Altered PPARγ expression inhibits myogenic differentiation in C2C12 skeletal muscle cells

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily known to regulate adipocyte differentiation. However, its role in skeletal muscle differentiation is not known. To investigate possible involvement of PPARγ in skeletal muscle differentiation, we modulated its expression in C2C12 mouse skeletal muscle cells by stable transfection with sense or antisense plasmid constructs of PPARγ cDNA. Phenotypic observations and biochemical analysis of different myogenic markers showed that altered expression of PPARγ inhibited the formation of myotubes, as well as expression of muscle-specific myogenic proteins including myogenin, MyoD and creatine kinase activity. Together, we show that critical expression of PPARγ is required for skeletal muscle cells differentiation. *These authors contributed equally to this work.     

Role of endogenous TGF-β family in myogenic differentiation of C2C12 cells

The present study evaluated endogenous activities and the role of BMP and transforming growth factor-β (TGF-β), representative members of the TGF-β family, during myotube differentiation in C2C12 cells. Smad phosphorylation at the C-terminal serines was monitored, since TGF-β family members signal via the phosphorylation of Smads in a ligand-dependent manner. Expression of phosphorylated Smad1/5/8, which is an indicator of BMP activity, was higher before differentiation, and rapidly decreased after differentiation stimulation. Differentiation-related changes were consistent with those in the expression of Ids, well-known BMP-responsive genes. Treatment with inhibitors of BMP type I receptors or noggin in C2C12 myoblasts down-regulated the expression of myogenic regulatory factors, such as Myf5 and MyoD, leading to impaired myotube formation. Addition of BMP-2 during the myoblast phase also inhibited myotube differentiation through the down-regulation of Myf5 and MyoD. In contrast to endogenous BMP activity, the phosphorylation of Smad2, a TGF-β-responsive Smad, was higher 8-16 days after differentiation stimulation. A-83-01, an inhibitor of TGF-β type I receptor, increased the expression of Myf5 and MyoD, and enhanced myotube formation. The present results reveal that endogenous activities of the TGF-β family are changed during myogenesis in a pathway-specific manner, and that the activities are required for myogenesis.     

High expression of integrin αvβ3 enables uptake of targeted fluorescent probes into ovarian cancer cells and tumors

The cell line OVCAR-4 was recently ranked as one of the most representative cell lines for high grade serous ovarian cancer (HGSOC). However, little work has been done to assess the susceptibility of OVCAR-4 cells and tumors to the more common types of molecular targeting. Proteome profiles suggest OVCAR-4 express high levels of integrin αvβ3 receptors. Using flow cytometry with fluorescent antibodies we determined that OVCAR-4 cells have high number of integrin αvβ3 receptors ([9.8?±?2.5]?×?10⁴/cell) compared to the well-characterized cell line U87-MG ([5.2?±?1.4]?×?10⁴/cell). However, OVCAR-4 cells also have roughly three times the surface area of U87-MG cells, so the average αvβ3 receptor density is actually lower (11?±?3 versus 18?±?6?receptors/µm²). A series of new fluorescent molecular probes was prepared with structures comprised of a deep-red squaraine fluorophore (～680?nm emission) covalently attached to zero, one, or two cyclic pentapeptide cRGD sequences for integrin targeting. Microscopy studies showed that uptake of the divalent probe into cultured OVCAR-4 cells was 2.2?±?0.4 higher than the monovalent probe, which in turn was 2.2?±?0.4 higher than the untargeted probe. This probe targeting trend was also seen in OVCAR-4 mouse tumor models. The results suggest that clinically relevant OVCAR-4 cells can be targeted using molecular probes based on αvβ3 integrin receptor antagonists such as the cRGD peptide. Furthermore, deep-red fluorescent cRGD-squaraine probes have potential as targeted stains of cancerous tissue associated with HGSOC in surgery and pathology settings.     

In vitro differentiation of mouse ES cells into hepatocytes with coagulation factorsⅧandⅨexpression profiles

Coagulation factorsⅡ,Ⅴ,Ⅶ,Ⅷ,ⅨandⅩare produced by hepatocytes. So factorsⅧandⅨdeficiencies, which result in hemophilia A and B, have the potential to respond to cellular replacement therapy. Embryonic stem (ES) cells provide a unique source for therapeutic applications. Here, E14 mouse ES cells have been induced into hepatocytes in vitro. Morphology revealed that ES-derived hepatic-like cells were round or polyhedral shaped with distinct boundary of individual cells, and some arranged in trabeculae. These cells expressed endodermal- or liver-specific mRNA——transthyretin (TTR),α1-anti-trypsin (AAT),α-fetoprotein (AFP), albumin (ALB), glucose-6- phoshpatase (G6P) and tyrosine aminotransferase (TAT). Approximately (85.1±0.5)% of the ES-derived cells was stained positive green with ICG uptake. These cells were also stained magenta as a result of PAS reaction. In this paper, expression of coagulation factorsⅧandⅨmRNA in the ES-derived cells is documented. Therefore, ES cells might be developed as substitute donor cells for the therapy of coagulation factor deficiencies.     

PGC-1α is associated with C2C12 Myoblast differentiation

PGC-1α has been implicated as an important mediator of functional capacity of skeletal muscle. However, the role of PGC-1α in myoblast differentiation remains unexplored. In the present study, we observed a significant up-regulation of PGC-1α expression during the differentiation of murine C2C12 myoblast. To understand the biological significance of PGC-1α up-regulation in myoblast differentiation, C2C12 cells were transfected with murine PGC-1α cDNA and siRNA targeting PGC-1α, respectively. PGC-1α over-expressing clones fused to form typical myotubes with higher mRNA level of myosin heavy chain isoform I (MyHCI) and lower MyHCIIX. No obvious differentiation was observed in PGC-1α-targeted siRNA-transfected cells with marked decrement of mRNA levels of MyHCI and MyHCIIX. Furthermore, PGC-1α increased the expression of MyoD and MyoG in C2C12 cells, which controlled the commitment of precursor cells to myotubes. These results indicate that PGC-1α is associated with myoblast differentiation and elevates MyoD and MyoG expression levels in C2C12 cells.     

IL-17A promotes transdifferentiation of mouse myoblast cells (C2C12) into adipocytes by increasing the expression of peroxisome proliferator-activated receptor γ through CAAT/enhancer binding protein β signaling

Purpose of work  

Helper 17 T (Th17) effector cells are a recently identified Th subset and possess a unique property that distinguishes them from Th1 and Th2 subsets. The functional role of Th17 effector cells involves inflammatory responses, including autoimmunity and infection of specific pathogens. Therefore, IL-17A and its receptors may play a key role in determining the progression of certain inflammatory reactions. However, the relationship between IL-17A and adipogenesis has not yet been examined. Therefore, in this study, the effect of IL-17A on the adipogenic transdifferentiation of mouse myoblast (C2C12) cells was examined.     

Effect of purified human interferon-α on the expression of differentiation antigens and mitogen reactivity of cultured human thymic cells

Human thymic cells were cultured in vitro either alone or with the addition of a highly purified preparation of human interferon-α. Immunofluorescence techniques using a series of monoclonal antibodies showed that 2-day cultured thymocytes express a more mature phenotype (low HTA 1, high T3 and HLA-A,B,C) than normal, uncultured thymocytes. Interferon addition to the cultures results in a strong increment in the number of HLA⁺ cells and in the total amount of HLA expressed by the cultured cells. Experiments with purified cell populations showed that the cortical, immature, thymocyte was the target cell for interferon action. Phytohemagglutinin responses—but not interleukin 2 responses—were diminished after pretreatment of thymic cells with interferon. We suggest that interferon may favor a pathway of intrathymic differentiation phenotypically characterized by a high content of Class I HLA antigens.     

Platelet-derived CXCL12 regulates monocyte function,survival, differentiation into macrophages and foam cells through differential involvement of CXCR4–CXCR7

Platelets store and release CXCL12 (SDF-1), which governs differentiation of hematopoietic progenitors into either endothelial or macrophage-foam cells. CXCL12 ligates CXCR4 and CXCR7 and regulates monocyte/macrophage functions. This study deciphers the relative contribution of CXCR4–CXCR7 in mediating the effects of platelet-derived CXCL12 on monocyte function, survival, and differentiation. CXCL12 and macrophage migration inhibitory factor (MIF) that ligate CXCR4–CXCR7 induced a dynamic bidirectional trafficking of the receptors, causing CXCR4 internalization and CXCR7 externalization during chemotaxis, thereby influencing relative receptor availability, unlike MCP-1. In vivo we found enhanced accumulation of platelets and platelet-macrophage co-aggregates in peritoneal fluid following induction of peritonitis in mice. The relative surface expression of CXCL12, CXCR4, and CXCR7 among infiltrated monocytes was also enhanced as compared with peripheral blood. Platelet-derived CXCL12 from collagen-adherent platelets and recombinant CXCL12 induced monocyte chemotaxis specifically through CXCR4 engagement. Adhesion of monocytes to immobilized CXCL12 and CXCL12-enriched activated platelet surface under static and dynamic arterial flow conditions were mediated primarily through CXCR7 and were counter-regulated by neutralizing platelet-derived CXCL12. Monocytes and culture-derived-M1–M2 macrophages phagocytosed platelets, with the phagocytic potential of culture-derived-M1 macrophages higher than M2 involving CXCR4–CXCR7 participation. CXCR7 was the primary receptor in promoting monocyte survival as exerted by platelet-derived CXCL12 against BH3-mimetic induced apoptosis (phosphatidylserine exposure, caspase-3 activation, loss of mitochondrial transmembrane potential). In co-culture experiments with platelets, monocytes predominantly differentiated into CD163⁺ macrophages, which was attenuated upon CXCL12 neutralization and CXCR4/CXCR7 blocking antibodies. Moreover, OxLDL uptake by platelets induced platelet apoptosis, like other platelet agonists TRAP and collagen-related peptide (CRP). CXCL12 facilitated phagocytosis of apoptotic platelets by monocytes and M1–M2 macrophages, also promoted their differentiation into foam cells via CXCR4 and CXCR7. Thus, platelet-derived CXCL12 could regulate monocyte-macrophage functions through differential engagement of CXCR4 and CXCR7, indicating an important role in inflammation at site of platelet accumulation.Platelets are central players in regulation of inflammation at the site of thrombosis.^{1, 2, 3} When platelets are activated they release a variety of pro-inflammatory mediators including the chemokine CXCL12 (SDF-1).^{4, 5, 6, 7} CXCL12 binds to its chemokine receptors CXCR4 and CXCR7 and regulates cell migration, adhesion and survival.^{8, 9, 10, 11}Recently, platelets have been recognized to store substantial amounts of CXCL12 in their alpha-granules and release the chemokine upon activation.^{5, 6} Platelet-derived CXCL12 propagates migration and subsequent differentiation of CD34⁺ progenitor cells^{5, 12} into either an endothelial or a macrophage/foam cell phenotype depending on the culture conditions.^{12, 13} Release of CXCL12 from platelets is enhanced in acute coronary syndromes and correlates with the number of circulating CD34⁺ progenitor cells and platelet/CD34⁺ co-aggregates.^{14, 15} Enhanced levels of platelet–CXCL12 are associated with preservation of left ventricular function following myocardial infarction in humans.¹⁶ Administration of recombinant CXCL12 preserves myocardial function following transient ischemia in mice.¹⁷Monocytes/macrophages have a critical role in vascular inflammation and disease progression of atherosclerosis.¹⁸ Monocytes express both CXCR4 and CXCR7 although their role in monocyte function is still incompletely understood.^{9, 19}In the present study, we explored the effect of platelet-derived CXCL12 on monocyte function and the differential role of CXCR4 and CXCR7 for monocyte function and differentiation. We found that both chemokine receptors have a decisive but differential role for platelet-dependent monocyte function.     

Mg2+ in β-TCP/Mg–Zn composite enhances the differentiation of human bone marrow stromal cells into osteoblasts through MAPK-regulated Runx2/Osx

Inducing the osteogenic differentiation from bone marrow stromal cells (BMSCs) might be a potent strategy for treating bone loss and nonunion during fracture and improving fracture healing. Among several signaling pathways involved, mitogen-activated protein kinases (MAPKs) have been reported to play a critical role. Magnesium (Mg)-based alloys, including Mg–Zn alloy, have been used clinically as implants in the musculoskeletal field and could promote BMSC osteogenic differentiation. However, the underlying mechanisms remain unclear. In this study, we produced Mg–Zn alloy consists of Mg and low concentrations of Zn, calcium carbonate, and β-tricalcium phosphate (β-TCP; manifesting process not shown), prepared Mg, Zn, and Mg–Zn extracts, and investigated the specific effects of these extracts on human BMSC (hBMSC) osteogenic differentiation and MAPK signaling. Mg extracts and Mg–Zn extracts could significantly promote the osteogenic differentiation of hBMSCs as manifested as increased alkaline phosphatase levels, enhanced calcium nodules formation, and increased messenger RNA expression and protein levels of osteogenesis markers, including BMPs, Col-I, Runx2, and Osx; in the meantime, Mg culture medium (CM) and Mg–Zn CM both significantly enhanced the activation of MAPK signaling in hBMSCs. By adding ERK1/2 signaling, p38 signaling, or JNK signaling inhibitor to Mg–Zn CM, or conducting p38 MAPK silence in hBMSCs, we revealed that these extracts might promote hBMSC osteogenic differentiation via p38 MAPK signaling and MAPK-regulated Runx2/Osx. In conclusion, Mg²⁺ in β-TCP/Mg–Zn extract promotes the osteogenic differentiation of hBMSCs via MAPK-regulated Runx2/Osx interaction.     

Artemisinic acid is a regulator of adipocyte differentiation and C/EBP δ expression

Adipocyte dysfunction is associated with the development of obesity. In this study, artemisinic acid, which was isolated from Artemisia annua L., inhibited adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs) and its mechanism of action was determined. The mRNA levels of peroxidase proliferation-activated receptor (PPAR) γ and CCAAT/enhancer binding protein (C/EBP) α, late adipogenic factors, were reduced by artemisinic acid. Moreover, the mRNA levels of the PPAR γ target genes lipoprotein lipase, CD36, adipocyte protein, and liver X receptor were down-regulated by artemisinic acid. Artemisinic acid reduced expression of the C/EBP δ gene without impacting C/EBP β. In addition, attempts to elucidate a possible mechanism underlying the artemisinic acid-mediated effects revealed that reduced expression of the C/EBP δ gene was mediated by inhibiting Jun N-terminal kinase (JNK). Additionally, artemisinic acid also reduced the expression of the adipogenesis-associated genes glucose transporter-4 and vascular endothelial growth factor. In addition to the interference of artemisinic acid with adipogenesis, artemisinic acid significantly attenuated tumor necrosis factor-α-induced secretion of interleukin-6 by undifferentiated hAMSCs, thus influencing insulin resistance and the inflammatory state characterizing obesity. Taken together, these findings indicate that inhibiting adipogenic differentiation of hAMSCs by artemisinic acid occurs primarily through reduced expression of C/EBP δ, which is mediated by the inhibition of JNK and suggest that aremisinic acid may be used as a complementary treatment option for obesity associated with metabolic syndrome.     

Concerted inhibition of HIF-1α and -2α expression markedly suppresses angiogenesis in cultured RPE cells

HIF-1α is known to play an important role in the induction of VEGF by hypoxia in retinal pigment epithelial (RPE) cells. However, the involvement of the other isoform, HIF-2α, in RPE cells remains unclear. Thus, the purpose of present study was to clarify the role of HIF-2α during induction of angiogenic genes in hypoxic RPE cells. When human RPE cells (ARPE-19) were cultured under hypoxic conditions, HIF-1α and HIF-2α proteins increased. This induced an increase in mRNA for VEGF, causing secretion of VEGF protein into the medium. This conditioned medium induced tube formation in human vascular endothelial cells (HUVEC). The increased expression of mRNA for VEGF in hypoxic RPE cells was partially inhibited by HIF-1α siRNA, but not by HIF-2α siRNA. However, co-transfection of HIF-1α siRNA and HIF-2α siRNA augmented downregulation of VEGF mRNA and protein in hypoxic RPE cells and inhibited formation of tube-like structures in HUVEC. GeneChip and PCR array analyses revealed that not only VEGF, but also expression of other angiogenic genes were synergistically downregulated by co-transfection of hypoxic RPE cells with HIF-1α and HIF-2α siRNAs. These findings suggest an important compensatory role for the HIF-2α isoform in the regulation of angiogenic gene expression. Thus, suppression of angiogenic genes for HIF-1α and HIF-2α may be a possible therapeutic strategy against retinal angiogenesis in Age-related macular degeneration (ARMD).     

Inhibitory effect of 30-kDa phytoglycoprotein on expression of TNF-α and COX-2 via activation of PKCα and ERK 1/2 in LPS-stimulated RAW 264.7 cells

Endothelial cells may play a potential role in cholesterol efflux from peripheral tissues to liver. Cholesterol efflux from cells is essential for activation of the reverse cholesterol transport pathway and cardiovascular health. One of the cholesterol transporters is steroidogenic acute regulatory protein (StAR) which promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system. The aim of the present study was to determine the effects of a niacin–chromium complex on aortas of hyperlipidemic rats and on the cholesterol efflux from aorta endothelial cells by examination under light and transmission electron microscopes and evaluating the StAR immunoreactivity, respectively. Aorta lipid peroxidation (LPO) and glutathione (GSH) levels were determined by spectrophotometric methods. After treating hyperlipidemic animals with the complex, the StAR immunoreactivity in endothelial cells increased to achieve cholesterol homeostasis and efflux. Combined treatment with niacin and chromium resulted in an inhibition in the mast cell secretion and a decrease in lipid vacuole size in unilocular adipose tissue surrounding aorta, as well as in a decrease in morphological degenerations observing in aorta of hyperlipidemic rats. Aorta LPO levels increased and GSH levels decreased in the hyperlipidemic group, whereas treatment with niacin and chromium reversed these effects. In conclusion, this study reveals that combined treatment with niacin and chromium prevents the morphological and biochemical changes observed in thoracic aorta of hyperlipidemic rats, and may regulate effectively cardiovascular diseases inducing an increase in StAR levels on endothelial cells.     

Involvement of TNF-α in differential gene expression pattern of CXCR4 on human marrow-derived mesenchymal stem cells

Cell therapy and tissue repair are used in a variety of diseases including tissue and organ transplantation, autoimmune diseases and cancers. Now mesenchymal stem cells (MSCs) are an attractive and promising source for cell-based therapy according to their individual characteristics. Soluble factors which are able to induce MSCs migration have a vital role in cell engraftment and tissue regeneration. Tumor necrosis factor α (TNF-α) is a major cytokine present in damaged tissues. We have investigated the pattern of gene expression of chemokine receptor CXCR4 in nine groups of human bone marrow-derived MSCs stimulated with TNF-α in different dose and time manner. Comparison of TNF-α treated with untreated MSCs revealed the highest expression level of CXCR4 after treatment with 1, and 10 ng/ml of TNF-α in 24 h, and the production of CXCR4 mRNA was regulated up to 216 and 512 fold, respectively. Our results demonstrated the differential gene expression pattern of chemokine receptor CXCR4 in human marrow-derived MSCs stimulated with inflammatory cytokine TNF-α. These findings suggest that in vitro control of both dose and time factors may be important in stem cell migration capacity, and perhaps in future-stem cell transplantation therapies.