Activation of p38 and JNK MAPK pathways abrogates requirement for new protein synthesis for phorbol ester mediated induction of select MMP and TIMP genes.
The human matrix metalloproteinase (MMP) gene family includes 24 genes whose regulated expression, together with that of four tissue inhibitors of metalloproteinases (TIMPs), is essential in tissue remodelling and cell signalling. Quantitative real-time-PCR (qPCR) analysis was used to evaluate the shared and unique patterns of control of these two gene families in human MRC-5 and WI-38 fibroblasts in response to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA). The requirement for ongoing translation was analysed using three protein synthesis inhibitors, anisomycin, cycloheximide and emetine. PMA induced MMP1, 3, 8, 9, 10, 12, 13, 14 and TIMP1 and TIMP3 RNAs after 4-8 h, and induction of all except MMP9 and TIMP3 was blocked by all protein synthesis inhibitors. However, even though all inhibitors effectively blocked translation, PMA-induction of MMP9 and TIMP3 was blocked by emetine but was insensitive to cycloheximide and anisomycin. Anisomycin alone induced MMP9 and TIMP3, along with MMP25 and MMP19. The extracellular signal-regulated kinases (ERKs)-1/2 were strongly activated by PMA, while anisomycin activated the c-Jun N-terminal kinase (JNK) and p38 pathways, and cycloheximide activated p38, but emetine had no effect on the stress-activated mitogen-activated protein kinase (MAPK) pathways. The involvement of the p38 and JNK pathways in the selective effects of anisomycin and cycloheximide on MMP/TIMP expression was supported by use of pharmacological inhibitors. These data confirm that most inducible MMPs and TIMP1 behave as "late" activated, protein synthesis-dependent genes in fibroblasts. However, the requirement of protein synthesis for PMA-induction of MMPs and TIMPs is not universal, since it is abrogated for MMP9 and TIMP3 by stimulation of the stress-activated MAPK pathways. The definition of clusters of co-regulated genes among the two gene families will aid in bioinformatic dissection of control mechanisms.
Sampieri
Clara L
CL
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Nuttall
Robert K
RK
Young
David A
DA
Goldspink
Deborah
D
Clark
Ian M
IM
Edwards
Dylan R
DR
D015153:Blotting, Western; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D058851:GPI-Linked Proteins; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020781:Matrix Metalloproteinase 1 / Q000235:genetics; D053506:Matrix Metalloproteinase 10 / Q000235:genetics; D020782:Matrix Metalloproteinases / Q000235:genetics*; D053504:Matrix Metalloproteinases, Membrane-Associated / Q000235:genetics; D053505:Matrix Metalloproteinases, Secreted / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008954:Models, Biological; D014176:Protein Biosynthesis / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics; D019714:Tissue Inhibitor of Metalloproteinases / Q000235:genetics*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D058851:GPI-Linked Proteins; D047428:Protein Kinase Inhibitors; D011500:Protein Synthesis Inhibitors; C088390:TIMP3 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D019717:Tissue Inhibitor of Metalloproteinase-3; D019714:Tissue Inhibitor of Metalloproteinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020782:Matrix Metalloproteinases; D053504:Matrix Metalloproteinases, Membrane-Associated; D053505:Matrix Metalloproteinases, Secreted; C108362:matrix metalloproteinase 19; C403928:matrix metalloproteinase 25; C511770:MMP10 protein, human; D053506:Matrix Metalloproteinase 10; C522360:MMP1 protein, human; D020781:Matrix Metalloproteinase 1; D013755:Tetradecanoylphorbol Acetate