All-trans retinoic acid and dexamethasone regulate phagocytosis-related gene expression and enhance dead cell uptake in C2C12 myoblast cells.

Extensive mechanical stress frequently causes micro-traumas in skeletal muscle, followed by a regeneration period. The effective removal of dead myofibers is a prerequisite for proper regeneration, and several cell types, including professional phagocytes, were reported to be active in this process. Myoblasts express several molecules of the phagocytic machinery, such as BAI1, stabilin-2, and TAM (Tyro3, Axl, Mertk) tyrosine kinase receptors, but these molecules were reported to serve primarily cell fusion and survival, and their role in the phagocytosis was not investigated. Therefore, we aimed to investigate the in vitro phagocytic capacity of the C2C12 mouse myoblast cell line. RNA sequencing data were analyzed to determine the level and changes of phagocytosis-related gene expression during the differentiation process of C2C12 cells. To study the phagocytic capacity of myoblasts and the effect of dexamethasone, all-trans retinoic acid, hemin, and TAM kinase inhibitor treatments on phagocytosis, C2C12 cells were fed dead thymocytes, and their phagocytic capacity was determined by flow cytometry. The effect of dexamethasone and all-trans retinoic acid on phagocytosis-related gene expression was determined by quantitative PCR. Both undifferentiated and differentiated cells engulfed dead cells being the undifferentiated cells more effective. In line with this, we observed that the expression of several phagocytosis-related genes was downregulated during the differentiation process. The phagocytosis could be increased by dexamethasone and all-trans retinoic acid and decreased by hemin and TAM kinase inhibitor treatments. Our results indicate that myoblasts not only express phagocytic machinery genes but are capable of efficient dead cell clearance as well, and this is regulated similarly, as reported in professional phagocytes.

Loss of adenosine A3 receptors accelerates skeletal muscle regeneration in mice following cardiotoxin-induced injury.

Skeletal muscle regeneration is a complex process orchestrated by multiple interacting steps. An increasing number of reports indicate that inflammatory responses play a central role in linking initial muscle injury responses to timely muscle regeneration following injury. The nucleoside adenosine has been known for a long time as an endogenously produced anti-inflammatory molecule that is generated in high amounts during tissue injury. It mediates its physiological effects via four types of adenosine receptors. From these, adenosine A3 receptors (A3Rs) are not expressed by the skeletal muscle but are present on the surface of various inflammatory cells. In the present paper, the effect of the loss of A3Rs was investigated on the regeneration of the tibialis anterior (TA) muscle in mice following cardiotoxin-induced injury. Here we report that regeneration of the skeletal muscle from A3R-/- mice is characterized by a stronger initial inflammatory response resulting in a larger number of transmigrating inflammatory cells to the injury site, faster clearance of cell debris, enhanced proliferation and faster differentiation of the satellite cells (the muscle stem cells), and increased fusion of the generated myoblasts. This leads to accelerated skeletal muscle tissue repair and the formation of larger myofibers. Though the infiltrating immune cells expressed A3Rs and showed an increased inflammatory profile in the injured A3R-/- muscles, bone marrow transplantation experiments revealed that the increased response of the tissue-resident cells to tissue injury is responsible for the observed phenomenon. Altogether our data indicate that A3Rs are negative regulators of injury-related regenerative inflammation and consequently also that of the muscle fiber growth in the TA muscle. Thus, inhibiting A3Rs might have a therapeutic value during skeletal muscle regeneration following injury.

Nur77 and PPARγ regulate transcription and polarization in distinct subsets of M2-like reparative macrophages during regenerative inflammation.

Macrophage polarization is a process whereby macrophages develop a specific phenotype and functional response to different pathophysiological stimuli and tissue environments. In general, two main macrophage phenotypes have been identified: inflammatory (M1) and alternatively activated (M2) macrophages characterized specifically by IL-1ß and IL-10 production, respectively. In the cardiotoxin-induced skeletal muscle injury model bone marrow-derived macrophages (BMDMs) play the central role in regulating tissue repair. Bone marrow-derived monocytes arriving at the site of injury differentiate first to M1 BMDMs that clear cell debris and trigger proliferation and differentiation of the muscle stem cells, while during the process of efferocytosis they change their phenotype to M2 to drive resolution of inflammation and tissue repair. The M2 population is formed from at least three distinct subsets: antigen presenting, resolution-related and growth factor producing macrophages, the latest ones expressing the transcription factor PPARγ. Nuclear receptor subfamily 4 group A member 1 (NR4A1; also termed Nur77) transcription factor is expressed as an early response gene, and has been shown to suppress the expression of pro-inflammatory genes during efferocytosis. Here we demonstrate that (1) Nur77 null BMDMs are characterized by elevated expression of PPARγ resulting in enhanced efferocytosis capacity; (2) Nur77 and PPARγ regulate transcription in different subsets of M2 skeletal muscle macrophages during muscle repair; (3) the loss of Nur77 prolongs M1 polarization characterized by increased and prolonged production of IL-1ß by the resolution-related macrophages normally expressing Nur77; whereas, in contrast, (4) it promotes M2 polarization detected via the increased number of IL-10 producing CD206+ macrophages generated from the PPARγ-expressing subset.

Transglutaminase 2 associated with PI3K and PTEN in a membrane-bound signalosome platform blunts cell death.

Atypically expressed transglutaminase 2 (TG2) has been identified as a poor prognostic factor in a variety of cancers. In this study, we evaluated the contribution of TG2 to the prolonged cell survival of differentiated acute promyelocytic leukaemia (APL) cells in response to the standard treatment with combined retinoic acid (ATRA) and arsenic trioxide (ATO). We report that one advantage of ATRA + ATO treatment compared to ATRA alone diminishes the amount of activated and non-activated CD11b/CD18 and CD11c/CD18 cell surface integrin receptors. These changes suppress ATRA-induced TG2 docking on the cytosolic part of CD18 ß2-integrin subunits and reduce cell survival. In addition, TG2 overexpresses and hyperactivates the phosphatidylinositol-3-kinase (PI3K), phospho-AKT S473, and phospho-mTOR S2481 signalling axis. mTORC2 acts as a functional switch between cell survival and death by promoting the full activation of AKT. We show that TG2 presumably triggers the formation of a signalosome platform, hyperactivates downstream mTORC2-AKT signalling, which in turn phosphorylates and inhibits the activity of FOXO3, a key pro-apoptotic transcription factor. In contrast, the absence of TG2 restores basic phospho-mTOR S2481, phospho-AKT S473, PI3K, and PTEN expression and activity, thereby sensitising APL cells to ATO-induced cell death. We conclude, that atypically expressed TG2 may serve as a hub, facilitating signal transduction via signalosome formation by the CD18 subunit with both PI3K hyperactivation and PTEN inactivation through the PI3K-PTEN cycle in ATRA-treated APL cells.

Retinoids Promote Mouse Bone Marrow-Derived Macrophage Differentiation and Efferocytosis via Upregulating Bone Morphogenetic Protein-2 and Smad3.

Clearance of apoptotic cells by bone marrow-derived macrophages differentiated from monocytes plays a central role in the resolution of inflammation, as the conversion of pro-inflammatory M1 macrophages to M2 macrophages that mediate the resolution process occurs during efferocytosis. Thus, proper efferocytosis is a prerequisite for proper resolution of inflammation, and failure in efferocytosis is associated with the development of chronic inflammatory diseases. Previous studies from our laboratory have shown that (13R)-all-trans-13,14-dihydroretinol (DHR), the product of retinol saturase, acting from day 4 of monocyte differentiation enhances the efferocytosis capacity of the resulted macrophages. Loss of retinol saturase in mice leads to impaired efferocytosis, and to development of autoimmunity. In the present paper, we report that in differentiating monocytes DHR, retinol, and all-trans retinoic acid all act directly on retinoic acid receptors and enhance the clearance of apoptotic cells by upregulating the expression of several efferocytosis-related genes. The effect of retinoids seems to be mediated by bone morphogenetic protein (BMP)-2, and the Smad3 transcription factor. In addition, retinoids also upregulate the expression of the vitamin D receptor and that of vascular endothelial growth factor A, indicating that altogether retinoids promote the generation of a pro-reparative M2 macrophage population during monocyte differentiation.

Involvement of phosphatidylserine receptors in the skeletal muscle regeneration: therapeutic implications.

Sarcopenia is a progressive loss of muscle mass and strength with a risk of adverse outcomes such as disability, poor quality of life, and death. Increasing evidence indicates that diminished ability of the muscle to activate satellite cell-dependent regeneration is one of the factors that might contribute to its development. Skeletal muscle regeneration following myogenic cell death results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibres. Satellite cell differentiation is not a satellite cell-autonomous process but depends on signals provided by the surrounding cells. Infiltrating macrophages play a key role in the process partly by clearing the necrotic cell debris, partly by producing cytokines and growth factors that guide myogenesis. At the beginning of the muscle regeneration process, macrophages are pro-inflammatory, and the cytokines produced by them trigger the proliferation and differentiation of satellite cells. Following the uptake of dead cells, however, a transcriptionally regulated phenotypic change (macrophage polarization) is induced in them resulting in their transformation into healing macrophages that guide resolution of inflammation, completion of myoblast differentiation, myoblast fusion and growth, and return to homeostasis. Impaired efferocytosis results in delayed cell death clearance, delayed macrophage polarization, prolonged inflammation, and impaired muscle regeneration. Thus, proper efferocytosis by macrophages is a determining factor during muscle repair. Here we review that both efferocytosis and myogenesis are dependent on the cell surface phosphatidylserine (PS), and surprisingly, these two processes share a number of common PS receptors and signalling pathways. Based on these findings, we propose that stimulating the function of PS receptors for facilitating muscle repair following injury could be a successful approach, as it would enhance efferocytosis and myogenesis simultaneously. Because increasing evidence indicates a pathophysiological role of impaired efferocytosis in the development of chronic inflammatory conditions, as well as in impaired muscle regeneration both contributing to the development of sarcopenia, improving efferocytosis should be considered also in its management. Again applying or combining those treatments that target PS receptors would be expected to be the most effective, because they would also promote myogenesis. A potential PS receptor-triggering candidate molecule is milk fat globule-EGF-factor 8 (MFG-E8), which not only stimulates PS-dependent efferocytosis and myoblast fusion but also promotes extracellular signal-regulated kinase (ERK) and Akt activation-mediated cell proliferation and cell cycle progression in myoblasts.

Regenerating Skeletal Muscle Compensates for the Impaired Macrophage Functions Leading to Normal Muscle Repair in Retinol Saturase Null Mice.

Skeletal muscle repair is initiated by local inflammation and involves the engulfment of dead cells (efferocytosis) by infiltrating macrophages at the injury site. Macrophages orchestrate the whole repair program, and efferocytosis is a key event not only for cell clearance but also for triggering the timed polarization of the inflammatory phenotype of macrophages into the healing one. While pro-inflammatory cytokines produced by the inflammatory macrophages induce satellite cell proliferation and differentiation into myoblasts, healing macrophages initiate the resolution of inflammation, angiogenesis, and extracellular matrix formation and drive myoblast fusion and myotube growth. Therefore, improper efferocytosis results in impaired muscle repair. Retinol saturase (RetSat) initiates the formation of various dihydroretinoids, a group of vitamin A derivatives that regulate transcription by activating retinoid receptors. Previous studies from our laboratory have shown that RetSat-null macrophages produce less milk fat globule-epidermal growth factor-factor-8 (MFG-E8), lack neuropeptide Y expression, and are characterized by impaired efferocytosis. Here, we investigated skeletal muscle repair in the tibialis anterior muscle of RetSat-null mice following cardiotoxin injury. Our data presented here demonstrate that, unexpectedly, several cell types participating in skeletal muscle regeneration compensate for the impaired macrophage functions, resulting in normal muscle repair in the RetSat-null mice.

Impaired Skeletal Muscle Development and Regeneration in Transglutaminase 2 Knockout Mice.

Skeletal muscle regeneration is triggered by local inflammation and is accompanied by phagocytosis of dead cells at the injury site. Efferocytosis regulates the inflammatory program in macrophages by initiating the conversion of their inflammatory phenotype into the healing one. While pro-inflammatory cytokines induce satellite cell proliferation and differentiation into myoblasts, growth factors, such as GDF3, released by healing macrophages drive myoblast fusion and myotube growth. Therefore, improper efferocytosis may lead to impaired muscle regeneration. Transglutaminase 2 (TG2) is a versatile enzyme participating in efferocytosis. Here, we show that TG2 ablation did not alter the skeletal muscle weights or sizes but led to the generation of small size myofibers and to decreased grip force in TG2 null mice. Following cardiotoxin-induced injury, the size of regenerating fibers was smaller, and the myoblast fusion was delayed in the tibialis anterior muscle of TG2 null mice. Loss of TG2 did not affect the efferocytic capacity of muscle macrophages but delayed their conversion to Ly6C-CD206+, GDF3 expressing cells. Finally, TG2 promoted myoblast fusion in differentiating C2C12 myoblasts. These results indicate that TG2 expressed by both macrophages and myoblasts contributes to proper myoblast fusion, and its ablation leads to impaired muscle development and regeneration in mice.

TAM kinase signaling is indispensable for proper skeletal muscle regeneration in mice.

Skeletal muscle regeneration following injury results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Infiltrating macrophages play an essential role in the process partly by clearing the necrotic cell debris, partly by producing cytokines that guide myogenesis. Infiltrating macrophages are at the beginning pro-inflammatory, but phagocytosis of dead cells induces a phenotypic change to become healing macrophages that regulate inflammation, myoblast fusion and growth, fibrosis, vascularization and return to homeostasis. The TAM receptor kinases Mer and Axl are known efferocytosis receptors in macrophages functioning in tolerogenic or inflammatory conditions, respectively. Here we investigated their involvement in the muscle regeneration process by studying the muscle repair following cardiotoxin-induced injury in Mer-/- mice. We found that Axl was the only TAM kinase receptor expressed on the protein level by skeletal muscle and C2C12 myoblast cells, while Mer was the dominant TAM kinase receptor in the CD45+ cells, and its expression significantly increased during repair. Mer ablation did not affect the skeletal muscle weight or structure, but following injury it resulted in a delay in the clearance of necrotic muscle cell debris, in the healing phenotype conversion of macrophages and consequently in a significant delay in the full muscle regeneration. Administration of the TAM kinase inhibitor BMS-777607 to wild type mice mimicked the effect of Mer ablation on the muscle regeneration process, but in addition, it resulted in a long-persisting necrotic area. Finally, in vitro inhibition of TAM kinase signaling in C2C12 myoblasts resulted in decreased viability and in impaired myotube growth. Our work identifies Axl as a survival and growth receptor in the mouse myoblasts, and reveals the contribution of TAM kinase-mediated signaling to the skeletal muscle regeneration both in macrophages and in myoblasts.

Retinol Saturase Knock-Out Mice are Characterized by Impaired Clearance of Apoptotic Cells and Develop Mild Autoimmunity.

Apoptosis and the proper clearance of apoptotic cells play a central role in maintaining tissue homeostasis. Previous work in our laboratory has shown that when a high number of cells enters apoptosis in a tissue, the macrophages that engulf them produce retinoids to enhance their own phagocytic capacity by upregulating several phagocytic genes. Our data indicated that these retinoids might be dihydroretinoids, which are products of the retinol saturase (RetSat) pathway. In the present study, the efferocytosis of RetSat-null mice was investigated. We show that among the retinoid-sensitive phagocytic genes, only transglutaminase 2 responded in macrophages and in differentiating monocytes to dihydroretinol. Administration of dihydroretinol did not affect the expression of the tested genes differently between differentiating wild type and RetSat-null monocytes, despite the fact that the expression of RetSat was induced. However, in the absence of RetSat, the expression of numerous differentiation-related genes was altered. Among these, impaired production of MFG-E8, a protein that bridges apoptotic cells to the αvß3/ß5 integrin receptors of macrophages, resulted in impaired efferocytosis, very likely causing the development of mild autoimmunity in aged female mice. Our data indicate that RetSat affects monocyte/macrophage differentiation independently of its capability to produce dihydroretinol at this stage.

Macrophages engulf apoptotic and primary necrotic thymocytes through similar phosphatidylserine-dependent mechanisms.

One of the major roles of professional phagocytes is the removal of dead cells in the body. We know less about the clearance of necrotic cells than apoptotic cell phagocytosis, despite the fact that both types of dead cells need to be cleared together and necrotic cells appear often in pathological settings. In the present study, we examined phagocytosis of heat- or H2O2-killed necrotic and apoptotic thymocytes by mouse bone marrow-derived macrophages (BMDMs) in vitro and found that the two cell types are engulfed at equal efficiency and compete with each other when added together to BMDMs. Phagocytosis of both apoptotic and necrotic thymocytes was decreased by (a) blocking phosphatidylserine on the surface of dying cells; (b) inhibition of Mer tyrosine kinase, Tim-4, integrin ß3 receptor signaling, or Ras-related C3 botulinum toxin substrate 1 activity; or (c) using BMDMs deficient for transglutaminase 2. Stimulation of liver X, retinoid X, retinoic acid or glucocorticoid nuclear receptors in BMDMs enhanced not only apoptotic, but also necrotic cell uptake. Electron microscopic analysis of the engulfment process revealed that the morphology of phagosomes and the phagocytic cup formed during the uptake of dying thymocytes is similar for apoptotic and necrotic cells. Our data indicate that apoptotic and necrotic cells are cleared via the same mechanisms, and removal of necrotic cells in vivo can be facilitated by molecules known to enhance the uptake of apoptotic cells.

Short-term high-fat meal intake alters the expression of circadian clock-, inflammation-, and oxidative stress-related genes in human skeletal muscle.

Dietary food, depending on timing, amount and composition can influence gene expression in various tissues. Here, we investigated the effect of high-fat meal diets of different compositions on the gene expression pattern of human skeletal muscle. Gene expression data of skeletal muscle samples from human volunteers prior and 4 h after the consumption of high lipid-containing meal consisting of either saturated-, monounsaturated- or polyunsaturated fatty acids were downloaded from the public repository. List of 843 differently expressed genes (DEGs) was generated. Functional analysis revealed that circadian rhythm-, inflammation- and oxidative stress-related genes are highly overrepresented among the DEGs. The magnitude of gene expression changes significantly increases with the saturation level of the dietary fatty acids and the majority of the DEGs are upregulated. We propose that, by altering circadian clock gene expression and inducing inflammation and oxidative stress, high lipid intake can contribute to muscle function decay in the long run.

Altered Gene Expression of Muscle Satellite Cells Contributes to Agerelated Sarcopenia in Mice.

BACKGROUND: During aging, muscle tissue undergoes profound changes which lead to a decline in its functional and regenerative capacity. We utilized global gene expression analysis and gene set enrichment analysis to characterize gene expression changes in aging muscle satellite cells. METHOD: Gene expression data; obtained from Affymetrix Mouse Genome 430 2.0 Array, for 14 mouse muscle satellite cell samples (5 young, 4 middle-aged, and 5 aged), were retrieved from public Gene Expression Omnibus repository. List of differentially expressed genes was generated based on 0.05 multiple-testing-adjusted p-value and 2-fold FC cut-off values. Functional profiling of genes was carried out using PANTHER Classification System. RESULTS: We have found several differentially expressed genes in satellite cells derived from aged mice compared to young ones. The gene expression changes increased progressively with time, and the majority of the differentially expressed genes were upregulated during aging. While the downregulated genes could not be correlated with specific biological processes the upregulated ones could be associated with muscle differentiation-, inflammation- or fibrosis-related processes. The latter two processes encompass the senescence-associated secretory phenotype for satellite cells which alters the tissue microenvironment and contributes to inflammation and fibrosis observed in aging muscle. CONCLUSION: Our analysis reveals that by altering gene expression pattern and expressing inflammatory mediators and extracellular matrix components, these cells can directly contribute to muscle wasting in aged mice.

The proteomic profile of a mouse model of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) develops as a complication of retinal detachment surgery and represents a devastating condition leading to serious vision loss. A good animal model that permits extensive functional studies and drug testing is crucial in finding better therapeutic modalities for PVR. A previously established mouse model, using dispase injection, was analyzed from the proteomic point of view, examining global protein profile changes by 2D electrophoresis, image analysis and HPLC-tandem mass spectrometry-based protein identification. The easy applicability of the mouse model was used to study the role of transglutaminase 2 (TG2) in PVR formation by proteomic examination of dispase-induced TG2 knockout vitreous samples. Our data demonstrate that, despite the altered appearance of crystallin proteins, the lack of TG2 did not prevent the development of PVR.

Transglutaminase 2 in human diseases.

Transglutaminase 2 (TG2) is an inducible transamidating acyltransferase that catalyzes Ca(2+)-dependent protein modifications. In addition to being an enzyme, TG2 also serves as a G protein for several seven transmembrane receptors and acts as a co-receptor for integrin ß1 and ß3 integrins distinguishing it from other members of the transglutaminase family. TG2 is ubiquitously expressed in almost all cell types and all cell compartments, and is also present on the cell surface and gets secreted to the extracellular matrix via non-classical mechanisms. TG2 has been associated with various human diseases including inflammation, cancer, fibrosis, cardiovascular disease, neurodegenerative diseases, celiac disease in which it plays either a protective role, or contributes to the pathogenesis. Thus modulating the biological activities of TG2 in these diseases will have a therapeutic value.

Anti-inflammatory Mechanisms Triggered by Apoptotic Cells during Their Clearance.

In the human body, billions of cells die by apoptosis every day. The subsequent clearance of apoptotic cells by phagocytosis is normally efficient enough to prevent secondary necrosis and the consequent release of cell contents that would induce inflammation and trigger autoimmunity. In addition, apoptotic cells generally induce an anti-inflammatory response, thus removal of apoptotic cells is usually immunologically silent. Since the first discovery that uptake of apoptotic cells leads to transforming growth factor (TGF)-ß and interleukin (IL)-10 release by engulfing macrophages, numerous anti-inflammatory mechanisms triggered by apoptotic cells have been discovered, including release of anti-inflammatory molecules from the apoptotic cells, triggering immediate anti-inflammatory signaling pathways by apoptotic cell surface molecules via phagocyte receptors, activating phagocyte nuclear receptors following uptake and inducing the production of anti-inflammatory soluble mediators by phagocytes that may act via paracrine or autocrine mechanisms to amplify and preserve the anti-inflammatory state. Here, we summarize our present knowledge about how these anti-inflammatory mechanisms operate during the clearance of apoptotic cells.

Altered expression of autophagy-related genes might contribute to glucocorticoid resistance in precursor B-cell-type acute lymphoblastic leukemia.

OBJECTIVES: Autophagy is an evolutionarily conserved process playing an important role in tumor cell's resistance to chemotherapy. Response to glucocorticoid (GC) treatment is out of the most important prognostic factors in childhood acute lymphoblastic leukemia (ALL); however, only few data are available connecting GC response and role of autophagy. Our aim was to investigate whether altered expression of autophagy-related genes contributes to GC-resistant phenotype in GC-sensitive and GC-resistant precursor B-cell-type (PBC) ALL cells. METHODS: Gene expression data were obtained from public database for 26 children diagnosed with PBC ALL either sensitive or resistant to in vitro prednisolone treatment. RESULTS: We have identified 36 autophagy-associated genes which were differently expressed, based on at least a twofold difference, GC-sensitive group as compared to GC-resistant one. Of the 36 genes, 10 were downregulated and 26 upregulated in the GC-resistant group. The average fold change values for the decreased and increased transcripts were -4.57 and 2.67, respectively. CONCLUSIONS: Our data imply that GC sensitivity might depend on the expression of several genes involved in regulation and execution of autophagy in a way that key autophagy inducers are downregulated while inhibitors of autophagy are upregulated in GC-resistant cells.

The metastatic tumor antigen 1-transglutaminase-2 pathway is involved in self-limitation of monosodium urate crystal-induced inflammation by upregulating TGF-ß1.

INTRODUCTION: Transglutaminase 2 (TG2), a protein crosslinking enzyme with multiple biochemical functions, has been connected to various inflammatory processes. In this study, the involvement of TG2 in monosodium urate (MSU) crystal-induced inflammation was studied. METHODS: Immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) were performed to detect TG2 expression in synovial fluid mononuclear cells (SFMCs) and synovial tissue from patients with gouty arthritis. MSU crystal-exposed RAW264.7 mouse macrophages were analyzed for interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), transforming growth factor ß1 (TGF-ß1) and TG2 expression by RT-PCR and enzyme-linked immunosorbent assay (ELISA). TG2 small interfering (si)-RNA-mediated silencing and overexpression in RAW264.7 cells were used to evaluate the involvement of TG2 in resolving MSU crystal-induced inflammation. The role of metastatic tumor antigen 1 (MTA1), a master chromatin modifier, was investigated by MTA1 si-RNA-mediated knockdown. In addition, the inflammatory responses were followed in wild type and TG2 null mice after being challenged with MSU crystals in an in vivo peritonitis model. RESULTS: TG2 expression was up-regulated in the synovium tissue and SFMCs from patients with gouty arthritis. The levels of MTA1, TG2, TGF-ß1, IL-1ß and TNF-α mRNAs were consistently increased in MSU crystal-stimulated RAW264.7 cells. si-MTA1 impaired the basal, as well as the MSU crystal-induced expression of TG2 and TGF-ß1, but increased that of IL-1ß and TNF-α. TG2 overexpression dramatically suppressed MSU crystal-induced IL-1ß and TNF-α, but significantly enhanced the TGF-ß1 production. Neutralizing TGF-ß antibodies or inhibition of the crosslinking activity of TG2 attenuated these effects. On the contrary, loss of TG2 resulted in a reduced TGF-ß, but in an increased IL-1ß and TNF-α production in MSU crystal-stimulated RAW264.7 cells and mouse embryonic fibroblasts (MEFs). MSU crystal-stimulated IL-1ß production was Janus kinase 2 (JAK2)-signaling dependent and TG2-induced TGF-ß suppressed the activity of it. Finally, TG2-deficient mice exhibited hyper inflammatory responses after being challenged with MSU crystals in an in vivo peritonitis model. CONCLUSIONS: These findings reveal an inherent regulatory role of the MTA1-TG2 pathway in the self-limitation of MSU crystal-induced inflammation via positively regulating the levels of active TGF-ß1 in macrophages that opposes the MSU crystal-induced JAK2-dependent pro-inflammatory cytokine formation.

Retinoids induce Nur77-dependent apoptosis in mouse thymocytes.

Nur77 is a transcription factor, which plays a determinant role in mediating T cell receptor-induced cell death of thymocytes. In addition to regulation of transcription, Nur77 contributes to apoptosis induction by targeting mitochondria, where it can convert Bcl-2, an anti-apoptotic protein into a proapoptotic molecule. Previous studies have demonstrated that retinoids are actively produced in the mouse thymus and can induce a transcription-dependent apoptosis in mouse thymocytes. Here we show that retinoic acids induce the expression of Nur77, and retinoid-induced apoptosis is completely dependent on Nur77, as retinoids were unable to induce apoptosis in Nur77 null thymocytes. In wild-type thymocytes retinoids induced enhanced expression of the apoptosis-related genes FasL, TRAIL, NDG-1, Gpr65 and Bid, all of them in a Nur77-dependent manner. The combined action of these proteins led to Caspase 8-dependent Bid cleavage in the mitochondria. In addition, we could demonstrate the Nur77-dependent induction of STAT1 leading to enhanced Bim expression, and the mitochondrial translocation of Nur77 leading to the exposure of the Bcl-2/BH3 domain. The retinoid-induced apoptosis was dependent on both Caspase 8 and STAT1. Our data together indicate that retinoids induce a Nur77-dependent cell death program in thymocytes activating the mitochondrial pathway of apoptosis.

Glucocorticoids enhance prolonged clearance of apoptotic cells by upregulating liver X receptor, peroxisome proliferator-activated receptor-δ and UCP2.

Efficient phagocytic clearance of apoptotic cells (efferocytosis) is essential to prevent the development of chronic inflammation and autoimmunity. Glucocorticoids are widely used in the therapy of chronic inflammatory diseases, and increasing evidence suggests that they act partly via enhancing efferocytosis by macrophages. Glucocorticoids were previously shown to promote both protein S- and MFG-E8-dependent efferocytosis. Since previous studies in our laboratory have demonstrated that glucocorticoids induce the expression of retinaldehyde dehydrogenases in macrophages, in the present experiments the possible involvement of retinoids in the glucocorticoid-induced efferocytosis was studied in mouse bone marrow derived macrophages. Here we show that glucocorticoids promote not only short-term, but also long-term clearance of apoptotic cells. Glucocorticoids seem to directly induce the expression of the phagocytosis-related genes MERTK, C1q, UCP2, and the transcription factor C/EBPß. C/EBPß contributes to the further induction of the phagocytosis-related genes, and is required for the induction of lipid sensing receptors LXRs, PPARδ, RARα, RXRα and RALDH1, the latter one in an LXR- and RARα-dependent manner. Glucocorticoid-induced enhancement in long-term efferocytosis was dependent on the induction of lipid sensing receptors known to be triggered by the lipid content of the engulfed cells to enhance phagocytic capacity. Retinoids did not affect the glucocorticoid-induced short term phagocytosis of apoptotic cells, but were required for the glucocorticoid-induced enhancement of efferocytosis during prolonged clearance of apoptotic cells by promoting efficient LXR and PPARδ upregulation. Our data indicate that retinoids could be considered as potential promoters of the efficacy of glucocorticoid treatment in inflammatory diseases.