mitochondria. Silencing of VDAC1 expression in human skin fibroblasts exposed to high glucose led to a less pronounced manifestation of all the signs of damage to mitochondria. Our data identify a mitochondria-related response to pharmacological and genetic suppression of VDAC activity in vascular cells in hyperglycemia and suggest the potential therapeutic value of targeting these channels for the treatment of diabetic vasculopathies.

Comparative Study of Cytotoxic and Membranotropic Properties of Betulinic Acid-F16 Conjugate on Breast Adenocarcinoma Cells (MCF-7) and Primary Human Fibroblasts.

The present study evaluates the cytotoxicity of a previously synthesized conjugate of betulinic acid (BA) with the penetrating cation F16 on breast adenocarcinoma (MCF-7) and human fibroblast (HF) cell lines, and also shows the mechanism underlying its membranotropic action. It was confirmed that the conjugate exhibits higher cytotoxicity compared to native BA at low doses also blocking the proliferation of both cell lines and causing cell cycle arrest in the G0/G1 phase. We show that the conjugate indeed has a high potential for accumulation in mitochondria, being visualized in these organelles, which is most pronounced in cancer cells. The effect of the conjugate was observed to be accompanied by ROS hyperproduction in both cancerous and healthy cells, despite the lower base level of ROS in the latter. Along with this, using artificial liposomes, we determined that the conjugate is able to influence the phase state of lipid membranes, make them more fluid, and induce nonspecific permeabilization contributing to the overall cytotoxicity of the tested agent. We conclude that the studied BA-F16 conjugate does not have significant selective cytotoxicity, at least against the studied breast cancer cell line MCF-7.

Identification of a Novel Small RNA Encoded in the Mouse Urokinase Receptor uPAR Gene (Plaur) and Its Molecular Target Mef2d.

Urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored receptor of urokinase (uPA), which is involved in brain development, nerve regeneration, wound healing and tissue remodeling. We have recently shown that Plaur, which encodes uPAR, is an early response gene in murine brain. Assumingly, diverse functions of Plaur might be attributed to hypothetical, unidentified microRNAs encoded within introns of the Plaur gene. Using a bioinformatic approach we identified novel small RNAs within the Plaur gene and named them Plaur-miR1-3p and Plaur-miR1-5p. We confirmed Plaur-dependent expression of Plaur-miR1-3p and Plaur-miR1-5p in the mouse brain and mouse neuroblastoma Neuro2a cells. Utilizing an in silico MR-microT algorithm in DianaTools we selected two target genes - Mef2d and Emx2 with the highest binding scores to small RNAs selected from identified Plaur-Pre-miR1. Furthermore, sequencing of mouse brain samples for Plaur-miR1-5p target genes revealed two more genes-Nrip3 and Snrnp200. The expression of Emx2, Mef2d, and Snrnp200 in the mouse brain and Mef2d and Snrnp200 in Neuro2a cells correlated with expression of Plaur and small RNAs-Plaur-miR1-3p and Plaur-miR1-5p. Finally, we demonstrated elevated MEF2D protein expression in the mouse brain after Plaur induction and displayed activating effects of Plaur-miR1-5p on Mef2d expression in Neuro2a cells using Luciferase reporter assay. In conclusion, we have identified Plaur-miR1-3p and Plaur-miR1-5p as novel small RNAs encoded in the Plaur gene. This finding expands the current understanding of Plaur function in brain development and functioning.

Impact of p53 knockdown on protein dataset of HaCaT cells.

The HaCaT line of immortalized non-tumor cells is a popular model of keratinocytes used for dermatological studies, in the practice of toxicological tests, and in the study of skin allergic reactions. These cells maintain a stable keratinocyte phenotype, do not require specific growth factors during cultivation, and respond to keratinocyte differentiation stimuli. HaCaT cells bear two mutant p53 alleles - R282Q and H179Y. At least two mechanisms of GOF (gain-of-function) of mutant p53 are known: it affects functions of p63/p73 by inhibiting their binding to DNA; or it binds to new DNA sites by interacting with other transcription factors (NF-Y, E2F1, NF-KB, VDR, p63). Proteins of the P53 family play an important role in the regulation of proliferation and differentiation processes of human keratinocytes. Proteomic study of HaCaT cells with TP53 gene knockdown provides new data for understanding the limitations of HaCaT cells when using them as an experimental model of normal human keratinocytes. In this article we present datasets obtained through the high-throughput shotgun proteomics analysis of human immortalized HaCaT keratinocytes and p53 knockdown HaCaT keratinocytes. As a protocol for proteomic profiling of cells, we used the approach of obtaining LC-MS/MS measurements followed by their processing with MaxQuant software (version 1.6.3.4). The "RAW" files were deposited to the ProteomeXchange with identifier PXD033538.

Urokinase Receptor uPAR Downregulation in Neuroblastoma Leads to Dormancy, Chemoresistance and Metastasis.

uPAR is a membrane receptor that binds extracellular protease urokinase, contributes to matrix remodeling and plays a crucial role in cellular adhesion, proliferation, survival, and migration. uPAR overexpression in tumor cells promotes mitogenesis, opening a prospective avenue for targeted therapy. However, uPAR targeting in cancer has potential risks. We have recently shown that uPAR downregulation in neuroblastoma promotes epithelial-mesenchymal transition (EMT), potentially associated with metastasis and chemoresistance. We used data mining to evaluate the role of uPAR expression in primary and relapsed human neuroblastomas. To model the decreased uPAR expression, we targeted uPAR using CRISPR/Cas9 and shRNA in neuroblastoma Neuro2a cells and evaluated their chemosensitivity in vitro as well as tumor growth and metastasis in vivo. We demonstrate that the initially high PLAUR expression predicts poor survival in human neuroblastoma. However, relapsed neuroblastomas have a significantly decreased PLAUR expression. uPAR targeting in neuroblastoma Neuro2a cells leads to p38 activation and an increased p21 expression (suggesting a dormant phenotype). The dormancy in neuroblastoma cells can be triggered by the disruption of uPAR-integrin interaction. uPAR-deficient cells are less sensitive to cisplatin and doxorubicin treatment and exhibit lower p53 activation. Finally, low uPAR-expressing Neuro2a cells formed smaller primary tumors, but more frequent metastasis in mice. To the best of our knowledge, this is the first study revealing the pathological role of dormant uPAR-deficient cancer cells having a chemoresistant and motile phenotype.

Hematopoietically-expressed homeobox protein HHEX regulates adipogenesis in preadipocytes.

Obesity is a key health problem and is associated with a high risk of type 2 diabetes and other metabolic diseases. Increased weight as well as dysregulation of adipocyte homeostasis are the main drivers of obesity. Pathological adipogenesis plays a central role in obesity-related complications such as type 2 diabetes, hypertension and others. Thus, an understanding of the molecular mechanisms involved in physiological and pathogenic adipogenesis can help to develop new strategies to prevent or cure obesity and related diseases. Previously, genetic polymorphisms in the HHEX gene that encodes the homeobox transcription factor HEX (PRH) were found to be associated with type 2 diabetes and high body mass index at birth by GWAS in distinct human populations. To understand whether HHEX has a regulatory function in adipogenesis, we performed RNAi-mediated knockdown of Hhex in preadipocyte cell line 3T3-L1 in vitro, and studied changes in the efficacy of adipogenesis. We found that Hhex knockdown blocks adipogenesis in preadipocytes in a dose-dependent manner and leads to a significant decrease of PPAR-gamma protein - the main regulator of adipogenesis. We also propose that Hhex can play an important role in adipocyte differentiation by affecting the level of the PPAR-gamma protein. Our study supports the claim that Hhex plays an important role in adipocyte differentiation program and can contribute to fat tissue homeostasis.

Functional Heterogeneity of Protein Kinase A Activation in Multipotent Stromal Cells.

Multipotent stromal cells (MSC) demonstrate remarkable functional heterogeneity; however, its molecular mechanisms remain largely obscure. In this study, we explored MSC response to hormones, which activate Gs-protein / cyclic AMP (cAMP) / protein kinase A (PKA) dependent signaling, at the single cell level using genetically encoded biosensor PKA-Spark. For the first time, we demonstrated that about half of cultured MSCs are not able to activate the cAMP/PKA pathway, possibly due to the limited availability of adenylyl cyclases. Using this approach, we showed that MSC subpopulations responding to various hormones largely overlapped, and the share of responding cells did not exceed 40%. Using clonal analysis, we showed that signaling heterogeneity of MSC could be formed de novo within 2 weeks.

Downregulation of uPAR promotes urokinase translocation into the nucleus and epithelial to mesenchymal transition in neuroblastoma.

The urokinase system is involved in a variety of physiological processes, such as fibrinolysis, matrix remodeling, wound healing, and regeneration. Upon binding to its cognate receptor urokinase-type plasminogen activator receptor (uPAR), urokinase-type plasminogen activator (uPA) catalyzes the conversion of plasminogen to plasmin and the activation of matrix metalloproteases. Apart from this, uPA-uPAR interaction can lead to the activation of transcription factors, mitogen-activated protein kinase signaling pathways and RTK cascades. Elevated expression of uPA and uPAR is markedly associated with cancer progression and metastasis and correlates with a poor prognosis in clinics. Targeting the urokinase system has proved to be effective in experimental models in vitro and in vivo, however, in clinics the inhibition of the uPA/uPAR system has fallen short of expectations, suggesting that the question of the functional relevance of uPA/uPAR system is far from being moot. Recently, using CRISPR/Cas9 technology, we have shown that uPAR knockout decreases the proliferation of neuroblastoma Neuro2a cells in vitro. In the present study we demonstrate that uPAR expression is essential for maintaining the epithelial phenotype in Neuro2a cells and that uPAR silencing promotes epithelial-mesenchymal transition (EMT) and increased cell migration. Accordingly, uPAR knockout results in the downregulation of epithelial markers (E-cadherin, occludin, and claudin-5) and in the increase of mesenchymal markers (N-cadherin, α-smooth muscle actin, and interleukin-6). In search of the molecular mechanism underlying these changes, we identified uPA as a key component. Two key insights emerged as a result of this work: in the absence of uPAR, uPA is translocated into the nucleus where it is presumably involved in the activation of transcription factors (nuclear factor κB and Snail) resulting in EMT. In uPAR-expressing cells, uPAR functions as a uPA "trap" that binds uPA on the cell surface and promotes controlled uPA internalization and degradation in lysosomes.

Optimization of CRISPR/Cas9 Technology to Knock Out Genes of Interest in Aneuploid Cell Lines.

IMPACT STATEMENT: Cell lines represent convenient models to elucidate specific causes of multigenetic and pluricausal diseases, to test breakthrough regenerative technologies. Most commonly used cell lines surpass diploid cells in their accessibility for delivery of large DNA molecules and genome editing, but the main obstacles for obtaining cell models with knockout-targeted protein from aneuploid cells are multiple allele copies and karyotype/phenotype heterogeneity. In the study, we report an original approach to CRISPR-/Cas9-mediated genome modification of aneuploid cell cultures to create functional cell models, achieving highly efficient targeted protein knockout and avoiding "clonal effect" (for the first time to our knowledge).

CRISPR/Cas9 nickase mediated targeting of urokinase receptor gene inhibits neuroblastoma cell proliferation.

Neuroblastoma is a tumor arising from pluripotent sympathoadrenal precursor cells of neural cell origin. Neuroblastoma is one of the most aggressive childhood tumors with highly invasive and metastatic potential. The increased expression of urokinase and its receptor is often associated with a negative prognosis in neuroblastoma patients. We have shown that targeting of the Plaur gene in mouse neuroblastoma Neuro 2A cells by CRISPR/Cas9n results in ~60% decrease in cell proliferation (p<0.05), reduction in the number of Ki-67 positive cells, caspase 3 activation and PARP-1 cleavage. Knockout of uPAR leads to downregulation of mRNA encoding full-length TrkC receptor, which is involved in p38MAPK and Akt signalling pathways. This finding provides a rationale to study a role of uPAR in neuroblastoma progression, since uPAR could be considered a potential therapeutic target in neuroblastoma treatment.

CRISPR/Cas9-mediated modification of the extreme C-terminus impairs PDGF-stimulated activity of Duox2.

Duox2 belongs to the large family of NADPH-oxidase enzymes that are implicated in immune response, vasoregulation, hormone synthesis, cell growth and differentiation via the regulated synthesis of H2O2 and reactive oxygen species. We and others have shown that Duox2 and H2O2 are involved in platelet-derived growth factor (PDGF) induced migration of fibroblasts. Now, using the CRISPR/Cas9-mediated genome editing we demonstrate that the extreme C-terminal region of Duox2 is required for PDGF-stimulated activity of Duox2 and H2O2 production. We generated the fibroblast cells that stably co-express the wild-type or C-terminally modified Duox2 and fluorescent H2O2 probe Hyper. We found that nonsense substitution of the last 23 amino acids in Duox2 results in complete loss of PDGF stimulation of intracellular H2O2 and fibroblast migration, yet these mutations have no effects on the expression of Duox2 and other NADPH-oxidases in cells. These findings illustrate for the first time that the extreme C-terminus of Duox2 is required for the functional activity of the enzyme. Furthermore, the conservative nature of the C-terminus suggests its role for activity in other NADPH-oxidases.