Le Yang

Bone Marrow-Derived Mesenchymal Stem Cells Differentiate to Hepatic Myofibroblasts by Transforming Growth Factor-β1 via Sphingosine Kinase/Sphingosine 1-Phosphate (S1P)/S1P Receptor Axis

Sphingosine kinase (SphK) is involved in numerous biological processes, including cell growth, proliferation, and differentiation. However, whether SphK participates in the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) to myofibroblasts has been unknown. In a carbon tetrachloride-treated mouse model, SphK1 was expressed in BMSCs in damaged liver. Furthermore, mRNA expression of both SphK1 and transforming growth factor β1 (TGF-β1) was significantly increased after liver injury, with a positive correlation between them. The SphK inhibitor SKI significantly blocked BMSC differentiation to myofibroblasts during liver injury (the proportion of BMSC-derived myofibroblasts decreased markedly, compared with no SKI treatment) and attenuated the extent of liver fibrosis. Using primary mouse BMSCs, we demonstrated that TGF-β1 induced BMSC differentiation to myofibroblasts, accompanied by the up-regulation of SphK1 and modulation of sphingosine 1-phosphate (S1P) receptor (S1PR) expression. Notably, pharmacological or siRNA-mediated inhibition of SphK1 abrogated the prodifferentiating effect of TGF-β1. Moreover, using either S1PR subtype-specific antagonists or specific siRNAs, we found that the prodifferentiating effect of TGF-β1 was mediated by S1PR(1) and S1PR(3). These data suggest that SphK1 activation by TGF-β1 leads to differentiation of BMSCs to myofibroblasts mediated by S1PR(1) and S1PR(3) up-regulation, thus providing new information on the mechanisms by which TGF-β1 gives rise to fibrosis and opening new perspectives for pharmacological treatment of liver fibrosis.

15‐deoxy‐Δ12,14‐prostaglandin J2 reduces recruitment of bone marrow–derived monocyte/macrophages in chronic liver injury in mice

15‐Deoxy‐Δ12,14‐Prostaglandin J2 (15d‐PGJ2), a natural peroxisome proliferator‐activated receptor gamma (PPAR‐γ) ligand, has been implicated as a new antiinflammatory compound with possible clinical applications. Based on this concept, this study was designed to evaluate the effects of 15d‐PGJ2 on bone marrow–derived monocyte/macrophage (BMM) migration, phagocytosis, and cytokine expression after liver injury using mouse models induced by cholestasis or carbon tetrachloride. Mice were lethally irradiated and received bone marrow transplants from enhanced green fluorescent protein transgenic mice. Our results showed that recruitment of BMM was significantly increased during chronic liver injury, and that 15d‐PGJ2 administration reduced BMM, but not neutrophil, dendritic, or T cell migration toward the damaged liver, involving reactive oxygen species generation and independently of PPAR‐γ. Moreover, 15d‐PGJ2 inhibited the phagocytic activity of BMM and down‐regulated inflammatory cytokine expression in vivo and in vitro. Accordingly, hepatic inflammation and fibrosis were strikingly ameliorated after 15d‐PGJ2 administration. Conclusion: Our findings strongly suggest the antiinflammation and antifibrogenic potential of 15d‐PGJ2 in chronic liver diseases. (HEPATOLOGY 2012;56:350–360)

Sphingosine 1-Phosphate Receptor 2 and 3 Mediate Bone Marrow-Derived Monocyte/Macrophage Motility in Cholestatic Liver Injury in Mice

Sphingosine 1-phosphate (S1P)/S1P receptor (S1PR) system has been implicated in the pathological process of liver injury. This study was designed to evaluate the effects of S1P/S1PR on bone marrow-derived monocyte/macrophage (BMM) migration in mouse models of cholestatic liver injury, and identify the signaling pathway underlying this process. S1PR1–3 expression in BMM was characterized by immunofluorescence, RT-PCR and Western blot. Cell migration was determined in Boyden chambers. In vivo, the chimera mice, which received BM transplants from EGFP-transgenic mice, received an operation of bile duct ligation (BDL) to induce liver injury with the administration of S1PR2/3 antagonists. The results showed that S1PR1–3 were all expressed in BMMs. S1P exerted a powerful migratory action on BMMs via S1PR2 and S1PR3. Furthermore, PTX and LY-294002 (PI3K inhibitor) prevented S1PR2/3-mediated BMM migration, and Rac1 activation by S1P was inhibited by JTE-013, CAY-10444 or LY294002. Administration of S1PR2/3 antagonists in vivo significantly reduced BMM recruitment in BDL-treated mice, and attenuated hepatic inflammation and fibrosis. In conclusion, S1P/S1PR2/3 system mediates BMM motility by PTX-PI3K-Rac1 signaling pathway, which provides new compelling information on the role of S1P/S1PR in liver injury and opens new perspectives for the pharmacological treatment of hepatic fibrosis.

Endocannabinoid System Contributes to Liver Injury and Inflammation by Activation of Bone Marrow-Derived Monocytes/Macrophages in a CB1-Dependent Manner.

Hepatic injury undergoes significant increases in endocannabinoidsand infiltrations of macrophages, yet the concrete mechanisms of changes in endocannabinoids and the functions of macrophage-expressed cannabinoid receptors (CBs) are unclear. Biosynthetic and degradative enzymes of endocannabinoids revealed a significant change in human fibrotic liver. Meanwhile, we showed dynamic changes of these enzymes and CBs (CB1 and CB2) from 1 to 56 d in carbon tetrachloride–induced murine liver injury. Biosynthetic enzymes (N-acylphosphatidyl-ethanolamine selective phospholipase D and diacylglycerol lipase-α) and CBs were markedly increased, whereas degradative enzymes (fatty acid amidohydrolase and monoacylglycerol lipase) were downregulated. Moreover, these enzymes intimately correlated with the fibrosis parameter [procollagen α1(III)]. Bone marrow–derived monocytes/macrophages (BMM) expressed CBs. Interestingly, CB1 but not CB2 mediated BMM migration through a Boyden chambers assay, and the effect depended on the G(α)i/o/RhoA/ROCK signaling pathway. ICR mice were lethally irradiated and received BM transplants from enhanced GFP transgenic mice. Four weeks later, mice of BM reconstruction were subjected to carbon tetrachloride–induced liver injury. In the chimeric murine model, we found that blockade of CB1 by administration of a CB1 antagonist inhibited the recruitment of BMM into injured liver using immunofluorescence staining and FACS, but it did not have effects on migration of T cells and dendritic cells without CB1 expression. Furthermore, activation of CB1 enhanced cytokine expression of BMM. In vivo, inhibition of CB1 attenuated the inflammatory cytokine level through real-time RT-PCR and cytometric bead array, ameliorating hepatic inflammation and fibrosis. In this study, we identify inactivation of BMM-expressed CB1 as a therapeutic strategy for reducing hepatic inflammation and fibrosis.

Cannabinoid receptor 1 but not 2 mediates macrophage phagocytosis by G(α)i/o /RhoA/ROCK signaling pathway.

Phagocytosis is critical to macrophages linking innate and adaptive immune reaction. Cannabinoid receptor 1 (CB1) and 2 (CB2) mediate immune modulation. However, the role of cannabinoid receptors in macrophage phagocytosis is undefined. In this study, we found that two murine macrophage lines (J774A.1 and RAW264.7) and peripheral blood macrophages all expressed CB1 and CB2 by immunofluorescence‐staining, real time RT‐PCR and Western blot. Macrophage phagocytic activity was determined by quantifying fluorescent intensity of the engulfed BioParticles or fluorescence‐activated cell sorting. mAEA (CB1 agonist) enhanced phagocytosis of macrophages, but JWH133 (CB2 agonist) had no influence. Pharmacological or genetic ablation of CB1 inhibited mAEA‐enhanced phagocytosis, while CB2 had no such effects. Meanwhile, activation of CB1 increased GTP‐bounding active form of small GTPase RhoA, but not Rac1 or Cdc42. AM281 (CB1 antagonist) and pertussis toxin (PTX, G(α) i/o protein inhibitor) decreased GTP‐bound RhoA protein level with mAEA. In addition, PTX, C3 Transferase (RhoA inhibitor) or Y27632 (Rho‐associated kinase ROCK inhibitor) attenuated CB1‐mediated phagocytosis. These results confirm that activation of CB1 regulates macrophage phagocytosis through G(α) i/o/RhoA/ROCK signaling pathway. Moreover, activation of CB1 induced significant up‐regulation of CB1 expression by real time RT‐PCR and Western blot analysis, but not CB2. It indicated the existence of a positive feedback between CB1 activation and CB1 expression. The up‐regulation of CB1 was RhoA‐independent but it may contribute to maintaining high phagocytic activity of macrophages for a longer time. In conclusion, CB1 mediates macrophage phagocytosis by G(α) i/o/RhoA/ROCK signal axis. These data further underline the role of CB1 in macrophage phagocytic process. J. Cell. Physiol. 230: 1640–1650, 2015.

Macrophage Migration Inhibitor Factor Upregulates MCP-1 Expression in an Autocrine Manner in Hepatocytes during Acute Mouse Liver Injury.

Macrophage migration inhibitor factor (MIF), a multipotent innate immune mediator, is an upstream component of the inflammatory cascade in diseases such as liver disease. Monocyte chemoattractant protein-1 (MCP-1), a highly representative chemokine, is critical in liver disease pathogenesis. We investigated the role of MIF in regulating hepatocytic MCP-1 expression. MIF and MCP-1 expression were characterized by immunochemistry, RT-PCR, ELISA, and immunoblotting in CCl4-treated mouse liver and isolated hepatocytes. MIF was primarily distributed in hepatocytes, and its expression increased upon acute liver injury. Its expression was also increased in injured hepatocytes, induced by LPS or CCl4, which mimic liver injury in vitro. MIF was expressed earlier than MCP-1, strongly inducing hepatocytic MCP-1 expression. Moreover, the increase in MCP-1 expression induced by MIF was inhibited by CD74- or CD44-specific siRNAs and SB203580, a p38 MAPK inhibitor. Further, CD74 or CD44 deficiency effectively inhibited MIF-induced p38 activation. MIF inhibitor ISO-1 reduced MCP-1 expression and p38 phosphorylation in CCl4-treated mouse liver. Our results showed that MIF regulates MCP-1 expression in hepatocytes of injured liver via CD74, CD44, and p38 MAPK in an autocrine manner, providing compelling information on the role of MIF in liver injury, and implying a new regulatory mechanism for liver inflammation.

Peroxisome Proliferator-Activated Receptor Gamma Negatively Regulates the Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells Toward Myofibroblasts in Liver Fibrogenesis

Background/Aims: Bone marrow-derived mesenchymal stem cells (BMSCs) have been confirmed to have capacity to differentiate toward hepatic myofibroblasts, which contribute to fibrogenesis in chronic liver diseases. Peroxisome proliferator-activated receptor gamma (PPARγ), a ligand-activated transcription factor, has gained a great deal of recent attention as it is involved in fibrosis and cell differentiation. However, whether it regulates the differentiation of BMSCs toward myofibroblasts remains to be defined. Methods: Carbon tetrachloride or bile duct ligation was used to induce mouse liver fibrosis. Expressions of PPARγ, α-smooth muscle actin, collagen α1 (I) and collagen α1 (III) were detected by real-time RT-PCR and Western blot or immunofluorescence assay. Results: PPARγ expression was decreased in mouse fibrotic liver. In addition, PPARγ was declined during the differentiation of BMSCs toward myofibroblasts induced by transforming growth factor β1. Activation of PPARγ stimulated by natural or synthetic ligands suppressed the differentiation of BMSCs. Additionally, knock down of PPARγ by siRNA contributed to BMSC differentiation toward myofibroblasts. Furthermore, PPARγ activation by natural ligand significantly inhibited the differentiation of BMSCs toward myofibroblasts in liver fibrogenesis and alleviated liver fibrosis. Conclusions: PPARγ negatively regulates the differentiation of BMSCs toward myofibroblasts, which highlights a further mechanism implicated in the BMSC differentiation.

Cannabinoid Receptor 1 Participates in Liver Inflammation by Promoting M1 Macrophage Polarization via RhoA/NF-κB p65 and ERK1/2 Pathways, Respectively, in Mouse Liver Fibrogenesis

Macrophage M1/M2 polarization mediates tissue damage and inflammatory responses. Cannabinoid receptor (CB) 1 participated in liver fibrogenesis by affecting bone marrow (BM)-derived monocytes/macrophages (BMMs) activation. However, the knowledge of whether CB1 is involved in the polarization of BMMs remains limited. Here, we found M1 gene signatures (including CD86, MIP-1β, tumor necrosis factor, IL-6, and inducible nitric oxide synthase) and the amount of M1 macrophages (CD86+ cells, gated by F4/80) were significantly elevated in carbon tetrachloride (CCl4)-induced mouse injured livers, while that of M2 type macrophages had little change by RT-qPCR and fluorescence-activated cell sorting (FACS). Our preceding study confirmed CB1 was involved in CCl4-induced liver fibrogenesis. Our results noted CB1 expression showed positive correlation with CD86. Blockade of CB1 by its antagonist or siRNA in vivo downregulated the mRNA and protein levels of M1 markers using RT-qPCR, western blot, and Cytometric Bead Array (CBA) assays, and reduced the proportion of M1 macrophages. Moreover, chimera mouse models, which received BM transplants from EGFP-transgenic mice or clodronate liposome injection mouse models, in which Kupffer cells were depleted, were performed to clarify the role of CB1 on the polarization of Kupffer cells and BMMs. We found that CB1 was especially involved in BMM polarization toward M1 phenotype but have no effect on that of Kupffer cells. The reason might due to the lower CB1 expression in Kupffer cells than that of BMMs. In vitro, we discovered CB1 was involved in the polarization of BMMs toward M1. Furthermore, CB1-induced M1 polarization was apparently impaired by PTX [G(α)i/o protein inhibitor], Y27632 (ROCK inhibitor), and PD98059 [extracellular signal-regulated kinase (ERK) inhibitor], while SB203580 (p38 inhibitor) and compound C (AMPK inhibitor) had no such effect. ACEA (CB1 agonist) activated G(α)i/o coupled CB1, then enlarged GTP-bound Rho and phosphor-ERK1/2, independently. NF-κB p65 nuclear translocation is also a marker of M1 phenotype macrophages. We found that CB1 switched on NF-κB p65 nuclear translocation only depending on G(α)i/o/RhoA signaling pathway. Conclusion CB1 plays a crucial role in regulating M1 polarization of BMMs in liver injury, depending on two independent signaling pathways: G(α)i/o/RhoA/NF-κB p65 and G(α)i/o/ERK1/2 pathways.

15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits Homing of Bone Marrow-Derived Mesenchymal Stem Cells Triggered by Chronic Liver Injury via Redox Pathway

It has been reported that bone marrow-derived mesenchymal stem cells (BMSCs) have capacity to migrate to the damaged liver and contribute to fibrogenesis in chronic liver diseases. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand for peroxisome proliferator-activated receptor gamma (PPARγ), is considered a new inhibitor of cell migration. However, the actions of 15d-PGJ2 on BMSC migration remain unknown. In this study, we investigated the effects of 15d-PGJ2 on the migration of BMSCs using a mouse model of chronic liver fibrosis and primary mouse BMSCs. Our results demonstrated that in vivo, 15d-PGJ2 administration inhibited the homing of BMSCs to injured liver by flow cytometric analysis and, in vitro, 15d-PGJ2 suppressed primary BMSC migration in a dose-dependent manner determined by Boyden chamber assay. Furthermore, the repressive effect of 15d-PGJ2 was blocked by reactive oxygen species (ROS) inhibitor, but not PPARγ antagonist, and action of 15d-PGJ2 was not reproduced by PPARγ synthetic ligands. In addition, 15d-PGJ2 triggered a significant ROS production and cytoskeletal remodeling in BMSCs. In conclusion, our results suggest that 15d-PGJ2 plays a crucial role in homing of BMSCs to the injured liver dependent on ROS production, independently of PPARγ, which may represent a new strategy in the treatment of liver fibrosis.

Cannabinoid Receptor 1 Mediates Homing of Bone Marrow-Derived Mesenchymal Stem Cells Triggered by Chronic Liver Injury.

Cannabinoid receptors (CBs) have been implicated in the pathogenesis of various liver diseases, including liver fibrosis. Our previous studies have demonstrated that after liver injury, mouse bone marrow‐derived mesenchymal stem cells (BMSCs) can migrate to the injured liver and differentiate to myofibroblasts, contributing to hepatic fibrogenesis. However, the role of CBs in the homing of BMSCs in liver injury is yet unclear. In this study, we found that both CB1 and CB2 were expressed in BMSCs. Migration assays were performed by transwell chambers. CB1 agonist ACEA promoted the migration of BMSCs, but CB2 agonist JWH133 had no effect. Pharmacological or genetic ablation of CB1 reduced ACEA‐induced migration, whereas CB2 did not. Moreover, activation of CB1 increased active GTP‐bound Rac1, RhoA, and Cdc42 protein levels. The elevated GTP‐bound Rac1 and RhoA protein levels were decreased by CB1 antagonist AM281 treatment, but not Cdc42. In addition, ACEA‐induced migration was suppressed by NSC23766 (Rac1 inhibitor) or C3 transferase (RhoA inhibitor), whereas MLS‐573151 (Cdc42 inhibitor) had no effect. Consistent with these data, Rac1 or RhoA knock‐down significantly blocked CB1‐mediated migration. Meanwhile, CB1‐mediated migration was associated with cytoskeletal remodeling. In vivo, administration of CB1 antagonist AM281 markedly inhibited the recruitment of BMSCs to the injured liver using fluorescence‐activated cell sorting. Furthermore, blockade of CB1 significantly attenuated liver fibrosis. In conclusion, our results suggest that CB1 plays a crucial role in liver fibrosis through mediating the homing of BMSCs to damaged liver, which may provide new insight into the pathogenesis and treatment of liver fibrosis. J. Cell. Physiol. 232: 110–121, 2017.