Progesterone differentially regulates the membrane-type matrix metalloproteinase-1 (MT1 -MMP) compartment of proMMP-2 activation in MG-63 cells.
Osteoblast-derived matrix metalloproteinases (MMPs) are considered to play a crucial role in bone formation and initiation of bone resorption by degrading the bone matrix. MMP-2 is constitutively secreted in a latent zymogen by osteoblasts, and requires the process of activation mediated by membrane-type matrix metalloproteinase-1 (MT1-MMP)/tissue inhibitor of metalloproteinase (TIMP-2) complex in the cell surface. Bone is one target tissue for progestins. In the present study, we observed the effects of progesterone on proMMP-2 activation and MT1-MMP expression, and also TIMP-2 levels in osteoblastic MG-63 cells. Gelatin zymograms and ELISA showed that progesterone have no effects on proMMP-2 activation. Using Western immunoblot analysis, we unexpectedly found that treatment with increasing doses of progesterone in MG-63 cells caused a dose-dependent increase in expression of MT1-MMP protein, and after 48h treatment, progesterone at 10(-8)M increased MT1-MMP protein level. Confocal immunohistochemistry analysis also confirmed that progesterone induced MT1-MMP expression in MG-63 cells. The results of Northern blot analysis showed that progesterone at 10(-8)M increased MT1-MMP protein levels after 48 h treatment. We also found that TIMP-2 levels were undetectable in MG-63 cells. In conclusion, progesterone increases MT1-MMP protein and mRNA levels in MG-63 cells, but has no effects on proMMP-2 activation, which is partly attributable to the undetectable levels of tissue inhibitor of metalloproteinase-2 (TIMP-2). Our studies suggest that TIMP-2 is involved in proMMP-2 activation, and regulation of MT1-MMP by progesterone may contribute to its actions on bone formation.
Luo
X H
XH
Institute of Endocrinology & Metabolism, The Second Affiliated Hospital, Hunan Medical University, Changsha, PR China. xianghangluo@21cn.com
Liao
E Y
EY
D015152:Blotting, Northern; D015153:Blotting, Western; D001859:Bone Neoplasms / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D018093:Gelatinases / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000378:metabolism*; D018613:Microscopy, Confocal; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D012516:Osteosarcoma / Q000378:metabolism; D011374:Progesterone / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004792:Enzyme Precursors; D012333:RNA, Messenger; D019716:Tissue Inhibitor of Metalloproteinase-2; D011374:Progesterone; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D018093:Gelatinases; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; C069860:progelatinase