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.

Key Role of Mesenchymal Stromal Cell Interaction with Macrophages in Promoting Repair of Lung Injury.

Lung macrophages (Mφs) are essential for pulmonary innate immunity and host defense due to their dynamic polarization and phenotype shifts. Mesenchymal stromal cells (MSCs) have secretory, immunomodulatory, and tissue-reparative properties and have shown promise in acute and chronic inflammatory lung diseases and in COVID-19. Many beneficial effects of MSCs are mediated through their interaction with resident alveolar and pulmonary interstitial Mφs. Bidirectional MSC-Mφ communication is achieved through direct contact, soluble factor secretion/activation, and organelle transfer. The lung microenvironment facilitates MSC secretion of factors that result in Mφ polarization towards an immunosuppressive M2-like phenotype for the restoration of tissue homeostasis. M2-like Mφ in turn can affect the MSC immune regulatory function in MSC engraftment and tissue reparatory effects. This review article highlights the mechanisms of crosstalk between MSCs and Mφs and the potential role of their interaction in lung repair in inflammatory lung diseases.

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.

Mesenchymal stromal (stem) cell therapy modulates miR-193b-5p expression to attenuate sepsis-induced acute lung injury.

Although mesenchymal stromal (stem) cell (MSC) administration attenuates sepsis-induced lung injury in pre-clinical models, the mechanism(s) of action and host immune system contributions to its therapeutic effects remain elusive. We show that treatment with MSCs decreased expression of host-derived microRNA (miR)-193b-5p and increased expression of its target gene, the tight junctional protein occludin (Ocln), in lungs from septic mice. Mutating the Ocln 3' untranslated region miR-193b-5p binding sequence impaired binding to Ocln mRNA. Inhibition of miR-193b-5p in human primary pulmonary microvascular endothelial cells prevents tumour necrosis factor (TNF)-induced decrease in Ocln gene and protein expression and loss of barrier function. MSC-conditioned media mitigated TNF-induced miR-193b-5p upregulation and Ocln downregulation in vitro When administered in vivo, MSC-conditioned media recapitulated the effects of MSC administration on pulmonary miR-193b-5p and Ocln expression. MiR-193b-deficient mice were resistant to pulmonary inflammation and injury induced by lipopolysaccharide (LPS) instillation. Silencing of Ocln in miR-193b-deficient mice partially recovered the susceptibility to LPS-induced lung injury. In vivo inhibition of miR-193b-5p protected mice from endotoxin-induced lung injury. Finally, the clinical significance of these results was supported by the finding of increased miR-193b-5p expression levels in lung autopsy samples from acute respiratory distress syndrome patients who died with diffuse alveolar damage.

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.

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.

Tumor necrosis factor-α induces claudin-3 upregulation in kidney tubular epithelial cells through NF-κB and CREB1.

Claudins are essential for tight junction formation and paracellular transport, and they affect key cellular events including proliferation and migration. The properties of tight junctions are dynamically modulated by a variety of inputs. We previously showed that the inflammatory cytokine tumor necrosis factor-α (TNFα), a major pathogenic factor in kidney disease, alters epithelial permeability by affecting the expression of claudin-1, -2, and -4 in kidney tubular cells. Here, we explored the effect of TNFα on claudin-3 (Cldn-3), a ubiquitous barrier-forming protein. We found that TNFα elevated Cldn-3 protein expression in tubular epithelial cells (LLC-PK1 and IMCD3) as early as 3 h post treatment. Bafilomycin A and bortezomib, inhibitors of lysosomal and proteasomes, respectively, reduced Cldn-3 degradation. However, TNFα caused a strong upregulation of Cldn-3 in the presence of bafilomycin, suggesting an effect independent from lysosomes. Blocking protein synthesis using cycloheximide prevented Cldn-3 upregulation by TNFα, verifying the contribution of de novo Cldn-3 synthesis. Indeed, TNFα elevated Cldn-3 mRNA levels at early time points. Using pharmacological inhibitors and siRNA-mediated silencing, we determined that the effect of TNFα on Cldn-3 was mediated by extracellular signal regulated kinase (ERK)-dependent activation of NF-κB and PKA-induced activation of CREB1. These two pathways were turned on by TNFα in parallel and both were required for the upregulation of Cldn-3. Because Cldn-3 was suggested to modulate cell migration and epithelial-mesenchymal transition (EMT), and TNFα was shown to affect these processes, Cldn-3 upregulation may modulate regeneration of the tubules following injury.

Immunofluorescent Staining of Claudin-2 in Cultured Kidney Tubular Cells.

Members of the claudin family of tight junction proteins regulate paracellular permeability and modulate cell signaling. During junction remodeling, these proteins are selectively inserted into or retrieved from the tight junctions, but the control and coordination of these processes remain incompletely understood. Visualization of claudins allows the assessment of changes in their localization and abundance. We use the described protocol to stain claudin-2, but it can also be adapted to stain any tight junction protein. We found that using methanol for fixing allows the best preservation of claudin-2 both at the membrane and in cytoplasmic vesicles. Staining is done using a claudin-2 specific primary and a fluorescently labelled secondary antibody, along with DAPI to label nuclei. The samples are then imaged using confocal microscopy, and a z-stack is obtained allowing visualization of both junctional and intracellular claudin-2. Total claudin-2 signal can be quantified after 3D reconstruction of the images using the Imaris software.

Measuring Cell Growth and Junction Development in Epithelial Cells Using Electric Cell-Substrate Impedance Sensing (ECIS).

Electric Cell-substrate Impedance Sensing (ECIS) is an automated method that can be used to quantify processes such as cell attachment, growth, migration and barrier functions (i.e., the properties of tight junctions). The method provides simultaneous information on cell number and tight junction function by detecting electric parameters of cells grown on electrodes. Samples are probed with small alternating current (AC) over a range of frequencies, and changes in capacitance and impedance are measured over time. Capacitance reflects the degree of electrode coverage by cells, that correlates with cell number, and can be used to assess cell proliferation or migration. Impedance values inform about barrier function. Obtaining real-time simultaneous information on these parameters is unique to this system and is of great value for addressing fundamental questions such as the role of tight junction proteins in cell growth and migration. This protocol describes the use of ECIS to follow cell growth and tight junction-dependent barrier generation in tubular epithelial cells. We used this method to explore how depleting claudin-2, a tight junction protein affects tubular cell growth and barrier function. During the process, cells are transfected with control or claudin-2-specific siRNA, and 24h later plated on electrodes. ECIS automatically collects information on cell growth and barrier as the monolayer develops. The data are initially analyzed using the ECIS software and exported into a graph software for further processing.

Claudin-2: Roles beyond Permeability Functions.

Claudin-2 is expressed in the tight junctions of leaky epithelia, where it forms cation-selective and water permeable paracellular channels. Its abundance is under fine control by a complex signaling network that affects both its synthesis and turnover in response to various environmental inputs. Claudin-2 expression is dysregulated in many pathologies including cancer, inflammation, and fibrosis. Claudin-2 has a key role in energy-efficient ion and water transport in the proximal tubules of the kidneys and in the gut. Importantly, strong evidence now also supports a role for this protein as a modulator of vital cellular events relevant to diseases. Signaling pathways that are overactivated in diseases can alter claudin-2 expression, and a good correlation exists between disease stage and claudin-2 abundance. Further, loss- and gain-of-function studies showed that primary changes in claudin-2 expression impact vital cellular processes such as proliferation, migration, and cell fate determination. These effects appear to be mediated by alterations in key signaling pathways. The specific mechanisms linking claudin-2 to these changes remain poorly understood, but adapters binding to the intracellular portion of claudin-2 may play a key role. Thus, dysregulation of claudin-2 may contribute to the generation, maintenance, and/or progression of diseases through both permeability-dependent and -independent mechanisms. The aim of this review is to provide an overview of the properties, regulation, and functions of claudin-2, with a special emphasis on its signal-modulating effects and possible role in diseases.

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.

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.

Nuclear Factor (Erythroid-Derived 2)-Like 2 Regulates the Hepatoprotective Effects of Remote Ischemic Conditioning in Hemorrhagic Shock.

AIMS: Remote ischemic conditioning (RIC) protects against organ ischemia/reperfusion injury in experimental and clinical settings. We have demonstrated that RIC prevents liver and lung inflammation/injury after hemorrhagic shock/resuscitation (S/R). In this study, we used a murine model of S/R to investigate the role of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in mediating hepatoprotection. RESULTS: The combination of RIC with S/R caused a synergistic rise in Nrf2 and its translocation to the nucleus in the liver. Increased activation of Nrf2 by RIC augmented heme oxygenase-1 (HO-1) and autophagy and exerted hepatoprotection, concurrent with reductions in S/R-induced TNF-α (tumor necrosis factor alpha) and IL-6 (interleukin-6). In Nrf2 knockout (KO) animals, RIC did not exert hepatoprotection, and it failed to upregulate HO-1 and autophagy. Further, resuscitating wildtype (WT) animals with blood from donor WT animals undergoing RIC was hepatoprotective, but not in Nrf2 KO recipient animals. Interestingly, RIC blood from Nrf2 KO donor animals was also not protective when used to resuscitate WT animals, suggesting a role for Nrf2 both in the afferent arm of RIC where protective factors are generated and also in the efferent arm where organ protection is exerted. Finally, RIC plasma prevented oxidant-induced zebrafish mortality, but not in Nrf2a morpholino knockdown fish. INNOVATION: Activation of Nrf2 is an essential mechanism underlying the hepatoprotective effects of RIC. Nrf2 appears to play a role in the afferent limb of RIC protection, as its absence precludes the generation of the protective humoral factors induced by RIC. CONCLUSION: Our studies demonstrate the critical role of Nrf2 in the ability of RIC to prevent organ injury after S/R.

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.

Mediated nuclear import and export of TAZ and the underlying molecular requirements.

Nucleocytoplasmic distribution of Yap/TAZ is regulated by the Hippo pathway and the cytoskeleton. While interactions with cytosolic and nuclear "retention factors" (14-3-3 and TEAD) are known to control their localization, fundamental aspects of Yap/TAZ shuttling remain undefined. It is unclear if translocation occurs only by passive diffusion or via mediated transport, and neither the potential nuclear localization and efflux signals (NLS, NES) nor their putative regulation have been identified. Here we show that TAZ cycling is a mediated process and identify the underlying NLS and NES. The C-terminal NLS, representing a new class of import motifs, is necessary and sufficient for efficient nuclear uptake via a RAN-independent mechanism. RhoA activity directly stimulates this import. The NES lies within the TEAD-binding domain and can be masked by TEAD, thereby preventing efflux. Thus, we describe a RhoA-regulated NLS, a TEAD-regulated NES and propose an improved model of nucleocytoplasmic TAZ shuttling beyond "retention".

Mesenchymal stem cells enhance NOX2-dependent reactive oxygen species production and bacterial killing in macrophages during sepsis.

Human mesenchymal stem/stromal cells (MSCs) have been reported to produce an M2-like, alternatively activated phenotype in macrophages. In addition, MSCs mediate effective bacterial clearance in pre-clinical sepsis models. Thus, MSCs have a paradoxical antimicrobial and anti-inflammatory response that is not understood.Here, we studied the phenotypic and functional response of monocyte-derived human macrophages to MSC exposure in vitroMSCs induced two distinct, coexistent phenotypes: M2-like macrophages (generally elongated morphology, CD163+, acute phagosomal acidification, low NOX2 expression and limited phagosomal superoxide production) and M1-like macrophages characterised by high levels of phagosomal superoxide production. Enhanced phagosomal reactive oxygen species production was also observed in alveolar macrophages from a rodent model of pneumonia-induced sepsis. The production of M1-like macrophages was dependent on prostaglandin E2 and phosphatidylinositol 3-kinase. MSCs enhanced human macrophage phagocytosis of unopsonised bacteria and enhanced bacterial killing compared with untreated macrophages. Bacterial killing was significantly reduced by blockade of NOX2 using diphenyleneiodonium, suggesting that M1-like cells are primarily responsible for this effect. MSCs also enhanced phagocytosis and polarisation of M1-like macrophages derived from patients with severe sepsis.The enhanced antimicrobial capacity (M1-like) and inflammation resolving phenotype (M2-like) may account for the paradoxical effect of these cells in sepsis in vivo.

Cell confluence regulates claudin-2 expression: possible role for ZO-1 and Rac.

Claudin-2 (Cldn-2) is a channel-forming tight junction (TJ) protein in the proximal tubules that mediates paracellular Na+ transport and has also emerged as a regulator of proliferation and migration. Expression of Cldn-2 is altered by numerous stimuli, but the underlying mechanisms remain incompletely understood. Here we show that Cldn-2 protein and mRNA expression were low in subconfluent tubular cells and increased during junction maturation. Cldn-1 or occludin did not exhibit similar confluence-dependence. Conversely, disruption of TJs by Ca2+ removal or silencing of zonula occludens-1 (ZO-1) or ZO-2 induced a large drop in Cldn-2 abundance. Immunofluorescent staining revealed a more uneven Cldn-2 staining in nascent, Cldn-1-positive TJs. Subconfluence and ZO-1 silencing augmented Cldn-2 degradation and reduced Cldn-2 promoter activity, suggesting that insertion into the TJs slows Cldn-2 turnover. Indeed, blocking endocytosis or lysosomal degradation increased Cldn-2 abundance. Cell confluence increased expression of the junctional adapters ZO-1 and -2, and the small GTPase Rac, and elevated Rac activity and p21-activated kinase (Pak) phosphorylation, suggesting that they might mediate confluence-dependent Cldn-2 regulation. Indeed, Rac silencing or Pak inhibition strongly reduced Cldn-2 protein abundance, which was likely the combined effect on turnover, as these interventions reduced Cldn-2 promoter activity and augmented Cldn-2 degradation. Taken together, our data suggest that TJ integrity and maturity, ZO-1 expression/TJ localization, and Rac/Pak control Cldn-2 degradation and synthesis. A feedback mechanism connecting Cldn-2 expression with junction remodeling, e.g., during wound healing, epithelial-mesenchymal transition, or tumor metastasis formation, may have important downstream effects on permeability, proliferation, and migration.

TGF-ß1 regulates the expression and transcriptional activity of TAZ protein via a Smad3-independent, myocardin-related transcription factor-mediated mechanism.

Hippo pathway transcriptional coactivators TAZ and YAP and the TGF-ß1 (TGFß) effector Smad3 regulate a common set of genes, can physically interact, and exhibit multilevel cross-talk regulating cell fate-determining and fibrogenic pathways. However, a key aspect of this cross-talk, TGFß-mediated regulation of TAZ or YAP expression, remains uncharacterized. Here, we show that TGFß induces robust TAZ but not YAP protein expression in both mesenchymal and epithelial cells. TAZ levels, and to a lesser extent YAP levels, also increased during experimental kidney fibrosis. Pharmacological or genetic inhibition of Smad3 did not prevent the TGFß-induced TAZ up-regulation, indicating that this canonical pathway is dispensable. In contrast, inhibition of p38 MAPK, its downstream effector MK2 (e.g. by the clinically approved antifibrotic pirferidone), or Akt suppressed the TGFß-induced TAZ expression. Moreover, TGFß elevated TAZ mRNA in a p38-dependent manner. Myocardin-related transcription factor (MRTF) was a central mediator of this effect, as MRTF silencing/inhibition abolished the TGFß-induced TAZ expression. MRTF overexpression drove the TAZ promoter in a CC(A/T-rich)6GG (CArG) box-dependent manner and induced TAZ protein expression. TGFß did not act by promoting nuclear MRTF translocation; instead, it triggered p38- and MK2-mediated, Nox4-promoted MRTF phosphorylation and activation. Functionally, higher TAZ levels increased TAZ/TEAD-dependent transcription and primed cells for enhanced TAZ activity upon a second stimulus (i.e. sphingosine 1-phosphate) that induced nuclear TAZ translocation. In conclusion, our results uncover an important aspect of the cross-talk between TGFß and Hippo signaling, showing that TGFß induces TAZ via a Smad3-independent, p38- and MRTF-mediated and yet MRTF translocation-independent mechanism.