Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2 (iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12 myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2 was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2 blockade by use of bromoenol lactone (10 microM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.

SummaryThe transcription factor Fli-1 is implicated in the pathogenesis of both murine and human lupus. Increased levels of Fli-1 mRNA were present in the peripheral blood lymphocytes from lupus patients; furthermore, transgenic overexpression of Fli-1 in normal mice resulted in the development of a lupus-like disease. Lupus nephritis is a major cause of death in both lupus patients as well as in animal models. In this study, we generated Fli-1 heterozygous knockout (Fli-1 +/-) NZM2410 mice (of which the wild-type is a widely used lupus murine model) that expressed decreased levels of Fli-1 and investigated the impact of Fli-1 expression on lupus nephritis development and survival. Ninety-three per cent of the Fli-1 +/-NZM2410 mice survived to the age of 52 weeks compared to only 35% of wild-type NZM2410 mice. Autoantibodies, including anti-dsDNA and anti-glomerular basement antigen, in Fli-1 +/-NZM2410 mice were statistically significantly lower when compared to wild-type NZM2410 mice at the ages of 30 and 34 weeks. Total B cell and activated B cell populations in the spleens from Fli-1 +/-NZM2410 mice were decreased significantly compared to wild-type NZM2410 mice. Fli-1 +/-NZM2410 mice also had remarkably diminished proteinuria and decreased renal pathological scores when compared with wild-type NZM2410 mice. Expression of early growth response 1 (Egr-1) was decreased significantly in the kidneys from Fli-1 +/-NZM2410 mice when compared to wildtype littermates. Our data indicate that expression of Fli-1 plays an important role in lupus disease development in NZM2410 mice.

SummaryThe transcription factor Fli-1 is implicated in the pathogenesis of both murine and human lupus. Decreased expression of Fli-1 in heterozygous (Fli-1 +/-) Murphy Roths Large (MRL)/lpr mice resulted in significantly lower kidney pathological scores and markedly increased survival. In this study, bone marrow (BM) transplantation was used to investigate the role of decreased expression of Fli-1 in haematopoietic versus non-haematopoietic cell lineages in autoimmune disease development. Wild-type (WT) MRL/lpr that received BM from Fli-1 +/-MRL/lpr mice had statistically significantly lower autoantibodies, less proteinuria, reduced renal disease and prolonged survival compared to WT MRL/lpr mice that received BM from WT MRL/lpr mice. Although not statistically significant, Fli-1 +/-MRL/lpr mice that received BM from WT MRL/lpr mice also had lower autoantibodies and improved survival compared to WT MRL/lpr mice that received BM from WT MRL/lpr mice. Our data indicate that expression of Fli-1 in haematopoietic cell lineages has a significant effect on disease development in MRL/lpr mice.

The Fli1 gene is a member of the ets family of transcription factors that is expressed in hematopoietic cells. Two-fold overexpression of Fli1 protein in transgenic mice results in the development of a lupus-like disease. Expression of Fli1 in systemic lupus erythematosus (SLE) patients and animal models of lupus is higher compared to normal controls. Here we examined the effects of reduced Fli1 expression on disease development in NZM2410 mice, an animal model of SLE. We generated Fli1 knockout heterozygous mice (Fli1+/-; Fli-1 homozygous knockout is embryonic lethal). Compared to wild-type (Fli1+/+) mice, we found that expression of Fli1 protein in Fli1+/- mice was reduced by half. Autoantibodies, Urinary albumin excretion and serum immunoglobulin concentrations were measured by ELISA. Fli1+/- NZM2410 mice survived significantly longer than Fli1+/+ NZM2410 mice. At the age of 52 weeks, 81% Fli1+/- NZM 2410 mice survived, whereas only 35% of Fli1+/+ mice survived. There were significant low urinary albumin excretions in Fli1+/- NZM2410 mice compared to Fli1+/+ NZM2410 mice at the age of 34 weeks. Compared with Fli1+/+ NZM2410 mice, Fli1+/- NZM2410 mice have significantly decreased levels of IgG anti-dsDNA and glomerular antigen (GA) antibodies at the age of 34 weeks. Our results suggest that the expression of Fli1 plays an important role in disease development in NZM2410 mice.