Abstract
The ADP-ribosyltransferase C3 exoenzyme from C. botulinum selectively inactivates Rho and is therefore often used as an inhibitor for investigations on Rho signaling. Previous studies of our group revealed that C3 inhibited cell proliferation in HT22 cells accompanied by increased transcriptional activities of Sp1 and c-Jun and reduced levels of cyclin D1, p21 and phosphorylated p38. By use of a p38α-deficient and a p38α-expressing control cell line, the impact of p38 on C3-mediated inhibition of cell proliferation and alterations on MAPK signaling was studied by growth kinetic experiments and Western blot analyses. The cell growth of p38α-expressing cells was impaired by C3, while the p38α-deficient cells did not exhibit any C3-induced effect. The activity of the MKK3/6-p38 MAPK signaling cascade as well as the phosphorylation of c-Jun and JNK was reduced by C3 exclusively in the presence of p38α. Moreover, the activity of upstream MAPKKK TAK1 was lowered in the p38α-expressing cells. These results indicated a resistance of p38α-deficient cells to C3-mediated inhibition of cell growth. This anti-proliferative effect was highly associated with the decreased activity of c-Jun and upstream p38 and JNK MAPK signaling as a consequence of the absence of p38α in these cells.
Highlights
The bacterial C3 exoenzyme from Clostridium botulinum C3-E174Q (C3) exoenzyme (C3) from Clostridium botulinum selectively inactivates RhoA, RhoB and RhoC by ADP-ribosylation [1,2,3], resulting in various cellular consequences, such as morphological alterations due to the disturbance of actin cytoskeleton and formation of contractile ring [4,5,6]
Art. 1, page 2 of 11 von Elsner et al: Anti-proliferative Effect of C3 Exoenzyme in Fibroblasts is Mediated by c-Jun Phosphorylation embryos [31, 32]
These results imply that the C3-mediated inhibition of cell proliferation in p38 −/− p38 mouse embryonic fibroblasts TAK1 (MEFs) strictly depended on RhoA inactivation and the presence of p38, but was independent of cyclin D1
Summary
The bacterial C3 exoenzyme (C3) from Clostridium botulinum selectively inactivates RhoA, RhoB and RhoC by ADP-ribosylation [1,2,3], resulting in various cellular consequences, such as morphological alterations due to the disturbance of actin cytoskeleton and formation of contractile ring [4,5,6]. C3 increases enzyme-independently the axonal growth and branching in primary hippocampal neurons [7]. Besides this neurotrophic effect, C3 effects apoptosis induction in various cell types. C3 and the enzyme-deficient mutant C3-E174Q, reduced the level of cell cycle regulator cyclin D1, increased the abundance of the negative cell cycle regulator RhoB and inhibited cell proliferation with varying extent in the murine hippocampal HT22 cells [12, 15]. Our group demonstrated a strong correlation between C3-induced inhibition of cell growth and the increased transcriptional activity of Sp1 accompanied by an elevated level of p21 in HT22 cells [16]. The main activation of MAPK canonically occurs via phosphorylation by upstream MAPK kinases, which in turn are phosphorylated and activated by MAPKK kinases such as MEKKs, ASK1 and TAK1 [22,23,24,25,26,27,28]
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