title: Pasteurella multocida toxin (PMT) upregulates CTGF which leads to mTORC1 activation in Swiss 3T3 cells.
abstract: Pasteurella multocida toxin (PMT) is a mitogenic protein that hijacks cellular signal transduction pathways via deamidation of heterotrimeric G proteins. We previously showed that rPMT activates mTOR signaling via a Gαq/11/PLCβ/PKC mediated pathway, leading in part to cell proliferation and migration. Herein, we show that mTOR and MAPK, but not membrane-associated tyrosine kinases, are activated in serum-starved 3T3 cells by an autocrine/paracrine substance(s) secreted into the conditioned medium following rPMT treatment. Surprisingly, this diffusible factor(s) is capable of activating mTOR and MAPK pathways even in MEF Gαq/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in rPMT-treated serum-starved 3T3 cells relative to untreated cells. These results were further confirmed using RT-PCR and Western blot analyses. In accord with rPMT-induced mTOR activation, upregulation of CTGF protein was observed in WT MEF, but not in Gαq/11 double knockout MEF cells. Although CTGF expression is regulated by TGFβ, rPMT did not activate TGFβ pathway. In addition, MEK inhibitors U0126 or PD98059, but not mTOR specific inhibitors, rapamycin and Torin 1, inhibited rPMT-induced upregulation of CTGF. Importantly, CTGF overexpression in serum-starved 3T3 cells using adenovirus led to phosphorylation of ribosomal protein S6, a downstream target of mTOR. However, despite the ability of CTGF to activate the mTOR pathway, upregulation of CTGF alone could not induce morphological changes as those observed in rPMT-treated cells. Our findings reveal that CTGF plays an important role, but there are additional factors involved in the mitogenic action of PMT.
authors: Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8012, USA. oubrahih@nhlbi.nih.gov
authors: Hammou
authors: H
authors: Oubrahim
authors: Allison
authors: A
authors: Wong
authors: Brenda A
authors: BA
authors: Wilson
authors: P Boon
authors: PB
authors: Chock
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D015398:Signal Transduction; D041701:Swiss 3T3 Cells; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D046912:Multiprotein Complexes; C062258:Pasteurella multocida toxin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11