Nevenka Juretić

ERK 1,2 and p38 pathways are involved in the proliferative stimuli mediated by urokinase in osteoblastic SaOS-2 cell line.

Bone metastases from prostate origin generate an osteoblastic reaction that is expressed in vitro by increased osteoblast proliferation. The urokinase‐like plasminogen activator (u‐PA) present in the media conditioned by tumoral prostatic cells acting as a ligand of the cellular membrane receptor (u‐PAR), has been identified as the specific factor that modulates this proliferative reaction. The present study represents an effort to unravel the intracellular pathway by which u‐PA activates osteoblastic proliferation and to evaluate the role of cellular receptor u‐PAR in this proliferative phenomenon. Our results show that in vitro u‐PA stimulates proliferation of SaOS‐2 osteoblastic cells by activating the MAP kinase route of ERK 1 and 2 and the p38 pathway. These results are in accordance with the inhibition of intermediate activation and cell proliferation by PD 098059 and SB 203580, specific inhibitors of MEK and p38, respectively. We also show that SaOS‐2 cells increase their proliferative response when cells are plated onto vitronectin, the second natural ligand of u‐PAR, and that culturing SaOS‐2 cells in the presence of u‐PA represents a stimuli for u‐PAR expression. On the basis of these results we propose that osteoblastic cells respond to the prostate‐derived u‐PA stimuli in a very efficient manner that includes the utilization of two different signaling routes and the stimulation of the expression of the u‐PA receptor.

Differential gene expression in skeletal muscle cells after membrane depolarization

Skeletal muscle is a highly plastic tissue with a remarkable capacity to adapt itself to challenges imposed by contractile activity. Adaptive response, that include hypertrophy and activation of oxidative mechanisms have been associated with transient changes in transcriptional activity of specific genes. To define the set of genes regulated by a depolarizing stimulus, we used 22 K mouse oligonucleotide microarrays. Total RNA from C2C12 myotubes was obtained at 2, 4, 18, and 24 h after high K+ stimulation. cDNA from control and depolarized samples was labeled with cyanine 3 or 5 dyes prior to microarray hybridization. Loess normalization followed by statistical analysis resulted in 423 differentially expressed genes using an unadjusted P‐value ≤ 0.01 as cut off. Depolarization affects transcriptional activity of a limited number of genes, mainly associated with metabolism, cell communication and response to stress. A number of genes related to Ca2+ signaling pathways are induced at 4 h, reinforcing the potential role of Ca2+ in early steps of signal transduction that leads to gene expression. Significant changes in the expression of molecules involved in muscle cell structure were observed; K+‐depolarization increased Tnni1 and Acta1 mRNA levels in both differentiated C2C12 and rat skeletal muscle cells in primary culture. Of these two, depolarization induced slow Ca2+ transients appear to have a role only in the regulation of Tnni1 transcriptional activity. We suggest that depolarization induced expression of a small set of genes may underlie Ca2+ dependent plasticity of skeletal muscle cells. J. Cell. Physiol. 210: 819–830, 2007.

Membrane depolarization induces calcium-dependent upregulation of Hsp70 and Hmox-1 in skeletal muscle cells

Heat shock proteins (HSPs) are a conserved family of cytoprotective polypeptides, synthesized by cells in response to stress. Hsp70 and heme oxygenase 1 (Hmox-1) are induced by a variety of cellular stressors in skeletal muscle, playing a role in long-term adaptations and muscle fibers regeneration. Though HSPs expression after exercise has been intensely investigated, the molecular mechanisms concerning Hsp70 and Hmox-1 induction are poorly understood. The aim of this work was to investigate the involvement of calcium in Hsp70 and Hmox-1 expression upon depolarization of skeletal muscle cells. We observed that depolarization of myotubes increased both mRNA levels and protein expression for Hsp70 and Hmox-1. Stimulation in the presence of intracellular calcium chelator BAPTA-AM resulted in a complete inhibition of Hsp70-induced expression. It is known that inositol-1,4,5-trisphophate (IP(3))-mediated slow Ca(2+) transients, evoked by membrane depolarization, are involved in the regulation of gene expression. Here we demonstrated that inhibition of IP(3)-dependent calcium signals decreased both Hsp70 mRNA induction and Hsp70 and Hmox-1 protein expression. Inhibitors of calcium-dependent protein kinase C also abolished Hsp70 mRNA induction. Our results provide evidence that membrane depolarization increases Hsp70 and Hmox-1 expression in cultured skeletal muscle cells, which the effect is critically dependent on Ca(2+) released from IP(3)-sensitive intracellular stores and that it involves PKC as an upstream effector in Hsp70 mRNA-induced expression.

Insulin-Dependent H2O2 Production Is Higher in Muscle Fibers of Mice Fed with a High-Fat Diet

Insulin resistance is defined as a reduced ability of insulin to stimulate glucose utilization. C57BL/6 mice fed with a high-fat diet (HFD) are a model of insulin resistance. In skeletal muscle, hydrogen peroxide (H2O2) produced by NADPH oxidase 2 (NOX2) is involved in signaling pathways triggered by insulin. We evaluated oxidative status in skeletal muscle fibers from insulin-resistant and control mice by determining H2O2 generation (HyPer probe), reduced-to-oxidized glutathione ratio and NOX2 expression. After eight weeks of HFD, insulin-dependent glucose uptake was impaired in skeletal muscle fibers when compared with control muscle fibers. Insulin-resistant mice showed increased insulin-stimulated H2O2 release and decreased reduced-to-oxidized glutathione ratio (GSH/GSSG). In addition, p47phox and gp91phox (NOX2 subunits) mRNA levels were also high (~3-fold in HFD mice compared to controls), while protein levels were 6.8- and 1.6-fold higher, respectively. Using apocynin (NOX2 inhibitor) during the HFD feeding period, the oxidative intracellular environment was diminished and skeletal muscle insulin-dependent glucose uptake restored. Our results indicate that insulin-resistant mice have increased H2O2 release upon insulin stimulation when compared with control animals, which appears to be mediated by an increase in NOX2 expression.

Abnormal distribution of inositol 1,4,5-trisphosphate receptors in human muscle can be related to altered calcium signals and gene expression in Duchenne dystrophy-derived cells

Inositol 1,4,5‐trisphosphate (IP3) receptors (IP3Rs) drive calcium signals involved in skeletal muscle excitation‐transcription coupling and plasticity; IP3R subtype distribution and downstream events evoked by their activation have not been studied in human muscle nor has their possible alteration in Duchenne muscular dystrophy (DMD). We studied the expression and localization of IP3R subtypes in normal and DMD human muscle and in normal (RCMH) and dystrophic (RCDMD) human muscle cell lines. In normal muscle, both type 1 IP3Rs (IP3R1) and type 2 IP3Rs (IP3R2) show a higher expression in type II fibers, whereas type 3 IP3Rs (IP3R3) show uniform distribution. In DMD biopsies, all fibers display a homogeneous IP3R2 label, whereas 24 ± 7% of type II fibers have lost the IP3R1 label. RCDMD cells show 5‐fold overexpression of IP3R2 and down‐regulation of IP3R3 compared with RCMH cells. A tetanic stimulus induces IP3‐dependent slow Ca2+ transients significantly larger and faster in RCDMD cells than in RCMH cells as well as significant ERK1/2 phosphorylation in normal but not in dystrophic cells. Excitation‐driven gene expression was different among cell lines; 44 common genes were repressed in RCMH cells and expressed in RCDMD cells or vice versa. IP3‐dependent Ca2+ release may play a significant role in DMD pathophysiology.—Cárdenas, C., Juretić, N., Bevilacqua, J. A., García, I. E., Figueroa, R., Hartley, R., Taratuto, A. L., Gejman, R., Riveros, N., Molgó, J., Jaimovich, E. Abnormal distribution of inositol 1,4,5‐trisphosphate receptors in human muscle can be related to altered calcium signals and gene expression in Duchenne dystrophy‐derived cells. FASEB J. 24, 3210–3221 (2010). www.fasebj.org

Electrical stimulation induces calcium-dependent up-regulation of neuregulin-1β in dystrophic skeletal muscle cell lines.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease originated by reduced or no expression of dystrophin, a cytoskeletal protein that provides structural integrity to muscle fibres. A promising pharmacological treatment for DMD aims to increase the level of a structural dystrophin homolog called utrophin. Neuregulin-1 (NRG-1), a growth factor that potentiates myogenesis, induces utrophin expression in skeletal muscle cells. Microarray analysis of total gene expression allowed us to determine that neuregulin-1β (NRG-1β) is one of 150 differentially expressed genes in electrically stimulated (400 pulses, 1 ms, 45 Hz) dystrophic human skeletal muscle cells (RCDMD). We investigated the effect of depolarization, and the involvement of intracellular Ca2+ and PKC isoforms on NRG-1β expression in dystrophic myotubes. Electrical stimulation of RCDMD increased NRG-1β mRNA and protein levels, and mRNA enhancement was abolished by actinomycin D. NRG-1β transcription was inhibited by BAPTA-AM, an intracellular Ca2+ chelator, and by inhibitors of IP3-dependent slow Ca2+ transients, like 2-APB, Ly 294002 and Xestospongin B. Ryanodine, a fast Ca2+ signal inhibitor, had no effect on electrical stimulation-induced expression. BIM VI (general inhibitor of PKC isoforms) and Gö 6976 (specific inhibitor of Ca2+-dependent PKC isoforms) abolished NRG-1β mRNA induction. Our results suggest that depolarization induced slow Ca2+ signals stimulate NRG-1β transcription in RCDMD cells, and that Ca2+-dependent PKC isoforms are involved in this process. Based on utrophin´s ability to partially compensate dystrophin disfunction, knowledge on the mechanism involved on NRG-1 up-regulation could be important for new therapeutic strategies design.

Interleukin-6 and neuregulin-1 as regulators of utrophin expression via the activation of NRG-1/ErbB signaling pathway in mdx cells ☆

Duchenne muscular dystrophy (DMD) is a neuromuscular disease originated by mutations in the dystrophin gene. A promising therapeutic approach deals with functional substitution of dystrophin by utrophin, a structural homolog that might be able to compensate dystrophin absence in DMD muscle fibers. It has been described that both interleukin-6 (IL-6) and neuregulin-1 (NRG-1; Heregulin-HRG) induce utrophin expression in skeletal muscle. We investigated a possible functional link among IL-6, NRG-1 and utrophin, in normal (C57) and dystrophic (mdx) skeletal muscle cells. Western Blot analysis allowed us to demonstrate that IL-6 (100ng/mL) induces NRG-1 receptor phosphorylation (ErbB2/ErbB3) in both cell types, in a process that depends on intracellular Ca2+ and metalloproteinase activity; it also induces a transient increase of ERK1 and GABPα phosphorylation only in dystrophic myotubes. Semiquantitative PCR showed that IL-6 treatment increases utrophin mRNA levels just in mdx myotubes. We observed that utrophin mRNA induction was abolished by BAPTA-AM (an intracellular Ca2+ chelator), GM6001 (a general metalloproteinase inhibitor), genistein (a general protein tyrosine kinase inhibitor), PD-158780 (an ErbB receptor tyrosine kinase inhibitor) and PD-98059 (a MEK inhibitor), whereas Ly-294002 and wortmannin (PI3K inhibitors) did not affect utrophin induction evoked by IL-6 in dystrophic myotubes. Our results suggest that IL-6 induces utrophin expression in mdx myotubes through activation of a NRG-1/ErbBs signaling cascade. Soluble NRG-1 elicited by proteolytic processing of transmembrane NRG-1 might induce ErbBs phosphorylation and ERK1/2 pathway activation, leading to utrophin up-regulation.

Altered Gene Expression Pathways in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is caused by the absence of functional dystrophin (Blake et al. 2002). Dystrophin is a cytoskeleton protein normally expressed in the inner face of the plasma membrane (Ahn and Kunkel 1993). In normal skeletal muscle, dystrophin is associated with a complex of glycoproteins known as dystrophin-associated proteins (DAPs), providing a linkage between the extracellular matrix (ECM) and cytoskeleton (Batchelor and Winder 2006). Lack of dystrophin in dystrophic muscle results in loss of the complex integrity and allegedly impairs the stability of the plasma membrane causing mechanical stress fragility, and an increase in Ca2+ permeability (Alderton and Steinhardt 2000). But the pathophysiology of muscular dystrophy is not only explained by this increased mechanical fragility and a role for dystrophin and DAPs has been suggested as being part of a protein signaling complex involved in cell survival (Rando 2001). In this chapter we discuss evidence of such a role, which may evidence possible interactions between dystrophin and proteins other than those involved in DAP and possible cell location of dystrophin in regions other than the sarcolemma cytoskeleton.