Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice.

Up until recently, methods for generating floxed mice either conventionally or by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) editing have been technically challenging, expensive and error-prone, or time-consuming. To circumvent these issues, several labs have started successfully using a small artificial intron to conditionally knockout (KO) a gene of interest in mice. However, many other labs are having difficulty getting the technique to work. The key problem appears to be either a failure in achieving correct splicing after the introduction of the artificial intron into the gene or, just as crucial, insufficient functional KO of the gene's protein after Cre-induced removal of the intron's branchpoint. Presented here is a guide on how to choose an appropriate exon and where to place the recombinase-regulated artificial intron (rAI) in that exon to prevent disrupting normal gene splicing while maximizing mRNA degradation after recombinase treatment. The reasoning behind each step in the guide is also discussed. Following these recommendations should increase the success rate of this easy, new, and alternative technique for producing tissue-specific KO mice.

Senescent Phenotype Induced by p90RSK-NRF2 Signaling Sensitizes Monocytes and Macrophages to Oxidative Stress in HIV-Positive Individuals.

BACKGROUND: The incidence of cardiovascular disease is higher in HIV-positive (HIV+) patients than it is in the average population, and combination antiretroviral therapy (cART) is a recognized risk factor for cardiovascular disease. However, the molecular mechanisms that link cART and cardiovascular disease are currently unknown. Our study explores the role of the activation of p90RSK, a reactive oxygen species-sensitive kinase, in engendering senescent phenotype in macrophages and accelerating atherogenesis in patients undergoing cART. METHODS: Peripheral whole blood from cART-treated HIV+ individuals and nontreated HIV-negative individuals was treated with H2O2 (200 µmol/L) for 4 minutes, and p90RSK activity in CD14+ monocytes was measured. Plaque formation in the carotids was also analyzed in these individuals. Macrophage senescence was determined by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. The involvement of p90RSK-NRF2 signaling in cART-induced senescence was assessed by p90RSK-specific inhibitor (FMK-MEA) or dominant-negative p90RSK (DN-p90RSK) and NRF2 activator (NRF2A). Further, the severity of atherosclerosis was determined in myeloid cell-specific wild-type and DN-p90RSK transgenic mice. RESULTS: Monocytes from HIV+ patients exhibited higher levels of p90RSK activity and were also more sensitive to reactive oxygen species than monocytes from HIV-negative individuals. A multiple linear regression analysis involving cART, Reynolds cardiovascular risk score, and basal p90RSK activity revealed that cART and basal p90RSK activity were the 2 significant determinants of plaque formation. Many of the antiretroviral drugs individually activated p90RSK, which simultaneously triggered all components of the macrophage senescent phenotype. cART inhibited antioxidant response element reporter activity via ERK5 S496 phosphorylation. NRF2A reversed the H2O2-induced overactivation of p90RSK in cART-treated macrophages by countering the induction of senescent phenotype. Last, the data obtained from our gain- or loss-of-function mice conclusively showed the crucial role of p90RSK in inducing senescent phenotype in macrophages and atherogenesis. CONCLUSIONS: cART increased monocyte/macrophage sensitivity to reactive oxygen species- in HIV+ individuals by suppressing NRF2-ARE activity via p90RSK-mediated ERK5 S496 phosphorylation, which coordinately elicited senescent phenotypes and proinflammatory responses. As such, our report underscores the importance of p90RSK regulation in monocytes/macrophages as a viable biomarker and therapeutic target for preventing cardiovascular disease, especially in HIV+ patients treated with cART.

Mechanotransduction properties of the cytoplasmic tail of PECAM-1.

BACKGROUND INFORMATION: Vascular endothelial cells (ECs) are a well-known cell system used in the study of mechanobiology. Using cultured ECs, we found that platelet EC adhesion molecule 1 (PECAM-1, CD31), a cell adhesion protein localised to regions of EC-EC contact, was rapidly tyrosine phosphorylated in ECs exposed to shear or cyclic stretch. Src-homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) binds phosphorylated PECAM-1 and activates the extracellular signal-regulated kinase1/2 (ERK1/2) signalling cascade, a known flow-activated signalling pathway. RESULTS: Although PECAM-1 tyrosine phosphorylation is characterised in ECs exposed to fluid shear stress, it is less well demonstrated in the cells stretched cyclically. Thus, we first show that PECAM-1 is tyrosine-phosphorylated in ECs cyclically stretched. We hypothesise that when an external force is applied to a monolayer of ECs, the force is directly transmitted to PECAM-1 which is then stretched and phosphorylation sites in its cytoplasmic domain are exposed and phosphorylated. This hypothesis requires the presence of any stretchable structure within the PECAM-1 cytoplasmic domain. Force spectroscopy measurements were performed with a construct containing cytoplasmic PECAM-1 domains inserted between I27 motifs, a recombinant string of the structural elements from titin. This strategy allowed us to identify the events in which a single molecule is being pulled and to detect the unravelling of the cytoplasmic domain of PECAM-1 by force. The response by PECAM-1 to mechanical loading was heterogeneous but with magnitudes as high as or higher than the naturally force bearing I27 domains. CONCLUSIONS: The PECAM-1 cytoplasmic domain has a structure that can be unfolded by externally applied force and this unfolding of PECAM-1 may be necessary for its phosphorylation, the first step of PECAM-1 mechanosignalling. SIGNIFICANCE: When EC monolayers are mechanically stimulated, the PECAM-1 found at EC contacts is phosphorylated. We have proposed that under these conditions, the cytoplasmic domain of PECAM-1 is unfolded, which then exposes a phosphorylation site, allowing it to be accessed. The stretch induced unfolding is essential to this model of PECAM-1 mechanosignalling. In this study, we investigate whether the cytoplasmic domain of PECAM-1 has a stretchable structure, and the results are in line with our hypothesis.

Endothelial activation and fibrotic changes are impeded by laminar flow-induced CHK1-SENP2 activity through mechanisms distinct from endothelial-to-mesenchymal cell transition.

Background: The deSUMOylase sentrin-specific isopeptidase 2 (SENP2) plays a crucial role in atheroprotection. However, the phosphorylation of SENP2 at T368 under disturbed flow (D-flow) conditions hinders its nuclear function and promotes endothelial cell (EC) activation. SUMOylation has been implicated in D-flow-induced endothelial-to-mesenchymal transition (endoMT), but the precise role of SENP2 in counteracting this process remains unclear. Method: We developed a phospho-specific SENP2 S344 antibody and generated knock-in (KI) mice with a phospho-site mutation of SENP2 S344A using CRISPR/Cas9 technology. We then investigated the effects of SENP2 S344 phosphorylation under two distinct flow patterns and during hypercholesteremia (HC)-mediated EC activation. Result: Our findings demonstrate that laminar flow (L-flow) induces phosphorylation of SENP2 at S344 through the activation of checkpoint kinase 1 (CHK1), leading to the inhibition of ERK5 and p53 SUMOylation and subsequent suppression of EC activation. We observed a significant increase in lipid-laden lesions in both the aortic arch (under D-flow) and descending aorta (under L-flow) of female hypercholesterolemic SENP2 S344A KI mice. In male hypercholesterolemic SENP2 S344A KI mice, larger lipid-laden lesions were only observed in the aortic arch area, suggesting a weaker HC-mediated atherogenesis in male mice compared to females. Ionizing radiation (IR) reduced CHK1 expression and SENP2 S344 phosphorylation, attenuating the pro-atherosclerotic effects observed in female SENP2 S344A KI mice after bone marrow transplantation (BMT), particularly in L-flow areas. The phospho-site mutation SENP2 S344A upregulates processes associated with EC activation, including inflammation, migration, and proliferation. Additionally, fibrotic changes and up-regulated expression of EC marker genes were observed. Apoptosis was augmented in ECs derived from the lungs of SENP2 S344A KI mice, primarily through the inhibition of ERK5-mediated expression of DNA damage-induced apoptosis suppressor (DDIAS). Summary: In this study, we have revealed a novel mechanism underlying the suppressive effects of L-flow on EC inflammation, migration, proliferation, apoptosis, and fibrotic changes through promoting CHK1-induced SENP2 S344 phosphorylation. The phospho-site mutation SENP2 S344A responds to L-flow through a distinct mechanism, which involves the upregulation of both mesenchymal and EC marker genes.

Sertoli Cell-Germ Cell Interactions Within the Niche: Paracrine and Juxtacrine Molecular Communications.

Male germ cell development depends on multiple biological events that combine epigenetic reprogramming, cell cycle regulation, and cell migration in a spatio-temporal manner. Sertoli cells are a crucial component of the spermatogonial stem cell niche and provide essential growth factors and chemokines to developing germ cells. This review focuses mainly on the activation of master regulators of the niche in Sertoli cells and their targets, as well as on novel molecular mechanisms underlying the regulation of growth and differentiation factors such as GDNF and retinoic acid by NOTCH signaling and other pathways.

Molecular basis of decreased Kir4.1 function in SeSAME/EAST syndrome.

SeSAME/EAST syndrome is a channelopathy consisting of a hypokalemic, hypomagnesemic, metabolic alkalosis associated with seizures, sensorineural deafness, ataxia, and developmental abnormalities. This disease links to autosomal recessive mutations in KCNJ10, which encodes the Kir4.1 potassium channel, but the functional consequences of these mutations are not well understood. In Xenopus oocytes, all of the disease-associated mutant channels (R65P, R65P/R199X, G77R, C140R, T164I, and A167V/R297C) had decreased K(+) current (0 to 23% of wild-type levels). Immunofluorescence demonstrated decreased surface expression of G77R, C140R, and A167V expressed in HEK293 cells. When we coexpressed mutant and wild-type subunits to mimic the heterozygous state, R199X, C140R, and G77R currents decreased to 55, 40, and 20% of wild-type levels, respectively, suggesting that carriers of these mutations may present with an abnormal phenotype. Because Kir4.1 subunits can form heteromeric channels with Kir5.1, we coexpressed the aforementioned mutants with Kir5.1 and found that currents were reduced at least as much as observed when we expressed mutants alone. Reduction of pH(i) from approximately 7.4 to 6.8 significantly decreased currents of all mutants except R199X but did not affect wild-type channels. In conclusion, perturbed pH gating may underlie the loss of channel function for the disease-associated mutant Kir4.1 channels and may have important physiologic consequences.

RAC1 Alterations Induce Acquired Dabrafenib Resistance in Association with Anaplastic Transformation in a Papillary Thyroid Cancer Patient.

BRAF-activating mutations are the most frequent driver mutations in papillary thyroid cancer (PTC). Targeted inhibitors such as dabrafenib have been used in advanced BRAF-mutated PTC; however, acquired resistance to the drug is common and little is known about other effectors that may play integral roles in this resistance. In addition, the induction of PTC dedifferentiation into highly aggressive KRAS-driven anaplastic thyroid cancer (ATC) has been reported. We detected a novel RAC1 (P34R) mutation acquired during dabrafenib treatment in a progressive metastatic lesion with ATC phenotype. To identify a potential functional link between this novel mutation and tumor dedifferentiation, we developed a cell line derived from the metastatic lesion and compared its behavior to isogenic cell lines and primary tumor samples. Our data demonstrated that RAC1 mutations induce changes in cell morphology, reorganization of F-actin almost exclusively at the cell cortex, and changes in cell adhesion properties. We also established that RAC1 amplification, with or without mutation, is sufficient to drive cell proliferation and resistance to BRAF inhibition. Further, we identified polyploidy of chromosome 7, which harbors RAC1, in both the metastatic lesion and its derived cell line. Copy number amplification and overexpression of other genes located on this chromosome, such as TWIST1, EGFR, and MET were also detected, which might also lead to dabrafenib resistance. Our study suggests that polyploidy leading to increased expression of specific genes, particularly those located on chromosome 7, should be considered when analyzing aggressive thyroid tumor samples and in further treatments.

Nucleus-mitochondria positive feedback loop formed by ERK5 S496 phosphorylation-mediated poly (ADP-ribose) polymerase activation provokes persistent pro-inflammatory senescent phenotype and accelerates coronary atherosclerosis after chemo-radiation.

The incidence of cardiovascular disease (CVD) is higher in cancer survivors than in the general population. Several cancer treatments are recognized as risk factors for CVD, but specific therapies are unavailable. Many cancer treatments activate shared signaling events, which reprogram myeloid cells (MCs) towards persistent senescence-associated secretory phenotype (SASP) and consequently CVD, but the exact mechanisms remain unclear. This study aimed to provide mechanistic insights and potential treatments by investigating how chemo-radiation can induce persistent SASP. We generated ERK5 S496A knock-in mice and determined SASP in myeloid cells (MCs) by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. Candidate SASP inducers were identified by high-throughput screening, using the ERK5 transcriptional activity reporter cell system. Various chemotherapy agents and ionizing radiation (IR) up-regulated p90RSK-mediated ERK5 S496 phosphorylation. Doxorubicin and IR caused metabolic changes with nicotinamide adenine dinucleotide depletion and ensuing mitochondrial stunning (reversible mitochondria dysfunction without showing any cell death under ATP depletion) via p90RSK-ERK5 modulation and poly (ADP-ribose) polymerase (PARP) activation, which formed a nucleus-mitochondria positive feedback loop. This feedback loop reprogramed MCs to induce a sustained SASP state, and ultimately primed MCs to be more sensitive to reactive oxygen species. This priming was also detected in circulating monocytes from cancer patients after IR. When PARP activity was transiently inhibited at the time of IR, mitochondrial stunning, priming, macrophage infiltration, and coronary atherosclerosis were all eradicated. The p90RSK-ERK5 module plays a crucial role in SASP-mediated mitochondrial stunning via regulating PARP activation. Our data show for the first time that the nucleus-mitochondria positive feedback loop formed by p90RSK-ERK5 S496 phosphorylation-mediated PARP activation plays a crucial role of persistent SASP state, and also provide preclinical evidence supporting that transient inhibition of PARP activation only at the time of radiation therapy can prevent future CVD in cancer survivors.

Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice.

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.

MAGI1 as a link between endothelial activation and ER stress drives atherosclerosis.

The possible association between the membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1) and inflammation has been suggested, but the molecular mechanisms underlying this link, especially during atherogenesis, remain unclear. In endothelial cells (ECs) exposed to disturbed flow (d-flow), p90 ribosomal S6 kinase (p90RSK) bound to MAGI1, causing MAGI1-S741 phosphorylation and sentrin/SUMO-specific protease 2 T368 phosphorylation-mediated MAGI1-K931 deSUMOylation. MAGI1-S741 phosphorylation upregulated EC activation via activating Rap1. MAGI1-K931 deSUMOylation induced both nuclear translocation of p90RSK-MAGI1 and ATF-6-MAGI1 complexes, which accelerated EC activation and apoptosis, respectively. Microarray screening revealed key roles for MAGI1 in the endoplasmic reticulum (ER) stress response. In this context, MAGI1 associated with activating transcription factor 6 (ATF-6). MAGI1 expression was upregulated in ECs and macrophages found in atherosclerotic-prone regions of mouse aortas as well as in the colonic epithelia and ECs of patients with inflammatory bowel disease. Further, reduced MAGI1 expression in Magi1-/+ mice inhibited d-flow-induced atherogenesis. In sum, EC activation and ER stress-mediated apoptosis are regulated in concert by two different types of MAGI1 posttranslational modifications, elucidating attractive drug targets for chronic inflammatory disease, particularly atherosclerosis.

The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells.

The ETS family of transcription factors is involved in several normal remodeling events and pathological processes including tumor progression. ETS transcription factors are divided into subfamilies based on the sequence and location of the ETS domain. ETV5 (Ets variant gene 5; also known as ERM) is a member of the PEA3 subfamily. Our meta-analysis of normal, benign, and malignant thyroid samples demonstrated that ETV5 expression is upregulated in papillary thyroid cancer and was predominantly associated with BRAF V600E or RAS mutations. However, the precise role of ETV5 in these lesions is unknown. In this study, we used the KTC1 cell line as a model for human advanced papillary thyroid cancer (PTC) because the cells harbor the heterozygous BRAF (V600E) mutation together with the C250T TERT promoter mutation. The role of ETV5 in PTC proliferation was tested using RNAi followed by high-throughput screening. Signaling pathways driving ETV5 expression were identified using specific pharmacological inhibitors. To determine if ETV5 influences the expression of epithelial-to-mesenchymal (EMT) markers in these cells, an EMT PCR array was used, and data were confirmed by qPCR and ChIP-qPCR. We found that ETV5 is critical for PTC cell growth, is expressed downstream of the MAPK pathway, and directly upregulates the transcription factor TWIST1, a known marker of intravasation and metastasis. Increased ETV5 expression could therefore be considered as a marker for advanced PTCs and a possible future therapeutic target.

Mechanotransduction in an extracted cell model: Fyn drives stretch- and flow-elicited PECAM-1 phosphorylation.

Mechanosensing followed by mechanoresponses by cells is well established, but the mechanisms by which mechanical force is converted into biochemical events are poorly understood. Vascular endothelial cells (ECs) exhibit flow- and stretch-dependent responses and are widely used as a model for studying mechanotransduction in mammalian cells. Platelet EC adhesion molecule 1 (PECAM-1) is tyrosine phosphorylated when ECs are exposed to flow or when PECAM-1 is directly pulled, suggesting that it is a mechanochemical converter. We show that PECAM-1 phosphorylation occurs when detergent-extracted EC monolayers are stretched, indicating that this phosphorylation is mechanically triggered and does not require the intact plasma membrane and soluble cytoplasmic components. Using kinase inhibitors and small interfering RNAs, we identify Fyn as the PECAM-1 kinase associated with the model. We further show that stretch- and flow-induced PECAM-1 phosphorylation in intact ECs is abolished when Fyn expression is down-regulated. We suggest that PECAM-1 and Fyn are essential components of a PECAM-1-based mechanosensory complex in ECs.