Myocardin-Related Transcription Factor Mediates Epithelial Fibrogenesis in Polycystic Kidney Disease.

Polycystic kidney disease (PKD) is characterized by extensive cyst formation and progressive fibrosis. However, the molecular mechanisms whereby the loss/loss-of-function of Polycystin 1 or 2 (PC1/2) provokes fibrosis are largely unknown. The small GTPase RhoA has been recently implicated in cystogenesis, and we identified the RhoA/cytoskeleton/myocardin-related transcription factor (MRTF) pathway as an emerging mediator of epithelium-induced fibrogenesis. Therefore, we hypothesized that MRTF is activated by PC1/2 loss and plays a critical role in the fibrogenic reprogramming of the epithelium. The loss of PC1 or PC2, induced by siRNA in vitro, activated RhoA and caused cytoskeletal remodeling and robust nuclear MRTF translocation and overexpression. These phenomena were also manifested in PKD1 (RC/RC) and PKD2 (WS25/-) mice, with MRTF translocation and overexpression occurring predominantly in dilated tubules and the cyst-lining epithelium, respectively. In epithelial cells, a large cohort of PC1/PC2 downregulation-induced genes was MRTF-dependent, including cytoskeletal, integrin-related, and matricellular/fibrogenic proteins. Epithelial MRTF was necessary for the paracrine priming of the fibroblast-myofibroblast transition. Thus, MRTF acts as a prime inducer of epithelial fibrogenesis in PKD. We propose that RhoA is a common upstream inducer of both histological hallmarks of PKD: cystogenesis and fibrosis.

Regulation of the RhoA exchange factor GEF-H1 by pro-fibrotic stimuli through a positive feedback loop involving RhoA, MRTF and Sp1.

RhoA and its effectors, the transcriptional coactivators Myocardin-Related Transcription Factor (MRTF) and Serum Response Factor (SRF), control epithelial phenotype and are indispensable for profibrotic epithelial reprogramming during fibrogenesis. Context-dependent control of RhoA and fibrosis-associated changes in its regulators, however, remain incompletely characterized. We previously identified the guanine nucleotide exchange factor GEF-H1 as a central mediator of RhoA activation in renal tubular cells exposed to inflammatory or fibrotic stimuli. Here we found that GEF-H1 expression and phosphorylation were strongly elevated in two animal models of fibrosis. In the Unilateral Ureteral Obstruction mouse kidney fibrosis model, GEF-H1 was upregulated predominantly in the tubular compartment. GEF-H1 was also elevated and phosphorylated in a rat pulmonary artery banding model of right ventricular fibrosis. Prolonged stimulation of LLC-PK1 tubular cells with tumor necrosis factor-α or transforming growth factor ß1 increased GEF-H1 expression and activated a luciferase-coupled GEF-H1 promoter. Knockdown and overexpression studies revealed that these effects were mediated by RhoA, cytoskeleton remodeling and MRTF, indicative of a positive feed-back cycle. Indeed, silencing endogenous GEF-H1 attenuated activation of the GEF-H1 promoter. Importantly, inhibition of MRTF using CCG-1423 prevented GEF-H1 upregulation in both animal models. MRTF-dependent increase in GEF-H1 was prevented by inhibition of the transcription factor Sp1, and mutating putative Sp1 binding sites in the GEF-H1 promoter eliminated its MRTF-dependent activation. Since the GEF-H1/RhoA axis is key for fibrogenesis, this novel MRTF/Sp1-dependent regulation of GEF-H1 abundance represents a potential target for reducing renal and cardiac fibrosis.

Therapeutic Effects of Mesenchymal Stromal Cells Require Mitochondrial Transfer and Quality Control.

Due to their beneficial effects in an array of diseases, Mesenchymal Stromal Cells (MSCs) have been the focus of intense preclinical research and clinical implementation for decades. MSCs have multilineage differentiation capacity, support hematopoiesis, secrete pro-regenerative factors and exert immunoregulatory functions promoting homeostasis and the resolution of injury/inflammation. The main effects of MSCs include modulation of immune cells (macrophages, neutrophils, and lymphocytes), secretion of antimicrobial peptides, and transfer of mitochondria (Mt) to injured cells. These actions can be enhanced by priming (i.e., licensing) MSCs prior to exposure to deleterious microenvironments. Preclinical evidence suggests that MSCs can exert therapeutic effects in a variety of pathological states, including cardiac, respiratory, hepatic, renal, and neurological diseases. One of the key emerging beneficial actions of MSCs is the improvement of mitochondrial functions in the injured tissues by enhancing mitochondrial quality control (MQC). Recent advances in the understanding of cellular MQC, including mitochondrial biogenesis, mitophagy, fission, and fusion, helped uncover how MSCs enhance these processes. Specifically, MSCs have been suggested to regulate peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α)-dependent biogenesis, Parkin-dependent mitophagy, and Mitofusins (Mfn1/2) or Dynamin Related Protein-1 (Drp1)-mediated fission/fusion. In addition, previous studies also verified mitochondrial transfer from MSCs through tunneling nanotubes and via microvesicular transport. Combined, these effects improve mitochondrial functions, thereby contributing to the resolution of injury and inflammation. Thus, uncovering how MSCs affect MQC opens new therapeutic avenues for organ injury, and the transplantation of MSC-derived mitochondria to injured tissues might represent an attractive new therapeutic approach.

Nuclear Import and Export of YAP and TAZ.

Yes-associated Protein (YAP) and its paralog Transcriptional Coactivator with PDZ-binding Motif (TAZ) are major regulators of gene transcription/expression, primarily controlled by the Hippo pathway and the cytoskeleton. Integrating an array of chemical and mechanical signals, they impact growth, differentiation, and regeneration. Accordingly, they also play key roles in tumorigenesis and metastasis formation. Their activity is primarily regulated by their localization, that is, Hippo pathway- and/or cytoskeleton-controlled cytosolic or nuclear sequestration. While many details of such prevailing retention models have been elucidated, much less is known about their actual nuclear traffic: import and export. Although their size is not far from the cutoff for passive diffusion through the nuclear pore complex (NPC), and they do not contain any classic nuclear localization (NLS) or nuclear export signal (NES), evidence has been accumulating that their shuttling involves mediated and thus regulatable/targetable processes. The aim of this review is to summarize emerging information/concepts about their nucleocytoplasmic shuttling, encompassing the relevant structural requirements (NLS, NES), nuclear transport receptors (NTRs, karyophererins), and NPC components, along with the potential transport mechanisms and their regulation. While dissecting retention vs. transport is often challenging, the emerging picture suggests that YAP/TAZ shuttles across the NPC via multiple, non-exclusive, mediated mechanisms, constituting a novel and intriguing facet of YAP/TAZ biology.

Endothelial ACKR1 is induced by neutrophil contact and down-regulated by secretion in extracellular vesicles.

Atypical chemokine receptor-1 (ACKR1), previously known as the Duffy antigen receptor for chemokines, is a widely conserved cell surface protein that is expressed on erythrocytes and the endothelium of post-capillary venules. In addition to being the receptor for the parasite causing malaria, ACKR1 has been postulated to regulate innate immunity by displaying and trafficking chemokines. Intriguingly, a common mutation in its promoter leads to loss of the erythrocyte protein but leaves endothelial expression unaffected. Study of endothelial ACKR1 has been limited by the rapid down-regulation of both transcript and protein when endothelial cells are extracted and cultured from tissue. Thus, to date the study of endothelial ACKR1 has been limited to heterologous over-expression models or the use of transgenic mice. Here we report that exposure to whole blood induces ACKR1 mRNA and protein expression in cultured primary human lung microvascular endothelial cells. We found that contact with neutrophils is required for this effect. We show that NF-κB regulates ACKR1 expression and that upon removal of blood, the protein is rapidly secreted by extracellular vesicles. Finally, we confirm that endogenous ACKR1 does not signal upon stimulation with IL-8 or CXCL1. Our observations define a simple method for inducing endogenous endothelial ACKR1 protein that will facilitate further functional studies.

Augmented Parkin-dependent mitophagy underlies the hepatoprotective effect of remote ischemic conditioning used prior to hemorrhagic shock.

BACKGROUND AND AIMS: Hemorrhagic shock-resuscitation (HSR) following trauma contributes to organ dysfunction by causing ischemia-reperfusion injury (IRI). We previously showed that 'remote ischemic preconditioning' (RIPC) exerted multi-organ protection from IRI. Maintenance of mitochondrial quality by clearance of dysfunctional mitochondria via mitophagy is vital in restoring organ integrity. We hypothesized that parkin-dependent mitophagy played a role in RIPC-induced hepatoprotection following HSR. METHODS: The hepatoprotective effect of RIPC in a murine model of HSR-IRI was investigated in wild type and parkin-/- animals. Mice were subjected to HSR ± RIPC and blood and organs were collected, followed by cytokine ELISAs, histology, qPCR, Western blots, and transmission electron microscopy. RESULTS: HSR increased hepatocellular injury, as measured by plasma ALT and liver necrosis, while antecedent RIPC prevented this injury; in parkin-/- mice, RIPC failed to exert hepatoprotection. The ability of RIPC to lessen HSR-induced rises in plasma IL-6 and TNFα, was lost in parkin-/- mice. While RIPC alone did not induce mitophagy, the application of RIPC prior to HSR caused a synergistic increase in mitophagy, this increase was not observed in parkin-/- mice. RIPC induced shifts in mitochondrial morphology favoring mitophagy in WT but not in parkin-/- animals. CONCLUSIONS: RIPC was hepatoprotective in WT mice following HSR but not in parkin-/- mice. Loss of protection in parkin-/- mice corresponded with the failure of RIPC plus HSR to upregulate the mitophagic process. Improving mitochondrial quality by modulating mitophagy, may prove to be an attractive therapeutic target in disease processes caused by IRI.

Advancing Discovery Research in Nephrology in Canada: A Conference Report From the 2021 Molecules and Mechanisms Mediating Kidney Health and Disease (M3K) Scientific Meeting and Investigator Summit.

Purpose of conference: New discoveries arising from investigations into fundamental aspects of kidney development and function in health and disease are critical to advancing kidney care. Scientific meetings focused specifically on fundamental biology of the kidney can facilitate interactions, support the development of collaborative groups, and accelerate translation of key findings. The Canadian fundamental kidney researcher community has lacked such a forum. On December 3 to 4, 2021, the first Molecules and Mechanisms Mediating Kidney Health and Disease (M3K) Scientific Meeting and Investigator Summit was held to address this gap with the goal of advancing fundamental kidney research nationally. The meeting was held virtually and was supported by a planning and dissemination grant from the Canadian Institutes of Health Research. Attendees included PhD scientists, nephrology clinician scientists, engineers, industry representatives, graduate students, medical residents, and fellows. Sources of information: This report was prepared from the scientific program, registration numbers, and details obtained from the online platform WHOVA, and summaries written by organizers and participants of the 2021 meeting. Methods: A 21-person team, consisting of the organizing committee members and participants from the meeting, was assembled. Key highlights of the meeting and future directions were identified and the team jointly assembled this report. Key findings: Participation in the meeting was strong, with more than 140 attendees across a range of disciplines. The program featured state-of-the-art presentations on diabetic nephropathy, the immune system, kidney development, and fibrosis, and was heavily focused on trainee presentations. The moderated "Investigator Summit" identified key barriers to research advancement and discussed strategies for overcoming them. These included establishment of a pan-Canadian fundamental kidney research network, development of key resources, cross-pollination with clinical nephrology, better reintegration into the Canadian Society of Nephrology, and further establishment of identity and knowledge translation. Limitations and implications: The 2021 M3K meeting represented a key first step in uniting fundamental kidney researchers in Canada. However, it was universally agreed that regular meetings were necessary to sustain this momentum. The proceedings of this meeting and future actions to sustain the M3K Scientific Meeting and Investigator Summit are presented in this article.

Objectif de la conférence: De nouvelles découvertes découlant des enquêtes sur les aspects fondamentaux du développement et de la fonction des reins en santé ou malades sont essentielles pour faire progresser les soins rénaux. Les réunions scientifiques axées spécifiquement sur la biologie fondamentale du rein peuvent faciliter les interactions, appuyer le développement de groupes de collaboration et accélérer l'application des principaux résultats. La communauté canadienne des chercheurs fondamentaux en néphrologie a manqué d'un tel forum. Les 3 et 4 décembre 2021, le premier Sommet des chercheurs et la réunion scientifique M3K (Molecules and Mechanisms Mediating Kidney Health and Disease) sur les molécules et les médiateurs de la santé et des maladies rénales ont eu lieu pour combler cette lacune; l'objectif était de faire progresser la recherche fondamentale en néphrologie à l'échelle nationale. La réunion s'est tenue virtuellement et était financée par une subvention de planification et de diffusion des Instituts de recherche en santé du Canada. Des doctorants, cliniciens-chercheurs en néphrologie, ingénieurs, représentants de l'industrie, étudiants diplômés, résidents en médecine et en surspécialisation figuraient parmi les participants. Sources: Ce rapport a été préparé à partir du program scientifique, des informations et des numéros d'inscription tirés de la plateforme en ligne WHOVA, et des résumés rédigés par les organisateurs et les participants à la réunion de 2021. Méthodologie: Une équipe de 20 personnes composée de membres du comité organisateur et de participants à la réunion a été formée. Les principaux points saillants de la réunion et les orientations futures ont été déterminés, puis l'équipe a rédigé conjointement le présent rapport. Principaux résultats: La réunion s'est avérée un succès; plus de 140 personnes provenant d'un large éventail de disciplines y ont participé. Le program comprenait des présentations de pointe sur la néphropathie diabétique, le système immunitaire, le développement des reins et la fibrose, et était fortement axé sur des présentations par des stagiaires. Le « Sommet des chercheurs ¼, animé par un modérateur, a permis de déterminer les principaux obstacles à l'avancement de la recherche et de discuter des stratégies pour les surmonter. Ces dernières incluent notamment la création d'un réseau pancanadien de recherche fondamentale en néphrologie, le développement de ressources clés, la pollinisation croisée avec la néphrologie clinique, une « meilleure réintégration dans la Société canadienne de néphrologie ¼ et la poursuite de l'établissement de l'identité et de l'application des connaissances. Limites et implications: La réunion M3K de 2021 a constitué une première étape clé dans l'unification des chercheurs fondamentaux en néphrologie au Canada. On a cependant universellement convenu que des réunions régulières étaient nécessaires pour maintenir cet élan. Le compte rendu de cette réunion ainsi que les actions futures pour soutenir la réunion scientifique M3K et le Sommet des chercheurs sont présentés dans le présent article.

ACTIVATION OF THE MITOCHONDRIAL ANTIVIRAL SIGNALING PROTEIN (MAVS) FOLLOWING LIVER ISCHEMIA/REPERFUSION AND ITS EFFECT ON INFLAMMATION AND INJURY.

ABSTRACT: Resuscitation of trauma patients after hemorrhagic shock causes global I/R, which may contribute to organ dysfunction. Oxidative stress resulting from I/R is known to induce signaling pathways leading to the production of inflammatory molecules culminating in organ dysfunction/injury. Our recent work demonstrated that oxidative stress was able to induce activation of the mitochondrial antiviral signaling protein (MAVS), a protein known to be involved in antiviral immunity, in an in vitro model. We therefore hypothesized that the MAVS pathway might be involved in I/R-induced inflammation and injury. The present studies show that MAVS is activated in vivo by liver I/R and in vitro in RAW 264.7 cells by hypoxia/reoxygenation (H/R). We utilized both in vivo (liver I/R in MAVS knockout mice) and in vitro (MAVS siRNA in RAW 264.7 cells followed by H/R) models to study the role of MAVS activation on downstream events. In vivo , we demonstrated augmented injury and inflammation in MAVS knockout mice compared with wild-type animals; as shown by increased hepatocellular injury, induction of hepatocyte apoptosis augmented plasma TNF-α levels. Further, in vitro silencing of MAVS by specific siRNA in RAW 264.7 and exposure of the cells to H/R caused activation of mitophagy. This may represent a compensatory response to increased liver inflammation. We conclude that activation of MAVS by hypoxia/reoxygenation dampens inflammation, potentially suggesting a novel target for intervention.

DDR1 associates with TRPV4 in cell-matrix adhesions to enable calcium-regulated myosin activity and collagen compaction.

Tissue fibrosis manifests as excessive deposition of compacted, highly aligned collagen fibrils, which interfere with organ structure and function. Cells in collagen-rich lesions often exhibit marked overexpression of discoidin domain receptor 1 (DDR1), which is linked to increased collagen compaction through the association of DDR1 with the Ca2+ -dependent nonmuscle myosin IIA (NMIIA). We examined the functional relationship between DDR1 and the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca2+ -permeable ion channel that is implicated in collagen compaction. Fibroblasts expressing high levels of DDR1 were used to model cells in lesions with collagen compaction. In these cells, the expression of the ß1 integrin was deleted to simplify studies of DDR1 function. Compared with DDR1 wild-type cells, high DDR1 expression was associated with increased Ca2+ influx through TRPV4, enrichment of TRPV4 in collagen adhesions, and enhanced contractile activity mediated by NMIIA. At cell adhesion sites to collagen, DDR1 associated with TRPV4, which enhanced DDR1-mediated collagen alignment and compaction. We conclude that DDR1 regulates Ca2+ influx through the TRPV4 channel to promote critical, DDR1-mediated processes that are important in lesions with collagen compaction and alignment.

Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers.

A new approach for improved RT-PCR is described. It is based on primers designed to form controlled stem-loop and homodimer configurations, hence the name 'double-bubble' primers. The primers contain three main regions for efficient RT-PCR: a 3' short overhang to allow reverse transcription, a stem region for hot start and a template-specific region for PCR amplification. As proof of principle, GAPDH, SARS-CoV-2 synthetic RNA and SARS-CoV-2 virus-positive nasopharyngeal swabs were used as templates. Additionally, these primers were used to positively confirm the N501Y mutation from nasopharyngeal swabs. Evidence is presented that the double-bubble primers offer fast, specific, robust and cost-effective improvement in RT-PCR amplification for detection of gene expression in general and for diagnostic detection and genotyping of SARS-CoV-2 in particular.

Myocardin-related transcription factor and serum response factor regulate cilium turnover by both transcriptional and local mechanisms.

Turnover of the primary cilium (PC) is critical for proliferation and tissue homeostasis. Each key component of the PC resorption machinery, the HEF1/Aurora kinase A (AurA)/HDAC6 pathway harbors cis-elements potentially targeted by the transcriptional co-activator myocardin-related transcription factor (MRTF) and/or its partner serum response factor (SRF). Thus we investigated if MRTF and/or SRF regulate PC turnover. Here we show that (1) both MRTF and SRF are indispensable for serum-induced PC resorption, and (2) they act via both transcriptional and local mechanisms. Intriguingly, MRTF and SRF are present in the basal body and/or the PC, and serum facilitates ciliary MRTF recruitment. MRTF promotes the stability and ciliary accumulation of AurA and facilitates SRF phosphorylation. Ciliary SRF interacts with AurA and HDAC6. MRTF also inhibits ciliogenesis. It interacts with and is required for the correct localization of the ciliogenesis modulator CEP290. Thus, MRTF and SRF are critical regulators of PC assembly and/or disassembly, acting both as transcription factors and as PC constituents.

MRTF: Basic Biology and Role in Kidney Disease.

A lesser known but crucially important downstream effect of Rho family GTPases is the regulation of gene expression. This major role is mediated via the cytoskeleton, the organization of which dictates the nucleocytoplasmic shuttling of a set of transcription factors. Central among these is myocardin-related transcription factor (MRTF), which upon actin polymerization translocates to the nucleus and binds to its cognate partner, serum response factor (SRF). The MRTF/SRF complex then drives a large cohort of genes involved in cytoskeleton remodeling, contractility, extracellular matrix organization and many other processes. Accordingly, MRTF, activated by a variety of mechanical and chemical stimuli, affects a plethora of functions with physiological and pathological relevance. These include cell motility, development, metabolism and thus metastasis formation, inflammatory responses and-predominantly-organ fibrosis. The aim of this review is twofold: to provide an up-to-date summary about the basic biology and regulation of this versatile transcriptional coactivator; and to highlight its principal involvement in the pathobiology of kidney disease. Acting through both direct transcriptional and epigenetic mechanisms, MRTF plays a key (yet not fully appreciated) role in the induction of a profibrotic epithelial phenotype (PEP) as well as in fibroblast-myofibroblast transition, prime pathomechanisms in chronic kidney disease and renal fibrosis.

Nucleocytoplasmic Shuttling of the Mechanosensitive Transcription Factors MRTF and YAP /TAZ.

Myocardin-related transcription factor (MRTF) and the paralogous Hippo pathway effectors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are transcriptional co-activators that play pivotal roles in myofibroblast generation and activation, and thus the pathogenesis of organ fibrosis. They are regulated by a variety of chemical and mechanical fibrogenic stimuli, primarily at the level of their nucleocytoplasmic shuttling. In this chapter we describe the tools and protocols that allow for exact, quantitative, and automated determination and analysis of the nucleocytoplasmic distribution of endogenous or heterologously expressed MRTF and YAP/TAZ, measured in large cell populations. Dynamic monitoring of nucleocytoplasmic ratios of transcription factors is a novel and important approach, suitable to address both the structural requirements and the regulatory mechanisms underlying transcription factor traffic and the consequent reprogramming of gene expression during fibrogenesis.

Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts.

We identify the focal adhesion protein kindlin-2 as player in a novel mechanotransduction pathway that controls profibrotic cardiac fibroblast to myofibroblast activation. Kindlin-2 is co-upregulated with the myofibroblast marker α-smooth muscle actin (α-SMA) in fibrotic rat hearts and in human cardiac fibroblasts exposed to fibrosis-stiff culture substrates and pro-fibrotic TGF-ß1. Stressing fibroblasts using ferromagnetic microbeads, stretchable silicone membranes, and cell contraction agonists all result in kindlin-2 translocation to the nucleus. Overexpression of full-length kindlin-2 but not of kindlin-2 missing a putative nuclear localization sequence (∆NLS kindlin-2) results in increased α-SMA promoter activity. Downregulating kindlin-2 with siRNA leads to decreased myofibroblast contraction and reduced α-SMA expression, which is dependent on CC(A/T)-rich GG(CArG) box elements in the α-SMA promoter. Lost myofibroblast features under kindlin-2 knockdown are rescued with wild-type but not ∆NLS kindlin-2, indicating that myofibroblast control by kindlin-2 requires its nuclear translocation. Because kindlin-2 can act as a mechanotransducer regulating the transcription of α-SMA, it is a potential target to interfere with myofibroblast activation in tissue fibrosis.

Avoidance of apoptotic death via a hyperploid salvage survival pathway after platinum treatment in high grade serous carcinoma cell line models.

The alkylating agent platinum is first-line chemotherapy treatment for high-grade serous carcinomas (HGSC) of tubal-ovarian origin. Platinum compounds cause DNA damage and induce apoptotic cell death in the bulk tumor population. However, subpopulations of tumor cells may exhibit diverging behaviors from the bulk tumor due to an alternate stress response that diverts tumor cells from apoptotic death. In this study, we identified a salvage survival pathway in which G2-arrested tumor cells bypassed apoptosis and progressed through aberrant mitotic events to then emerge as a distinct subpopulation of viable large hyperploid cells but with uncertain long-term propagation potential. Platinum-induced large hyperploid cells were flow sorted and showed rare regrowth capacity as compared to their more proficiently regenerating non-hyperploid counterparts. However, detailed time-lapse microscopy provided direct evidence that these hyperploid cells were mitotically active and could divide successfully to produce viable daughter cells. The hyperploid survival response was observed across different cell lines and utilization of this survival pathway was dependent on the strength of the G2-M checkpoint. Conceivably, this salvage survival strategy may contribute to increased genomic diversity of the regenerating tumor cell line through a coupled hyperploidization and de-polyploidization process that may be relevant for drug resistance.

Claudin-2 suppresses GEF-H1, RHOA, and MRTF, thereby impacting proliferation and profibrotic phenotype of tubular cells.

The tight junctional pore-forming protein claudin-2 (CLDN-2) mediates paracellular Na+ and water transport in leaky epithelia and alters cancer cell proliferation. Previously, we reported that tumor necrosis factor-α time-dependently alters CLDN-2 expression in tubular epithelial cells. Here, we found a similar expression pattern in a mouse kidney injury model (unilateral ureteral obstruction), consisting of an initial increase followed by a drop in CLDN-2 protein expression. CLDN-2 silencing in LLC-PK1 tubular cells induced activation and phosphorylation of guanine nucleotide exchange factor H1 (GEF-H1), leading to Ras homolog family member A (RHOA) activation. Silencing of other claudins had no such effects, and re-expression of an siRNA-resistant CLDN-2 prevented RHOA activation, indicating specific effects of CLDN-2 on RHOA. Moreover, kidneys from CLDN-2 knockout mice had elevated levels of active RHOA. Of note, CLDN-2 silencing reduced LLC-PK1 cell proliferation and elevated expression of cyclin-dependent kinase inhibitor P27 (P27KIP1) in a GEF-H1/RHOA-dependent manner. P27KIP1 silencing abrogated the effects of CLDN-2 depletion on proliferation. CLDN-2 loss also activated myocardin-related transcription factor (MRTF), a fibrogenic RHOA effector, and elevated expression of connective tissue growth factor and smooth muscle actin. Finally, CLDN-2 down-regulation contributed to RHOA activation and smooth muscle actin expression induced by prolonged tumor necrosis factor-α treatment, because they were mitigated by re-expression of CLDN-2. Our results indicate that CLDN-2 suppresses GEF-H1/RHOA. CLDN-2 down-regulation, for example, by inflammation, can reduce proliferation and promote MRTF activation through RHOA. These findings suggest that the initial CLDN-2 elevation might aid epithelial regeneration, and CLDN-2 loss could contribute to fibrotic reprogramming.

The Ion Transporter NKCC1 Links Cell Volume to Cell Mass Regulation by Suppressing mTORC1.

mTORC1 regulates cellular growth and is activated by growth factors and by essential amino acids such as Leu. Leu enters cells via the Leu transporter LAT1-4F2hc (LAT1). Here we show that the Na+/K+/2Cl- cotransporter NKCC1 (SLC12A2), a known regulator of cell volume, is present in complex with LAT1. We further show that NKCC1 depletion or deletion enhances LAT1 activity, as well as activation of Akt and Erk, leading to activation of mTORC1 in cells, colonic organoids, and mouse colon. Moreover, NKCC1 depletion reduces intracellular Na+ concentration and cell volume (size) and mass and stimulates cell proliferation. NKCC1, therefore, suppresses mTORC1 by inhibiting its key activating signaling pathways. Importantly, by linking ion transport and cell volume regulation to mTORC1 function, NKCC1 provides a long-sought link connecting cell volume (size) to cell mass regulation.

Profibrotic epithelial phenotype: a central role for MRTF and TAZ.

Epithelial injury is a key initiator of fibrosis but - in contrast to the previous paradigm - the epithelium in situ does not undergo wide-spread epithelial-mesenchymal/myofibroblast transition (EMT/EMyT). Instead, it assumes a Profibrotic Epithelial Phenotype (PEP) characterized by fibrogenic cytokine production. The transcriptional mechanisms underlying PEP are undefined. As we have shown that two RhoA/cytoskeleton-regulated transcriptional coactivators, Myocardin-related transcription factor (MRTF) and TAZ, are indispensable for EMyT, we asked if they might mediate PEP as well. Here we show that mechanical stress (cyclic stretch) increased the expression of transforming growth factor-ß1 (TGFß1), connective tissue growth factor (CTGF), platelet-derived growth factor and Indian Hedgehog mRNA in LLC-PK1 tubular cells. These responses were mitigated by siRNA-mediated silencing or pharmacological inhibition of MRTF (CCG-1423) or TAZ (verteporfin). RhoA inhibition exerted similar effects. Unilateral ureteral obstruction, a murine model of mechanically-triggered kidney fibrosis, induced tubular RhoA activation along with overexpression/nuclear accumulation of MRTF and TAZ, and increased transcription of the above-mentioned cytokines. Laser capture microdissection revealed TAZ, TGFß1 and CTGF induction specifically in the tubular epithelium. CCG-1423 suppressed total renal and tubular expression of these proteins. Thus, MRTF regulates epithelial TAZ expression, and both MRTF and TAZ are critical mediators of PEP-related epithelial cytokine production.

Hemorrhagic Shock/Resuscitation Reduces the M2 Phenotype of Alveolar Macrophages: A Potential Mechanism Contributing to Increased LPS-Induced Lung Injury.

Major hemorrhage is a significant contributor to the morbidity and mortality resulting from traumatic injury. In addition to its role in in early mortality, hemorrhagic shock followed by resuscitation (HS/R) is known to initiate immunological events that contribute to the development of organ dysfunction. The pathogenesis of acute lung injury following HS/R involves macrophage activation. Recent studies have shown that macrophage function may in part be regulated by polarization toward classical M1 pro-inflammatory cells or alternatively activated anti-inflammatory M2 cells. We hypothesized that alteration in the M1/M2 phenotypic balance of alveolar macrophages in the lung may contribute to a pro-inflammatory state following HS/R. Using a murine model, we show that HS/R causes a rapid reduction in surface cluster of differentiation (CD)206 and CD36, markers of M2 cells, as well as in CD206 messenger ribonucleic acid (mRNA). M1 markers including surface CD80 and tumour necrosis factor alpha and inducible nitric oxide synthase mRNA were increased, albeit in a somewhat delayed time course. The prostaglandin 5-deoxyDelta12,14 prostaglandin J2 (15d-PGJ2), known to polarize cells toward M2, restored levels of M2 macrophages toward control and prevented lung injury, as assessed by bronchoalveolar protein content. Adoptive cell transfer of in vitro M2 polarized macrophages also reduced lung inflammation/injury following hemorrhagic shock. Together, these studies demonstrate that HS/R increases M1/M2 ratio, predominantly by lowering M2 cells, and thus enhances the proinflammatory state. Various strategies aimed at promoting M2 polarization may lessen the magnitude of inflammation and injury. This represents a novel approach to the prevention/treatment of lung injury in critically ill trauma patients.

Sunitinib induces early histomolecular changes in a subset of renal cancer cells that contribute to resistance.

Sunitinib is the standard-of-care, first-line treatment for advanced renal cell carcinoma (RCC). Characteristics of treatment-resistant RCC have been described; however, complex tumor adaptation mechanisms obstruct the identification of significant operators in resistance. We hypothesized that resistance is a late manifestation of early, treatment-induced histomolecular alterations; therefore, studying early drug response may identify drivers of resistance. We describe an epithelioid RCC growth pattern in RCC xenografts, which emerges in sunitinib-sensitive tumors and is augmented during resistance. This growth modality is molecularly and morphologically related to the RCC spheroids that advance during in vitro treatment. Based on time-lapse microscopy, mRNA and microRNA screening, and tumor behavior-related characteristics, we propose that the spheroid and adherent RCC growth patterns differentially respond to sunitinib. Gene expression analysis indicated that sunitinib promoted spheroid formation, which provided a selective survival advantage under treatment. Functional studies confirm that E-cadherin is a key contributor to the survival of RCC cells under sunitinib treatment. In summary, we suggest that sunitinib-resistant RCC cells exist in treatment-sensitive tumors and are histologically identifiable.-Lichner, Z., Saleeb, R., Butz, H., Ding, Q., Nofech-Mozes, R., Riad, S., Farag, M., Varkouhi, A. K., dos Santos, C. C., Kapus, A., Yousef, G. M. Sunitinib induces early histomolecular changes in a subset of renal cancer cells that contribute to resistance.