Protease-activated receptor-1 expression in rat microglia after trimethyltin treatment.

In the nervous system, protease-activated receptors (PARs), which are activated by thrombin and other extracellular proteases, are expressed widely at both neuronal and glial levels and have been shown to be involved in several brain pathologies. As far as the glial receptors are concerned, previous experiments performed in rat hippocampus showed that expression of PAR-1, the prototypic member of the PAR family, increased in astrocytes both in vivo and in vitro following treatment with trimethyltin (TMT). TMT is an organotin compound that induces severe hippocampal neurodegeneration associated with astrocyte and microglia activation. In the present experiments, the authors extended their investigation to microglial cells. In particular, by 7 days following TMT intoxication in vivo, confocal immunofluorescence revealed an evident PAR-1-related specific immunoreactivity in OX-42-positive microglial cells of the CA3 and hilus hippocampal regions. In line with the in vivo results, when primary rat microglial cells were treated in vitro with TMT, a strong upregulation of PAR-1 was observed by immunocytochemistry and Western blot analysis. These data provide further evidence that PAR-1 may be involved in microglial response to brain damage.

Expression pattern and sub-cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U-73122 in rat astrocytoma cells.

Phosphoinositide specific phospholipase C (PI-PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI-PLC family display different expression and/or sub cellular distribution under non-physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellular distribution of PI-PLCs compared to untreated cells. Special attention require PI-PLC beta3 and PI-PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U-73122 treatment. The meaning of these modifications is unclear, also because the use of this N-aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells.

Protease-activated receptor-1 regulates cytokine production and induces the suppressor of cytokine signaling-3 in microglia.

Protease-activated receptors (PARs) are cleaved and activated by thrombin and other extracellular proteases which are released during tissue trauma and inflammation. PAR-1 is the prototypic member of the PAR family and has been shown to be upregulated in several brain pathologies being expressed by neurons and glial cells. The present experiments show that the administration of the PAR-1 activating peptides (TRAP6 and TFLLR) inhibits the production of the pro-inflammatory cytokines TNF-alpha and IL-6 in microglial cells treated with lipopolysaccharide (LPS) while promoting the release of the anti-inflammatory cytokine IL-10. Conversely, the addition of the specific PAR-2 agonist SLIGRL had no effect on the amount of cytokines released following LPS treatment. Consistent with these data PAR-1, but not PAR-2, stimulation upregulates the expression of the suppressor of cytokine signaling-3 (SOCS-3). The present data support the hypothesis that in microglia PAR-1 may be involved in the regulation of inflammatory reactions modulating the balance between pro- and anti-inflammatory cytokines possibly through SOCS induction.

Variegation of the phenotype induced by the Gata1low mutation in mice of different genetic backgrounds.

All mice harboring the X-linked Gata1low mutation in a predominantly CD1 background are born anemic and thrombocytopenic. They recover from anemia at 1 month of age but remain thrombocytopenic all their life and develop myelofibrosis, a syndrome similar to human idiopathic myelofibrosis, at 12 months. The effects of the genetic background on the myelofibrosis developed by Gata1low mice was assessed by introducing the mutation, by standard genetic approaches, in the C57BL/6 and DBA/2 backgrounds and by analyzing the phenotype of the different mutants at 12 to 13 (by histology) and 16 to 20 (by cytofluorimetry) months of age. Although all the Gata1low mice developed fibrosis at 12 to 13 months, variegations were observed in the severity of the phenotype expressed by mutants of different backgrounds. In C57BL/6 mice, the mutation was no longer inherited in a Mendelian fashion, and fibrosis was associated with massive osteosclerosis. Instead, DBA/2 mutants, although severely anemic, expressed limited fibrosis and osteosclerosis and did not present tear-drop poikilocytes in blood or extramedullary hemopoiesis in liver up to 20 months of age. We propose that the variegation in myelofibrosis expressed by Gata1low mutants of different strains might represent a model to study the variability of the clinical picture of the human disease.

Effects of insulin-like growth factor I and II on prostaglandin synthesis and plasminogen activator activity in human umbilical vein endothelial cells.

IGFs seem to contribute to the endothelial dysfunction observed in some vascular diseases. Because locally increased IGFs levels were detected in the preeclamptic fetoplacental unit, we hypothesized their involvement in the dysregulation of fibrinolysis and vascular tone typically observed in the fetoplacental compartment in this pregnancy disease. Therefore, in human umbilical vein endothelial cells (HUVECs), the potential effect of IGFs on the synthesis of plasminogen activators (PAs), PA inibitor-1 (PAI-1), and vasodilator and vasoconstrictor prostaglandins (PGs) was investigated. Moreover, in HUVECs treated with IGFs, the expression of cyclooxygenase (COX)-2, the rate-limiting enzyme in PG synthesis, was evaluated.HUVECs were treated for 24 h with IGFs (1-100 ng/ml) or IL-1beta (0.1 ng/ml). PA, PAI-1, and COX-2 mRNA was determined by RT-PCR and PG release and PA activity by RIA and colorimetric assay, respectively.We demonstrated an inhibition of urokinase-type PA activity and a 50% reduction of urokinase-type PA mRNA in HUVECs treated with IGFs. No effect was seen on PAI-1. Finally, both IGFs significantly decreased all PGs tested and COX-2 mRNA, whereas, as expected, IL-1beta had an opposite effect. In conclusion, our results suggest for IGFs a potential involvement in the endothelial dysfunction observed in preeclamptic fetoplacental unit.

Expression of dystrophin-associated proteins during neuronal differentiation of P19 embryonal carcinoma cells.

The dystrophin gene that is defective in Duchenne muscular dystrophy shows a complex transcriptional control based on several promoters driving independent cell-type-specific expression of different isoforms. Dystrophin isoforms together with dystroglycan, a transmembrane protein which in turn binds to extracellular matrix, are the core of a complex of proteins, the dystrophin-associated protein (DAP) complex, which also comprises cytoplasmic elements like dystrobrevin. Whereas the molecular organization of DAP complex in muscle is well documented, the composition of a similar complex in the nervous system remains largely unknown. We followed by competitive PCR the expression of DAP complex components during retinoic acid (RA)-induced neuronal differentiation of P19 cells. Transcripts for the full-length dystrophin, Dp427, and the short isoform, Dp71, as well as for alpha-dystrobrevin 2 increased in parallel with days in culture after RA stimulation, while dystroglycan, alpha-dystrobrevin 1 and 3, and beta-dystrobrevin were constitutively expressed. The upregulation of some of the components of the dystrophin complex during neuronal maturation suggests functional flexibility of the complex in the nervous system, where specific associations between different isoforms of DAP complex components could possibly organize distinct DAP complex-like complexes.