HAb18G/CD147 enhances the secretion of matrix metalloproteinases (MMP) via cGMP/NO-sensitive capacitative calcium entry (CCE) and accordingly attenuates adhesion ability of fibroblasts.
The present study examined the effect of hepatoma-associated antigen HAb18G (homologous to CD147) expression on the NO/cGMP-regulated Ca2+ mobilization to induce matrix metalloproteinases (MMP) production and attenuate adhesion ability of mouse fibroblast NIH/3T3 cells. HAb18G/CD147 cDNA was transfected into fibroblast 3T3 cells to obtain a cell line stably expressing HAb18G/CD147, t3T3, as demonstrated by immunofluorescence staining and flow cytometry assays. 8-Bromo-cGMP inhibited the thapsigargin-induced Ca2+ entry in 3T3 cells, whereas an inhibitor of protein kinase G, KT5823 (1 microM), led to an increase in Ca2+ entry. Expression of HAb18G/CD147 in t3T3 cells decreased the inhibitory response to cGMP. A similar effect on the Ca2+ entry was observed in 3T3 cells in response to an NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca2+ entry was also reduced in HAb18G/CD147-expressing t3T3 cells, indicating a role for HAb18G/CD 147 in NO/cGMP-regulated Ca2+ entry. Results of gelatin zymography assays showed that addition of extracellular Ca2+ induced MMP (MMP-2, MMP-9) release and activation in a dose-dependent manner, and expression of HAb18G/CD147 enhanced the secretion of MMP-2 and MMP-9 in 3T3 cells. 8-Bromo-cGMP and SNAP reduced the production of MMP in 3T3 cells but not in t3T3 with HAb18G/CD147 expression. RT-PCR experiments substantiated that the expression of MMP-2 and MMP-9 mRNA in HAb18G/CD 147-expressing t3T3 cell was significantly greater than that in 3T3 cells. Experiments investigating adhesion potentials demonstrated that HAb18G/CD147-expressing t3T3 cells pretreated with Ca2+ attached to Matrigel-coated culture plates significantly less efficiently than 3T3 cells. The proportion of attached cells could be increased by treatment with 8-bromo-cGMP and SNAP in 3T3 cells, but not in t3T3. These results suggest that HAb18G/CD147 attenuates adhesion potentials in fibroblasts by enhancing the secretion of MMP through NO/cGMP-sensitive capacitative Ca2+ entry.
Huang
Yong
Y
Cell Engineering Research Centre & Department of Cell Biology, The Fourth Military Medical University, Xi'an 710032, PR China.
Jiang
Jianli
J
Dou
Kefeng
K
Chen
Zhinan
Z
D016475:3T3 Cells; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism / Q000502:physiology*; D051926:Basigin; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism*; D005434:Flow Cytometry; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D041681:NIH 3T3 Cells; D009569:Nitric Oxide / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
D015703:Antigens, CD; C497887:Bsg protein, mouse; D018076:DNA, Complementary; D051926:Basigin; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D020782:Matrix Metalloproteinases; D006152:Cyclic GMP; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester