Interaction of CREB and PGC-1α Induces Fibronectin Type III Domain-Containing Protein 5 Expression in C2C12 Myotubes.

BACKGROUND/AIMS: Fibronectin type III domain-containing protein 5 (FNDC5), also known as irisin, is a myokine secreted from muscle in response to exercise. However, the molecular mechanisms that regulate FNDC5 expression and the functional significance of irisn in skeletal muscle remain unknown. In this study, we explored the potential pathways that induce FNDC5 expression and delineated the metabolic effects of irisin on skeletal muscle. METHODS: C2C12 myotubes were treated with drugs at various concentrations and durations. The expression and activation of genes were measured by real-time polymerase chain reaction (qRT-PCR) and Western blotting. Oxidative phosphorylation was quantified by measuring the oxygen consumption rate (OCR). RESULTS: We found that the exercise-mimicking treatment (cAMP, forskolin and isoproterenol) increased Fndc5 expression in C2C12 myotubes. CREB over-expressed C2C12 myotubes displayed higher Fndc5 expression. CREB over-expression also promoted peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) expression. PGC-1α-induced Fndc5 expression was blocked when the dominant negative form of CREB (S133A) was present. PGC-1α mutation (S570A) also decreased Fndc5 expression. Immunoprecipitation showed that overexpressed PGC-1α complexed with CREB in HEK293 cells. C2C12 myotubes treated with forskolin also increased endogenous CREB and PGC-1α binding. Functionally, irisin treatment increased mitochondrial respiration, enhanced ATP production, promoted fatty acid oxidation but decreased glycolysis in myotubes. CONCLUSION: Our observation indicates that cAMP-mediated PGC-1α/CREB interaction triggers Fndc5 expression, which acts as an autocrine/paracrine to shape the metabolic phenotype of myotubes.

DR5-mediated DISC controls caspase-8 cleavage and initiation of apoptosis in human glioblastomas.

To explore the molecular mechanisms by which glioblastomas are resistant to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), we examined TRAIL signalling pathways in the tumours. TRAIL has four membrane-anchored receptors, death receptor 4/5 (DR4/5) and decoy receptor 1/2 (DcR1/2). Of these receptors, only DR5 was expressed consistently in glioblastoma cell lines and tumour tissues, ruling out the role of DcR1/2 in TRAIL resistance. Upon TRAIL binding, DR5 was homotrimerized and recruited Fas-associated death domain (FADD) and caspase-8 for the assembly of death-inducing signalling complex (DISC) in the lipid rafts of the plasma membrane. In the DISC, caspase-8 was cleaved and initiated apoptosis by cleaving downstream caspases in TRAIL-sensitive glioblastoma cells. In TRAIL-resistant cells, however, DR5-mediated DISC was modified by receptor-interacting protein (RIP), cellular FADD-like interleukin-1beta-converting enzyme inhibitory protein (c-FLIP) and phosphoprotein enriched in diabetes or in astrocyte-15 (PED/PEA-15). This DISC modification occurred in the non-raft fractions of the plasma membrane and resulted in the inhibition of caspase-8 cleavage and activation of nuclear factor-kappaB (NF-kappaB). Treatment of resistant cells with parthenolide, an inhibitor of inhibitor of kappaB (I-kappaB), eliminated TRAIL-induced NF-kappaB activity but not TRAIL resistance. In contrast, however, targeting of RIP, c-FLIP or PED/PEA-15 with small interfering RNA (siRNA) led to the redistribution of the DISC from non-rafts to lipid rafts and eliminated the inhibition of caspase-8 cleavage and thereby TRAIL resistance. Taken together, this study indicates that the DISC modification by RIP, c-FLIP and PED/PEA-15 is the most upstream event in TRAIL resistance in glioblastomas.

ICAM-5 modulates cytokine/chemokine production in the CNS during the course of herpes simplex virus type 1 infection.

Chemokines are important in HSE development in the CNS but underlying regulatory events are unknown. Two-hybrid binding assays identified that intercellular adhesion molecule 5 (ICAM-5), an immune modulator in the CNS, interacted with neurovirulence factor, UOL, of HSV-1. Viral load and interleukin levels were similar in UOL deletion virus (DeltaUOL), and wild type virus infected mouse brains. However, higher numbers of lymphocytes, but unaltered soluble ICAM-5 and chemokine levels were detected in DeltaUOL infected mouse brains. In contrast, lower lymphocyte numbers, reduced soluble ICAM-5, and higher chemokine levels were detected in wild type virus infected brains. Our results suggest that ICAM-5 plays a critical role in modulating chemokine production in the CNS.

Cloning, characterization and promoter analysis of the common carp IGF-II gene.

Insulin-like growth factors (IGFs) belong to a family of growth factors with structural homology to proinsulin. Up till now, no specific details regarding the transcriptional regulation by autocrine, paracrine or endocrine effector molecules in vivo have been described for the IGF-II gene. This is in big contrast to IGF-I gene transcription which has been studied more extensively. To better understand how the IGF-II gene is controlled at the gene transcription level, we have isolated the common carp IGF-II gene together with the 5'-flanking region by genomic library screening. The mature IGF-II protein was encoded by exon 2 and exon 3. Transient transfection of the 5'-flanking region containing a TATA box-like sequence into cultured eukaryotic cells revealed that it is a strong promoter with definitive tissue specificity. Nucleotides between -301 and -62 in the promoter are essential to drive the basal IGF-II gene expression; whereas nucleotides between -891 and -416 in the promoter are responsible for the growth hormone activation. Using electrophoretic mobility shift assay and yeast one-hybrid screening, it was demonstrated that alpha1-antitrypsin could bind specifically to the nucleotide position -301 to -262 of the gene promoter. Co-transfection studies revealed that the over-expression of alpha1-antitrypsin increased the IGF-II promoter activity by 3.4-fold, further confirming that alpha1-antitrypsin acts as a trans-acting factor on the IGF-II promoter.

Down-regulation of goldfish (Carassius auratus) prolactin gene expression by dopamine and thyrotropin releasing hormone.

Prolactin (PRL) is the most versatile hormone identified with multiple functions from osmoregulation in euryhaline fish to lactation in mammals. However, little is known about the basic physiological control of PRL in stenohaline freshwater fish. In this study, goldfish is used as a model for the study of PRL gene expression in stenohaline fish. We report herein the identification of the goldfish PRL (gfPRL) genomic sequence which possesses five exons with its 5'-flanking gene promoter region characterized in vitro using GH3 and GH4 cell-lines. Dopamine and thyrotropin releasing hormone were found to down-regulate the transcription of this gfPRL gene promoter in vitro. This was further confirmed by the decrease of PRL mRNA levels in vitro (in goldfish pituitary primary cells) and in vivo (intra-peritoneal injection) following the administrations of dopamine and thyrotropin releasing hormone. The Pit-1 binding sites of gfPRL are highly conserved with a consensus DNA sequence of 5'TATNCAT-3', as confirmed with the electrophoretic mobility shift assay using nuclear extract from the GH3 cell-line, may be responsible for the control of gfPRL gene promoter.

Lipid rafts and nonrafts mediate tumor necrosis factor related apoptosis-inducing ligand induced apoptotic and nonapoptotic signals in non small cell lung carcinoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is capable of inducing apoptosis in non-small cell lung carcinoma (NSCLC). However, many of the human NSCLC cell lines are resistant to TRAIL, and TRAIL treatment of the resistant cells leads to the activation of nuclear factor-kappaB (NF-kappaB) and extracellular signal-regulated kinase 1/2 (ERK1/2). TRAIL can induce apoptosis in TRAIL-sensitive NSCLC cells through the induction of death-inducing signaling complex (DISC) assembly in lipid rafts of plasma membrane. In the DISC, caspase-8 is cleaved and initiates TRAIL-induced apoptosis. In contrast, TRAIL-DISC assembly in the nonraft phase of the plasma membrane leads to the inhibition of caspase-8 cleavage and NF-kappaB and ERK1/2 activation in TRAIL-resistant NSCLC cells. Receptor-interacting protein (RIP) and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP) mediates the DISC assembly in nonrafts and selective knockdown of either RIP or c-FLIP with interfering RNA redistributes the DISC from nonrafts to lipid rafts, thereby switching the DISC signals from NF-kappaB and ERK1/2 activation to caspase-8-initiated apoptosis. Chemotherapeutic agents inhibit c-FLIP expression, thereby enhancing the DISC assembly in lipid rafts for caspase-8-initiated apoptosis. These studies indicate that RIP and c-FLIP-mediated assembly of the DISC in nonrafts is a critical upstream event in TRAIL resistance and thus targeting of either RIP or c-FLIP may lead to the development of novel therapeutic strategies that can overcome TRAIL resistance in human NSCLC.

Effects of chronic cysteamine treatment on growth enhancement and insulin-like growth factor I and II mRNA levels in common carp tissues.

Insulin-like growth factors (IGF) belong to a family of growth factors with structural homology to proinsulin. In our previous studies, we found that both IGF-I and IGF-II gene expression showed growth hormone (GH) dependence in the brain and liver of juvenile common carp when treated in vivo with GH for a short time. This present work aimed to study the effects of both the short-term and long-term GH induction of IGF gene expression using cysteamine (CSH) and fasting/re-feeding. CSH is an agent that can deplete somatostatin to increase circulating GH level. IGF mRNA levels in the flesh (muscle) and liver of common carp were determined using real-time PCR. The chronic treatment of common carp with CSH was carried out for 63 d, with growth enhancement of the treated fish noted. Hepatic IGF-I and IGF-II mRNA levels increased in a dose-dependent manner with short-term CSH treatment, whereas IGF-I decreased and IGF-II increased in the liver after chronic CSH treatment. IGF-I and IGF-II mRNA levels in muscle were found to be elevated with the high-dose, long-term CSH treatment. Under the experimentally induced catabolic states of fasting, both hepatic IGF-I and IGF-II gene expression were significantly reduced, whereas they showed no change in muscle. After re-feeding, the hepatic expression of IGF-I in liver and muscle rebounded significantly. The hepatic IGF-II expression level showed no rebound after re-feeding, but the IGF-II level in muscle rebounded to the level of the fed group after re-feeding.

Human astrocytes are resistant to Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis.

Human astrocytes express Fas yet are resistant to Fas-induced apoptosis. Here, we report that calcium/calmodulin-dependent protein kinase II (CaMKII) is constitutively activated in human astrocytes and protects the cells from apoptotic stimulation by Fas agonist. Once stimulated, Fas recruits Fas-associated death domain and caspase-8 for the assembly of the death-inducing signaling complex (DISC); however, caspase-8 cleavage is inhibited in the DISC. Inhibition of CaMKII kinase activity inhibits the expression of phosphoprotein enriched astrocytes-15 kDa/phosphoprotein enriched in diabetes (PEA-15/PED) and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP), thus releasing their inhibition of caspase-8 cleavage. Inhibition of PEA-15/PED or c-FLIP by small interfering RNA sensitizes human astrocytes to Fas-induced apoptosis. In contrast, inhibition of CaMKII, PEA-15, or c-FLIP does not affect the sensitivity of human astrocytes to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL death receptors (DR4, DR5) are weakly expressed at mRNA, protein, and cell surface levels and thus fail to mediate the assembly of the DISC in human astrocytes. Overexpression of DR5 restores TRAIL signaling pathways and sensitizes the human astrocytes to TRAIL-induced apoptosis if CaMKII kinase activity or expression of PEA-15 and c-FLIP is inhibited; the results suggest that CaMKII-mediated pathways prevent TRAIL-induced apoptosis in human astrocytes under conditions in which TRAIL death receptors are upregulated. This study has therefore identified the molecular mechanisms that protect normal human astrocytes from apoptosis induced by Fas ligand and TRAIL.

Stimulation of growth hormone secretion from seabream pituitary cells in primary culture by growth hormone secretagogues is independent of growth hormone transcription.

The action of a number of growth hormone secretagogues (GHS) on growth hormone (GH) secretion and gene expression was studied in a primary culture of pituitary cells isolated from the black seabream Acanthopagrus schlegeli. The peptide GHS employed included growth hormone-releasing peptide (GHRP)-2, ipamorelin, and human ghrelin. The nonpeptide GHS employed included the benzolactam GHS L692,585 and the spiropiperidine GHS L163,540. Secreted GH was measured in the culture medium by an enzyme-linked immunosorbent assay (ELISA) method using a specific antibody against seabream GH. The GH mRNA content in the incubated cells was assessed by reverse transcription polymerase chain reaction (RT-PCR) using a pair of gene-specific primers designed from the cloned black seabream GH cDNA sequence. A dose-dependent stimulation of GH release was demonstrated by all the GHS tested, except human ghrelin, with EC(50) values in the nanomolar range. Simultaneous measurement of GH mRNA levels in the incubated seabream pituitary cells indicated that the GHS-stimulated increase in GH secretion was not paralleled by corresponding changes in GH gene expression. In contrast to the situation previously reported in the rat, no change in GH gene expression was noticed in the seabream pituitary cells even though the time of stimulation by GHS was increased up to 48 h, confirming that the GHS-stimulated GH secretion in seabream is independent of GH gene transcription.

Seabream growth hormone receptor: molecular cloning and functional studies of the full-length cDNA, and tissue expression of two alternatively spliced forms.

A full-length clone of the growth hormone receptor (GHR) was isolated from a cDNA library constructed from the liver of black seabream (Acanthopagrus schlegeli). The seabream GHR (sbGHR) cDNA sequence encodes a transmembrane protein of 640 amino acids (aa) possessing the characteristic motifs and architectural design of GHRs of other species. When compared to the other fish GHRs, it is most homologous to another marine fish species, the turbot, where the aa identity is 79.3%. But the sbGHR sequence is more remotely related to the goldfish GHR (51.6% aa identity) and the salmonid GHRs (approximately 46-48% aa identities). Phylogenetic comparison with other known GHRs indicates that the fish GHRs constitute a distinct group among the different vertebrate classes. The aa identities between sbGHR and other GHRs are low, being around 40% with mammalian GHRs, around 45% with avian and reptilian GHRs, and less than 35% with Xenopus GHR. CHO cells transfected with the sbGHR cDNA can be stimulated to proliferate by recombinant seabream growth hormone (sbGH). In addition, the transfected cells can transactivate a co-expressed mammalian serine protease inhibitor (Spi) 2.1 promoter upon stimulation by sbGH. These functional assays indicated that the fish receptor can interact with its homologous ligand to evoke the downstream post-receptor events. Reverse transcription-polymerase chain reaction (RT-PCR) and genomic PCR using a pair of gene-specific primers revealed the expression of two alternatively spliced forms of sbGHR in various tissues of the fish. A 93-bp intron, unique to the sbGHR gene and not found in any other known GHR genes, is alternatively spliced to give rise to two forms of receptor mRNA transcripts. The two forms of the receptor are differentially expressed among the different tissues of the fish.

PCR-cloning and gene expression studies in common carp (Cyprinus carpio) insulin-like growth factor-II.

Insulin-like growth factor-II (IGF-II) is a member of a growth factor family related to fetal growth in mammals but its physiological role has not been clearly identified in fish. In teleosts, the basic mechanism of the growth hormone (GH)-IGF axis is known to be operative but in a different manner. For instance, IGF-I exhibits GH dependence whereas for IGF-II, its GH dependence varies in different fish species. In this study, we used polymerase chain reaction (PCR) to obtain a common carp IGF-II (ccIGF-II) cDNA fragment and methods of rapid amplification of cDNA ends (RACEs) to obtain a full-length ccIGF-II sequence. The ccIGF-II encodes for a predicted amino acid sequence showing identities of 70.6%, 68.7%, 63.4% and 35% in comparison with salmon, barramundi, tilapia and human IGF-II, respectively. The nucleotide identity between the open reading frame (ORF) of the ccIGF-II and ccIGF-I cDNA sequence is only 36.2%. Distribution of ccIGF-II mRNA levels in common carp tissues was also studied; ccIGF-II expressed in hepatopancreas, heart, and many other tissues in adult carps are similar to the levels of ccIGF-I except in gills and testis. ccIGF-II levels were significantly higher than that of ccIGF-I in most juvenile tissues except in hepatopancreas, where ccIGF-I was higher (threefold) than that of ccIGF-II. The levels of ccIGF-I were also higher than ccIGF-II in carp larvae, from pre-hatched stage to day 30 post-hatching. Injection of porcine GH (pGH) increased the IGF-I and IGF-II mRNA levels in the hepatopancreas and brain of juvenile carps. However, hepatic IGF-I mRNA levels were induced more than IGF-II by pGH, whereas ccIGF-II levels gave a higher response than IGF-I in the brain in response to GH induction.