Tudor Badea

Sublytic C5b-9 induces proliferation of human aortic smooth muscle cells Role of mitogen activated protein kinase and phosphatidylinositol 3-kinase

Proliferation of vascular smooth muscle cells contributes to initimal hyperplasia during atherogenesis, but the factors regulating their proliferation are not well known. In the present study we report that sublytic C5b-9 assembly induced proliferation of differentiated human aortic smooth muscle cells (ASMC) in culture. Cell cycle re-entry occurred through activation of cdk4, cdk2 kinase and the reduction of p21 cell cycle inhibitor. We also investigated if C5b-9 cell cycle induction is mediated through activation of mitogen activated protein kinase (MAPK) pathways. Extracellular signal regulated kinase (ERK) 1 activity was significantly increased, while c-jun NH2-terminal kinase (JNK) 1 and p38 MAPK activity were only transiently increased. Pretreatment with wortmannin inhibits ERK1 activation by C5b-9, suggesting the involvement of phosphatidylinositol 3-kinase (PI 3-kinase). Both PI 3-kinase and p70 S6 kinase were activated by C5b-9 but not by C5b6. C5b-9 induced DNA synthesis was abolished by pretreatment with inhibitors of ERK1 and PI 3-kinase, but not by p38 MAPK. These data indicated that ERK1 and PI 3-kinase play a major role in C5b-9 induced ASMC proliferation.

Molecular Cloning and Characterization of RGC-32, a Novel Gene Induced by Complement Activation in Oligodendrocytes

Sublytic complement activation on oligodendrocytes (OLG) down-regulates expression of myelin genes and induces cell cycle in culture. Differential display (DD) was used to search for new genes whose expression is altered in response to complement and that may be involved in cell cycle activation. DD bands showing either increased or decreased mRNA expression in response to complement were identified and designated ResponseGenes to Complement (RGC) 1–32.RGC-1 is identical with heat shock protein 105,RGC-2 with poly(ADP-ribose) polymerase, andRGC-10 with IP-10. A new gene, RGC-32, that encodes a protein of 137 amino acids was cloned. RGC-32 has no homology with other known proteins, and contains no motif that would indicate its function. In OLG, the mRNA expression was increased by complement activation and by terminal complement complex assembly. RGC-32 protein was localized in the cytoplasm and co-immunoprecipitated with cdc2 kinase. Overexpression of RGC-32 increased DNA synthesis in OLGxC6 glioma cell hybrids. These results suggest thatRGC-32 may play a role in cell cycle activation.

Terminal complement complexes induce cell cycle entry in oligodendrocytes through mitogen activated protein kinase pathway

Sublytic complement attack through C5b-9 assembly induces oligodendrocytes (OLG) to express proto-oncogenes and to enter the cell cycle from resting G0/G1 phase to S phase. We have investigated whether cell cycle induction by C5b-9 is mediated by mitogen activated protein kinase (MAPK) pathway in OLG. C5b-9 but not C5b6 induced activation of both ERK1 and c-jun NH2 terminal kinases 1 (JNK1) in OLG. The increased ERK1 and JNK1 activities are transient, reaching a maximum around 20 min following exposure to C5b-9. Activation of Raf-1 and MEK1, upstream kinases of ERK1, was shown by increased Raf-1 kinase activity in anti-Raf-1 immunoprecipitates of OLG treated with C5b-9 and ERK1 activity that can be inhibited by PD098,059, a specific MEK1 inhibitor. Requirement for the ERK1 pathway in DNA synthesis was then evaluated using PD098,059. Enhanced DNA synthesis induced by serum complement was completely abolished when OLG were pretreated with PD098,059. On the other hand, c-fos mRNA expression induced by complement was inhibited only 50% by PD098,059, while the c-jun mRNA level was not affected by this MEK1 inhibitor. Interestingly, p70 S6 kinase, an important ribosomal kinase in mitogenesis, was also activated by C5b-9. These findings indicated that the MAPK pathways appears to play a major role in inducing OLG to enter the S phase of the cell cycle from the resting G1/G0 phase.

Tyrosine phosphorylation and activation of Janus kinase 1 and STAT3 by sublytic C5b-9 complement complex in aortic endothelial cells.

The pathway involving Janus kinase (JAK) and signal transducers and activators of transcription (STATs) plays an important role in differentiation and proliferation of cells initiated by receptor activation. In the present study we identified the JAK and STAT proteins activated by C5b-9 in human aortic endothelial cells (AEC). JAK1 but not JAK2 was tyrosine phosphorylated in response to sublytic C5b-9. STAT3 was rapidly tyrosine phosphorylated also by C5b-9. Pertussis toxin inhibited the C5b-9 induced JAK1 activation. However, phosphorylation of STAT3 was not inhibited by Pertussis toxin, although C5b-9 induced a time-dependent nuclear translocation of STAT3. These observations indicated that JAK1 is phosphorylated by C5b-9 through activation of trimeric G proteins of the Gi/Go family. Raf-1 and ERK1 were also activated by C5b-9 in human AEC in a G protein dependent manner. Therefore, JAK1 activity may be involved in activation of Raf-1 and ERK1 via G proteins activated by C5b-9. This study demonstrates the ability of membrane-inserted C5b-9 to activate JAK1 and STAT3 proteins, thus defining new signalling pathway by which C5b-9 may regulate gene activation.

Sublytic Terminal Complement Attack on Myotubes Decreases the Expression of mRNAs Encoding Muscle‐Specific Proteins

Abstract: Activation of inflammatory and cytotoxic complement effectors that include the C5b‐9 complex plays an important pathogenic role in myasthenia gravis, an inflammatory autoimmune disease of the muscle. Altered muscle‐specific gene expression has been observed in experimental myasthenic rats. In this study, we have examined the effect of sublytic C5b‐9 on myotubes differentiated from C2C12 myoblasts, by generating C5b‐9 with C7‐deficient serum with or without C7. Within 2 h, C7‐deficient serum plus C7, compared with C7‐deficient serum alone, induced markedly decreased levels of mRNAs encoding α‐actin, troponin I slow twitch isoform, acetylcholine receptor α, and muscle aldolase A, whereas the heat shock protein 83 mRNA level remained constant, by northern analysis. Because the half‐life of the acetylcholine receptor α was estimated to be >8 h, the C5b‐9 effect was, in part, due to enhanced mRNA decay. Because C5b‐9 also induced c‐jun mRNA and reduced the myoD mRNA level, a possible inhibition of muscle gene transcription by C5b‐9 was examined in myotubes transfected with troponin promoter‐luciferase gene constructs. Luciferase activity was reduced to 50% in response to C5b‐9 at 2 h. Thus, C5b‐9 appears to inhibit the muscle‐specific gene expression by stimulating mRNA decay and by decreasing the transcription process. The data also indicate a possible pathogenic role of C5b‐9 in immune‐mediated inflammatory muscle disorders in which complement activation has been implicated.