P130Cas Src-Binding and Substrate Domains Have Distinct Roles in Sustaining Focal Adhesion Disassembly and Promoting Cell Migration

The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas −/− mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation

5-HT2B antagonism arrests non-canonical TGF-β1-induced valvular myofibroblast differentiation

Transforming growth factor-β1 (TGF-β1) induces myofibroblast activation of quiescent aortic valve interstitial cells (AVICs), a differentiation process implicated in calcific aortic valve disease (CAVD). The ubiquity of TGF-β1 signaling makes it difficult to target in a tissue specific manner; however, the serotonin 2B receptor (5-HT2B) is highly localized to cardiopulmonary tissues and agonism of this receptor displays pro-fibrotic effects in a TGF-β1-dependent manner. Therefore, we hypothesized that antagonism of 5-HT2B opposes TGF-β1-induced pathologic differentiation of AVICs and may offer a druggable target to prevent CAVD. To test this hypothesis, we assessed the interaction of 5-HT2B antagonism with canonical and non-canonical TGF-β1 pathways to inhibit TGF-β1-induced activation of isolated porcine AVICs in vitro. Here we show that AVIC activation and subsequent calcific nodule formation is completely mitigated by 5-HT2B antagonism. Interestingly, 5-HT2B antagonism does not inhibit canonical TGF-β1 signaling as identified by Smad3 phosphorylation and activation of a partial plasminogen activator inhibitor-1 promoter (PAI-1, a transcriptional target of Smad3), but prevents non-canonical p38 MAPK phosphorylation. It was initially suspected that 5-HT2B antagonism prevents Src tyrosine kinase phosphorylation; however, we found that this is not the case and time-lapse microscopy indicates that 5-HT2B antagonism prevents non-canonical TGF-β1 signaling by physically arresting Src tyrosine kinase. This study demonstrates the necessity of non-canonical TGF-β1 signaling in leading to pathologic AVIC differentiation. Moreover, we believe that the results of this study suggest 5-HT2B antagonism as a novel therapeutic approach for CAVD that merits further investigation

THE CONNECTION OF THE BEHAVIOR ASSYMMETRY WITH THE FUNCTIONS OF THE HYPOPHYSIAL-ADRENOCORTICAL AND REPRODUCTIVE SYSTEMS

The object of investigation: she-rats of the Wistar line, the calves and cows of the black-motley race. For the first time, the dependence between the asymmetry of the spontaneous motor reactions in the T-labyrinth and the dynamics of the estral cycle has been revealed. It has been established, that the maximum degree of asymmetry (the left-side is preferable) is being detected in the stage of the proestrus. The arguments of influence of the corticosteroids on the behavior asymmetry have been obtained. The dynamics of the behavior asymmetry in the early ontogenesis of the mature-born animals has been described. The development of the left-side motor reactions of the animals brings to the exceeding of its reproductive possibilities, and the development of the right-side motor reactions facilitates the stability increase to the effect of the acute stress. The results can be placed in the base of the recommendations for the cattle-breeding and medicineAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Implementation of CRISPR/Cas editing analysis tool ICE to detect multiple edited alleles in mice

There is high demand for the development of experimental mouse models to study physiological processes and human diseases. CRISPR/Cas technology has offered a relatively efficient way to generate modifications in the mouse genome. However, this methodology often creates multiple, diverse genetic alterations, even when a strategy for a very specific targeted mutation is implemented. It is also rare that F0 mice will have single modified allele, and commonly, resultant mice are mosaic, containing multiple genomic modifications. In order to reduce the time and resources required to screen F0 mice and to detect somatic mosaicism, we implemented use of the computational analysis tool ICE. ICE is a CRISPR editing tool developed to analyze genomic modification in pooled cultured cells, where multiple genomic modifications can be expected. We applied this tool to determine its value in predicting genomic modifications in vivo using CRISPR/Cas. We tested one experimental case where sgRNA guided Cas9 created a double stranded DNA break in the Dio3 gene, and a ssDNA oligo provided as a template to insert a genetic tag. Preliminary data indicate a good correlation between genomic modifications detected by conventional Sanger sequencing of clones and those predicted by ICE. By increasing the number of direct comparisons between ICE predictions and actual sequence results, we aim to develop a reliable protocol to detect and evaluate efficiency of genomic modifications in F0 mice with assistance of computational methods

Notch signaling regulates perivascular adipose tissue (PVAT) function during diet-induced obesity

Background: Obesity is an established risk factor for cardiovascular diseases (CVD) and possibly shares molecular and cellular disease mechanisms as CVD due to their comorbidity. As a component of vasculature, perivascular adipose tissue (PVAT) has been recognized as a critical regulator of vascular function due to its anatomical proximity to the vascular wall. Study in PVAT thus help us understand cross talks between adipose tissue and cardiovascular system to re-evaluate clinical strategies for prevention and treatment of type 2 diabetes and CVD. Recent studies have shown that in addition to development, Notch/Rbp-jk signaling plays a crucial role in regulating metabolic homeostasis. Knockout of Notch signaling components was reported to induce beige phenotypes of white adipose tissue (Bi et al., 2014). Methods: We studied how Notch/Rbp-jk signaling and potential downstream pathways regulate health of PVAT using in vitro model and in vivo conditional knockout mice. PVAT derived stromal vascular fractions were differentiated into adipocytes and gene expression of Notch signaling component was further assessed. We also generated mouse models with adipose tissue specific knockout of Rbpj genes using Adipoq-Cre driver and examined their physiology, histology, and expression of metabolic and vascular relaxation pathway components as compared to control non-Cre mice. Results: Our data showed that Notch signaling was activated in PVAT during high fat diet (HFD) treatment. We found expression of Notch signaling component including RBPJ-k was increased during differentiation process of PVAT. We also found that knockout of Notch signaling in PVAT reduces obese phenotype of PVAT including adipocyte hypertrophy during HFD treatment. Moreover, PVAT of RBPJ conditional knock-out mice showed altered gene expression in the vasorelaxation pathway including eNOS signaling; and this potentially works through PI3/AKT pathways. Conclusion: Notch signaling plays important roles in regulating metabolic homeostasis of adipose tissue including PVAT. In addition, Notch signaling could potentially regulates PVATmediated vasorelaxation through energy metabolism pathway

Heterozygous caveolin-3 mice show increased susceptibility to palmitate-induced insulin resistance.

Insulin resistance and diabetes are comorbidities of obesity and affect one in 10 adults in the United States. Despite the high prevalence, the mechanisms of cardiac insulin resistance in obesity are still unclear. We test the hypothesis that the insulin receptor localizes to caveolae and is regulated through binding to caveolin-3 (CAV3). We further test whether haploinsufficiency forCAV3 increases the susceptibility to high-fat-induced insulin resistance. We used in vivo and in vitro studies to determine the effect of palmitate exposure on global insulin resistance, contractile performance of the heart in vivo, glucose uptake in the heart, and on cellular signaling downstream of theIR We show that haploinsufficiency forCAV3 increases susceptibility to palmitate-induced global insulin resistance and causes cardiomyopathy. On the basis of fluorescence energy transfer (FRET) experiments, we show thatCAV3 andIRdirectly interact in cardiomyocytes. Palmitate impairs insulin signaling by a decrease in insulin-stimulated phosphorylation of Akt that corresponds to an 87% decrease in insulin-stimulated glucose uptake inHL-1 cardiomyocytes. Despite loss of Akt phosphorylation and lower glucose uptake, palmitate increased insulin-independent serine phosphorylation ofIRS-1 by 35%. In addition, we found lipid induced downregulation ofCD36, the fatty acid transporter associated with caveolae. This may explain the problem the diabetic heart is facing with the simultaneous impairment of glucose uptake and lipid transport. Thus, these findings suggest that loss ofCAV3 interferes with downstream insulin signaling and lipid uptake, implicatingCAV3 as a regulator of theIRand regulator of lipid uptake in the heart

Notch Signaling Regulates Mouse Perivascular Adipose Tissue Function via Mitochondrial Pathways

Perivascular adipose tissue (PVAT) regulates vascular function by secreting vasoactive substances. In mice, Notch signaling is activated in the PVAT during diet-induced obesity, and leads to the loss of the thermogenic phenotype and adipocyte whitening due to increased lipid accumulation. We used the Adiponectin-Cre () strain to activate a ligand-independent Notch1 intracellular domain transgene () to drive constitutive Notch signaling in the adipose tissues (). We previously found that constitutive activation of Notch1 signaling in the PVAT phenocopied the effects of diet-induced obesity. To understand the downstream pathways activated by Notch signaling, we performed a proteomic analysis of the PVAT from control versus mice. This comparison identified prominent changes in the protein signatures related to metabolism, adipocyte homeostasis, mitochondrial function, and ferroptosis. PVAT-derived stromal vascular fraction cells were derived from our mouse strains to study the cellular and molecular phenotypes during adipogenic induction. We found that cells with activated Notch signaling displayed decreased mitochondrial respiration despite similar levels of adipogenesis and mitochondrial number. We observed variable regulation of the proteins related to mitochondrial dynamics and ferroptosis, including PHB3, PINK1, pDRP1, and the phospholipid hydroperoxidase GPX4. Mitochondria regulate some forms of ferroptosis, which is a regulated process of cell death driven by lipid peroxidation. Accordingly, we found that Notch activation promoted lipid peroxidation and ferroptosis in PVAT-derived adipocytes. Because the PVAT phenotype is a regulator of vascular reactivity, we tested the effect of Notch activation in PVAT on vasoreactivity using wire myography. The aortae from the mice had increased vasocontraction and decreased vasorelaxation in a PVAT-dependent and age-dependent manner. Our data provide support for the novel concept that increased Notch signaling in the adipose tissue leads to PVAT whitening, impaired mitochondrial function, increased ferroptosis, and loss of a protective vasodilatory signal. Our study advances our understanding of how Notch signaling in adipocytes affects mitochondrial dynamics, which impacts vascular physiology

Notch Signaling Regulates Mouse Perivascular Adipose Tissue Function via Mitochondrial Pathways

Perivascular adipose tissue (PVAT) regulates vascular function by secreting vasoactive substances. In mice, Notch signaling is activated in the PVAT during diet-induced obesity, and leads to the loss of the thermogenic phenotype and adipocyte whitening due to increased lipid accumulation. We used the Adiponectin-Cre (Adipoq-Cre) strain to activate a ligand-independent Notch1 intracellular domain transgene (N1ICD) to drive constitutive Notch signaling in the adipose tissues (N1ICD;Adipoq-Cre). We previously found that constitutive activation of Notch1 signaling in the PVAT phenocopied the effects of diet-induced obesity. To understand the downstream pathways activated by Notch signaling, we performed a proteomic analysis of the PVAT from control versus N1ICD;Adipoq-Cre mice. This comparison identified prominent changes in the protein signatures related to metabolism, adipocyte homeostasis, mitochondrial function, and ferroptosis. PVAT-derived stromal vascular fraction cells were derived from our mouse strains to study the cellular and molecular phenotypes during adipogenic induction. We found that cells with activated Notch signaling displayed decreased mitochondrial respiration despite similar levels of adipogenesis and mitochondrial number. We observed variable regulation of the proteins related to mitochondrial dynamics and ferroptosis, including PHB3, PINK1, pDRP1, and the phospholipid hydroperoxidase GPX4. Mitochondria regulate some forms of ferroptosis, which is a regulated process of cell death driven by lipid peroxidation. Accordingly, we found that Notch activation promoted lipid peroxidation and ferroptosis in PVAT-derived adipocytes. Because the PVAT phenotype is a regulator of vascular reactivity, we tested the effect of Notch activation in PVAT on vasoreactivity using wire myography. The aortae from the N1ICD;Adipoq-Cre mice had increased vasocontraction and decreased vasorelaxation in a PVAT-dependent and age-dependent manner. Our data provide support for the novel concept that increased Notch signaling in the adipose tissue leads to PVAT whitening, impaired mitochondrial function, increased ferroptosis, and loss of a protective vasodilatory signal. Our study advances our understanding of how Notch signaling in adipocytes affects mitochondrial dynamics, which impacts vascular physiology

Evaluating whether RAB27a loss impacts PVAT and aorta morphology during a HFD

Perivascular adipose tissue (PVAT) is a specialized adipose depot that surrounds the vasculature. In healthy conditions, PVAT exhibits a thermogenic phenotype, denoted by high metabolic activity, while exhibiting a lipid storage phenotype under obese conditions. While the thermogenic PVAT promotes anti-inflammation and vasodilation of blood vessels, the lipid storage phenotype increases the progression of cardiovascular disease by inducing inflammation and vasoconstriction. Ras related protein Rab-27A (RAB27a) is a trafficking protein that regulates exosome secretion and we have shown its expression is upregulated in the PVAT of obese mice. However, whether changes in RAB27a expression during obesity mediates the phenotypic change in PVAT is unclear. We hypothesize that loss of RAB27a expression during obesity will preserve the thermogenic phenotype of PVAT. To test our hypothesis, we quantified lipid accumulation differences in multiple adipose depots between wildtype and Rab27a global null mice fed a high fat diet (HFD) or sucrose-controlled diet (SCD). Additionally, changes in vascular morphology between our groups was examined by quantifying the total, lumenal, and medial area of transverse slices of thoracic aorta. To evaluate the impact of these changes on vasoreactivity, wire myography studies were completed to quantify changes in vascular contraction. These data show that diet, not the loss of RAB27a, affects lipid accumulation. However, Rab27a null mice have a smaller lumenal, medial, and total area of the aorta, regardless of diet. Additionally, Rab27a null mice fed a HFD showed increased contraction compared to control groups. These data suggest that RAB27a is a regulator of vascular contractility