Investigators in the Section on Metabolic Regulation previously revealed that PMT, a potent mitogen known to activate phospholipase C-beta-1, Jak-Stat, and Rho kinase pathways, down regulates caspase-12 mRNA and its protein level. In addition we found that PMT induces protein synthesis, ATP synthesis, and cell spreading in serum-deprived Swiss 3T3 cells. The mechanism of PMT-induced protein synthesis proceeds, in part, by a G-alpha q-dependent activation of mTORC1, a key Ser-Thr kinase conserved from yeast to mammals known to be involved in the regulation of protein synthesis, cell growth, proliferation, and autophagy in a nutrient- and energy-responsive manner. Further study reveals that PMT-induced mTORC1 activation proceeds via the MEK/ERK1/2 pathway. To investigate the upstream molecular events behind PMT-induced ERK activation, we found that, after 24h PMT treatment, serum-starved 3T3 cells generate a diffusible factor(s) which leads to ERK activation. To identify the putative factors secreted by PMT-treated cells that might be responsible for ERK activation, mRNA from PMT-treated and non-treated control cells were analyzed using Affymetrix GeneChips methods. We found 1774 probesets (1185 genes) up-regulated and 1599 probesets (1110 genes) down-regulated due to PMT treatment when compared with data from control cells. The mRNA with the greatest mean elevation in PMT-treated cells was the connective tissue growth factor (CTGF) mRNA, which was elevated about 140-fold relative to that of the control non-treated cells. The PMT-induced CTGF mRNA was further confirmed with RT-PCR experiments. Furthermore, using immunoblot analysis, we showed a huge increase in CTGF protein in both cell lysate and conditioned media obtained from PMT-treated cells. The addition of rapamycin, an inhibitor of the mTOR signaling pathway activated by PMT, for 24h did not significantly change the levels of CTGF mRNA and protein. However, treatment with U0126, the specific inhibitor of MEK, which is the upstream kinase of ERK signaling pathway, shows an inhibitory effect on PMT-induced CTGF protein levels, but not its CTGF mRNA, in both conditioned media and PMT-treated whole cell lysate. Interestingly, treatment of serum-starved 3T3 cells with recombinant CTGF protein for 24h leads to a sustained increase in the phosphorylation of ERK1/2. Thus, CTGF constitutes the primary candidate for a PMT-induced secreted factor involved in the activation of ERK. Furthermore, it was previously shown that some PMT-induced cellular effects are mediated via G-alpha q but not by the highly related G-alpha 11 protein. To study the role of G-alpha q on the elevation of CTGF mRNA and protein induced by PMT, we employed a fibroblast cell line derived from mouse embryos deficient in both G-alpha q and G-alpha 11. Our results showed that quiescent wild type MEF cells, when treated with PMT for 24h, showed an increase in CTGF protein levels in both PMT-treated whole cell lysate and conditioned media in comparison to that of the control non-treated cells. However, we did not observe any stimulatory effect due to PMT treatment on CTGF protein levels in fibroblasts derived from mice deficient in both G-alpha q and G-alpha 11. This result is in accordance with the fact that PMT fails to induce ERK1/2 phosphorylation in MEF cells deficient in both G-alpha q and G-alpha 11, and inhibition of ERK1/2 suppresses PMT-induced CTGF protein leves.
| Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A et al. (2013) Pasteurella multocida toxin (PMT) upregulates CTGF which leads to mTORC1 activation in Swiss 3T3 cells. Cell Signal 25:1136-48 |
| Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A et al. (2013) Mammalian target of rapamycin complex 1 (mTORC1) plays a role in Pasteurella multocida toxin (PMT)-induced protein synthesis and proliferation in Swiss 3T3 cells. J Biol Chem 288:2805-15 |
