Upregulation of FNDC5 gene expression in C2C12 cells after single and combined treatments of resveratrol and ATRA.

BACKGROUND: Irisin is a newly discovered myokine that secreted from skeletal muscle cells. Several studies showed that irisin involves in thermogenesis and increases the expression of browning markers such as uncoupling protein-1 that in turns induces the conversion of white adipose tissue to brown fat. Resveratrol (Res) and all-trans retinoic acid (ATRA) can also upregulate the expression of thermogenesis genes. In the present study, the effects of single and combined treatments of Res and ATRA on fibronectin type III domain containing 5 (FNDC5) gene expression was explored. METHODS: The mouse myoblasts, C2C12 cells, were seeded in 6-well plastic plates and cultured in DMEM media. After differentiation, in a pilot study, C2C12 myotubes were treated with different concentrations of Res and ATRA for 12 h. The best result was obtained by treatment of 1and 25 µM of Res and 1 µM of ATRA. Then the main study was continued by single and combined treatment of these compounds at chosen concentration. After treatments, total RNA was extracted from C2C12 cells. Complementary DNA (cDNA) was generated by the cDNA synthesis kit and FNDC5 mRNA expression was evaluated by the real-time PCR method. RESULTS: The FNDC5 gene expression in C2C12 myotubes of alone-treated with 1 µM, 25 µM Res and 10 µM ATRA did not change compared to vehicle group. However, in combination-treated the expression of FNDC5 gene was significantly increased compared to vehicle group. CONCLUSION: This is the first evidence that Res and ATRA can regulate FNDC5 gene expression in C2C12 myotubes. More investigations are necessary to explore the therapeutic effects of these nutrients in obesity, diabetes, cardiac and neurovascular disease.

Exposure to diethylhexyl phthalate (DEHP) and monoethylhexyl phthalate (MEHP) promotes the loss of alveolar epithelial phenotype of A549 cells.

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer that is metabolized to mono(2-ethylhexyl) phthalate (MEHP). Inhalation is an important exposure route for both phthalates, and their effects on lungs include inflammation, alteration of postnatal maturation (alveolarization), enlarged airspaces and cell differentiation changes, suggesting that alveolar epithelial cells-2 (AEC) are targets of phthalates. This study evaluated the cell progression, epithelial and mesenchymal markers, including surfactant secretion in A549 cells (AEC) that were exposed to DEHP (1-100 µM) or MEHP (1-50 µM) for 24-72 h. The results showed an increased cell proliferation at all concentrations of each phthalate at 24 and 48 h. Cell migration showed a concentration-dependent increase at 24 and 48 h of exposure to either phthalate and enlarged structures were seen. Decreased levels of both surfactants (SP-B/SP-C) were observed after the exposure to either phthalate at 48 h, and of SP-C positive cells exposed to MEHP, suggesting a loss of the epithelial phenotype. While a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal marker fibronectin were observed following exposure to either phthalate. Our results showed that DEHP and MEHP altered the structure and migration of A549 cells and promoted the loss of the epithelial phenotype.

Glucocorticoid receptor positively regulates transcription of FNDC5 in the liver.

Irisin is secreted by skeletal muscle during exercise and influences energy and metabolic homeostasis. This hormone is a cleaved and secreted fragment of fibronectin type III domain-containing 5 (FNDC5). Elucidation of the FNDC5 gene regulation mechanism is necessary to clarify the function of irisin as a potential therapeutic target in human metabolic diseases. Thus, we investigated the genetic and epigenetic mechanisms that regulate expression of the FNDC5 gene. FNDC5 mRNA was strong expressed in major energy-dependent human tissues, including heart, brain, liver, and skeletal muscle. Promoter analysis of the FNDC5 gene revealed that the core promoter region of the FNDC5 gene contained one CpG island that was located just upstream of the transcriptional start site for variants 2 and 3. Treatment with the histone deacetylase inhibitor sodium butyrate and the demethylating agent 5-azacytidine increased mRNA expression of FNDC5 in Huh7 cells. Prediction of transcription factor binding sites suggested that the glucocorticoid receptor was involved in the regulation of FNDC5 expression, and indeed, cortisol treatment increased mRNA expression of FNDC5 in Huh7 cells. Collectively, these findings offer insight into the genetic and epigenetic regulation of FNDC5, providing the initial steps required for understanding the role of irisin in the metabolic homeostasis.

Stimulatory effects of advanced glycation endproducts (AGEs) on fibronectin matrix assembly.

Advanced glycation endproducts (AGEs) are a heterogeneous group of compounds that form via non-enzymatic glycation of proteins throughout our lifespan and at a higher rate in certain chronic diseases such as diabetes. AGEs contribute to the progression of fibrosis, in part by stimulating cellular pathways that affect gene expression. Long-lived ECM proteins are targets for non-enzymatic glycation but the question of whether the AGE-modified ECM leads to excess ECM accumulation and fibrosis remains unanswered. In this study, cellular changes due to AGE accretion in the ECM were investigated. Non-enzymatic glycation of proteins in a decellularized fibroblast ECM was achieved by incubating the ECM in a solution of methylglyoxal (MGO). Mass spectrometry of fibronectin (FN) isolated from the glycated matrix identified twenty-eight previously unidentified MGO-derived AGE modification sites including functional sites such as the RGD integrin-binding sequence. Mesangial cells grown on the glycated, decellularized matrix assembled increased amounts of FN matrix. Soluble AGE-modified bovine serum albumin (BSA) also stimulated FN matrix assembly and this effect was reduced by function-blocking antibodies against the receptor for AGE (RAGE). These results indicate that cells respond to AGEs by increasing matrix assembly and that RAGE is involved in this response. This raises the possibility that the accumulation of ECM during the progression of fibrosis may be enhanced by cell interactions with AGEs on a glycated ECM.

Basic FGF or VEGF gene therapy corrects insufficiency in the intrinsic healing capacity of tendons.

Tendon injury during limb motion is common. Damaged tendons heal poorly and frequently undergo unpredictable ruptures or impaired motion due to insufficient innate healing capacity. By basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) gene therapy via adeno-associated viral type-2 (AAV2) vector to produce supernormal amount of bFGF or VEGF intrinsically in the tendon, we effectively corrected the insufficiency of the tendon healing capacity. This therapeutic approach (1) resulted in substantial amelioration of the low growth factor activity with significant increases in bFGF or VEGF from weeks 4 to 6 in the treated tendons (p < 0.05 or p < 0.01), (2) significantly promoted production of type I collagen and other extracellular molecules (p < 0.01) and accelerated cellular proliferation, and (3) significantly increased tendon strength by 68-91% from week 2 after AAV2-bFGF treatment and by 82-210% from week 3 after AAV2-VEGF compared with that of the controls (p < 0.05 or p < 0.01). Moreover, the transgene expression dissipated after healing was complete. These findings show that the gene transfers provide an optimistic solution to the insufficiencies of the intrinsic healing capacity of the tendon and offers an effective therapeutic possibility for patients with tendon disunion.

Intravenous Administration of Endothelial Colony-Forming Cells Overexpressing Integrin ß1 Augments Angiogenesis in Ischemic Legs.

When injected directly into ischemic tissue in patients with peripheral artery disease, the reparative capacity of endothelial progenitor cells (EPCs) appears to be limited by their poor survival. We, therefore, attempted to improve the survival of transplanted EPCs through intravenous injection and gene modification. We anticipated that overexpression of integrin ß1 will enable injected EPCs to home to ischemic tissue, which abundantly express extracellular matrix proteins, the ligands for integrins. In addition, integrin ß1 has an independent angiogenesis-stimulating function. Human endothelial colony-forming cells (ECFCs; late-outgrowth EPCs) were transduced using a lentiviral vector encoding integrin ß1 (ITGB1) or enhanced green fluorescent protein (GFP). We then locally or systemically injected phosphate-buffered saline or the genetically modified ECFCs (GFP-ECFCs or ITGB1-ECFCs; 1 × 10(5) cells each) into NOD/Shi-scid, IL-2Rγnull mice whose right femoral arteries had been occluded 24 hours earlier. Upregulation of extracellular matrix proteins, including fibronectin, was apparent in the ischemic legs. Four weeks later, blood perfusion of the ischemic limb was significantly augmented only in the ITGB1-ECFC group. Scanning electron microscopy of vascular casts revealed increases in the perfused blood vessels in the ischemic legs of mice in the ITGB1-ECFC group and significant increases in the density of both capillaries and arterioles. Transplanted ECFC-derived vessels accounted for 28% ± 4.2% of the vessels in the ITGB1-ECFC group, with no cell fusion. Intravenous administration of ECFCs engineered to home to ischemic tissue appears to efficiently mediate therapeutic angiogenesis in a mouse model of peripheral artery disease. Significance: The intravenous administration of endothelial colony-forming cells (ECFCs) genetically modified to overexpress integrin ß1 effectively stimulated angiogenesis in ischemic mouse hindlimbs. Transplanted ECFCs were observed in the ischemic leg tissue, even at the chronic stage. Moreover, the cells appeared functional, as evidenced by the improved blood flow. The cell type used (ECFCs), the route of administration (intravenous, not directly injected into the affected area), and the use of ligand-receptor interactions (extracellular matrix and integrins) for homing represent substantial advantages over previously reported cell therapies for the treatment of peripheral artery disease.

Insulin-like growth factor 1 increases apical fibronectin in blastocysts to increase blastocyst attachment to endometrial epithelial cells in vitro.

STUDY QUESTION: Does insulin-like growth factor 1 (IGF1) increase adhesion competency of blastocysts to increase attachment to uterine epithelial cells in vitro? SUMMARY ANSWER: IGF1 increases apical fibronectin on blastocysts to increase attachment and invasion in an in vitro model of implantation. WHAT IS KNOWN ALREADY: Fibronectin integrin interactions are important in attachment of blastocysts to uterine epithelial cells at implantation. STUDY DESIGN, SIZE, DURATION: Mouse blastocysts (hatched or near completion of hatching) were cultured in serum starved (SS) medium with varying treatments for 24, 48 or 72 h. Treatments included 10 ng/ml IGF1 in the presence or absence of the PI3 kinase inhibitor LY294002, an IGF1 receptor (IGF1R) neutralizing antibody or fibronectin. Effects of treatments on blastocysts were measured by attachment of blastocysts to Ishikawa cells, blastocyst outgrowth and fibronectin and focal adhesion kinase (FAK) localization and expression. Blastocysts were randomly allocated into control and treatment groups and experiments were repeated a minimum of three times with varying numbers of blastocysts used in each experiment. FAK and integrin protein expression on Ishikawa cells was quantified in the presence or absence of IGF1. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fibronectin expression and localization in blastocysts was studied using immunofluorescence and confocal microscopy. Global surface expression of integrin αvß3, ß3 and ß1 was measured in Ishikawa cells using flow cytometry. Expression levels of phosphorylated FAK and total FAK were measured in Ishikawa cells and blastocysts by western blot and image J analysis. Blastocyst outgrowth was quantified using image J analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The presence of IGF1 significantly increased mouse blastocyst attachment to Ishikawa cells compared with SS conditions (P < 0.01). IGF1 treatment resulted in distinct apical fibronectin staining on blastocysts, which was reduced by the PI3 kinase inhibitor LY294002. This coincided with a significant increase in blastocyst outgrowth in the presence of IGF1 (P < 0.01) or fibronectin (P < 0.001), which was abolished by LY294002 (P < 0.001). Apical expression of integrin αvß3, ß3 and ß1 in Ishikawa cells was unaltered by IGF1. However, IGF1 increased phosphorylated FAK (P < 0.05) and total FAK expression in Ishikawa cells. FAK signalling is linked to integrin activation and can affect the integrins' ability to bind and recognize extracellular matrix proteins such as fibronectin. Treatment of blastocysts with IGF1 before co-culture with Ishikawa cells increased their attachment (P < 0.05). This effect was abolished in the presence of LY294002 (P < 0.001) or an IGF1R neutralizing antibody (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This study uses an in vitro model of attachment that uses mouse blastocysts and human endometrial cells. This involves a species crossover and although this use has been well documented as a model for attachment (as human embryo numbers are limited) the results should be interpreted carefully. WIDER IMPLICATIONS OF THE FINDINGS: This study presents mechanisms by which IGF1 improves attachment of blastocysts to Ishikawa cells and documents for the first time how IGF1 can increase adhesion competency in blastocysts. Failure of the blastocyst to implant is the major cause of human assisted reproductive technology (ART) failure. As growth factors are absent during embryo culture, their addition to embryo culture medium is a potential avenue to improve IVF success. In particular, IGF1 could prove to be a potential treatment for blastocysts before transfer to the uterus in an ART setting.

Characterization of the metabolic effects of irisin on skeletal muscle in vitro.

AIMS: This work explored the effects of irisin on metabolism, gene expression and mitochondrial content in cultured myocytes. METHODS: C2C12 myocytes were treated with various concentrations of irisin for various durations. Glycolysis and oxidative metabolism were quantified by measurement of extracellular acidification and oxygen consumption, respectively. Metabolic gene expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and mitochondrial content was assessed by flow cytometry and confocal microscopy. RESULTS: Cells treated with irisin exhibited significantly increased oxidative metabolism. Irisin treatment also significantly increased mitochondrial uncoupling at various doses and durations. Lastly, treatment with irisin also significantly elevated metabolic gene expression including peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), irisin, glucose transporter 4 (GLUT4) and mitochondrial uncoupling protein 3 (UCP3) leading to increased mitochondrial biogenesis. CONCLUSIONS: Our observations are the first to document increased metabolism in myocytes through irisin-mediated induction of mitochondrial biogenesis and uncoupling with corresponding gene expression. These observations support the need for further investigation into the therapeutic and pharmacological effects of irisin, as well as development of irisin-based therapy.

Epigallocatechin-3-gallate (EGCG) downregulates EGF-induced MMP-9 in breast cancer cells: involvement of integrin receptor α5ß1 in the process.

PURPOSE: Epidermal growth factor receptor (EGFR/ErbB1) is a transmembrane protein with tyrosine kinase activity activated mainly by ligand, EGF. Matrix metalloproteinases (MMPs) are a family of proteinases that catalyses the destruction of ECM, among which MMP-9 has important role in tumor cell invasion. Secretion of MMP-9 is stimulated by a variety of factors, EGFR being significant. Epigallocatechin-3-gallate (EGCG) is a major polyphenol of green tea that inhibits cell proliferation and invasion. Here, we study the effect of EGFR alone and in collaboration with fibronectin on the status of MMP-9 in human breast cancer cell MDA-MB-231 and its molecular mechanism; study the role of EGCG on the induced MMP-9; and elucidate the signaling molecules involved in the process. METHODS: We performed zymography, immunoblots, real-time RT-PCR, cell adhesion assay, siRNA studies, and electrophoretic mobility shift assay to demonstrate the findings. RESULT: EGF induces MMP-9 activity and expression; FAK, PI3 K, and ERK are mainly involved in the process. EGF also causes the transactivation of MMP-9 gene by increasing the DNA binding activity of the transcription factors. EGCG downregulates EGF-induced MMP-9 expression by inhibiting the involved regulatory kinases. EGF collaborates with fibronectin to create a synergistic response, and EGCG inhibits the synergistic response in MDA-MB-231. CONCLUSION: The study demonstrates the requirement of cross talk between cell matrix adhesion molecules and growth factor receptors to improve biological responses and shows FAK/ERK as the pivotal point of this convergence in human breast carcinoma cell line MDA-MB-231. We also establish EGCG as the potential anti-tumor agent in human breast carcinoma.

The effect of growth factors, cytokines, and extracellular matrix proteins on fibronectin production in human vascular smooth muscle cells.

PURPOSE: Although 60% to 80% of the mature intimal hyperplastic plaque is composed of extracellular matrix (ECM) proteins, little is known about the factors that stimulate smooth muscle cells (SMCs) to produce these proteins. A major component of the ECM protein is fibronectin. Thus we studied fibronectin production and its response to various growth factors, cytokines, and other ECM proteins that are released at the time of vascular injury. METHODS: Quiescent cultured human SMCs were stimulated for varying intervals with increasing concentrations of agonist. Fibronectin in the cell medium was assayed by immunoblotting with a fibronectin-specific antibody. RESULTS: After 72 hours of stimulation, transforming growth factor-beta (10 ng/mL) had the most profound effect on fibronectin production (9.6- +/- 2.1-fold; P <.05), followed by epidermal growth factor (100 ng/mL; 5.0- +/- 0.1-fold; P <.05, for both). Surprisingly, the platelet-derived growth factors (AA, AB, and BB) and fibroblast growth factor did not stimulate fibronectin production. Among the matrix proteins studied, only collagen type I (20 microg/mL) stimulated fibronectin production (1.9- +/- 0.1-fold; P <.05), whereas collagen type IV and laminin had no effect. The contractile protein angiotensin II (100 ng/mL) was a weak stimulant of fibronectin (1.6- +/- 0.2-fold; P <.05). Time course studies of fibronectin production up to 72 hours revealed kinetics that varied with each agonist. Transforming growth factor-beta stimulated significant early production of fibronectin, whereas fibronectin production in response to epidermal growth factor and collagen type I was initially modest but increased with time. The effect of angiotensin II did not become evident until 72 hours. CONCLUSION: Cytokines, growth factors, and matrix proteins have varying quantitative effects on ECM protein production by human vascular SMCs. Knowledge of the factors that influence ECM protein production may allow for the design of specific inhibitors that can prevent intimal hyperplasia.