Truncated active matrix metalloproteinase-8 gene expression in HepG2 cells is active against native type I collagen.
OBJECTIVE
Excess type I collagen accumulation is a major feature of fibrotic diseases such as liver cirrhosis. Reversion of this disease has not been fully accomplished. Physiologically, collagen is degraded by interstitial collagenases, neutrophil collagenase (MMP-8) being the most active against type I collagen. Introduction of MMP-8 gene into liver cells could be an advantageous tool to potentiate fibrosis degradation.


METHODS
We cloned latent and active MMP-8 genes in prokaryotic and eukaryotic expression vectors and an adenoviral vector. Transfection of MMP-8 in HepG2 was effectuated by CaPO4, polylysine-lactose (P-L) and adenoviral transduction, and cells and culture supernatant were harvested 72 h after transfection for analysis of MMP-8 expression by reverse transcription-polymerase chain reaction and collagenolytic activity.


CONCLUSIONS
We show that a truncated neutrophil collagenase (tMMP-8) lacking a portion of the carboxy terminus and with an intact aminoterminus (latent; l-tMMP-8) or a truncated amino terminus (active; a-tMMP-8) has enzymatic activity against native type I collagen, and the activity was inhibited by EDTA, 1,10-phenanthroline and TIMP-1. Both MMP-8 mRNA (latent and active) were detected by polymerase chain reaction in cells transfected with CaPO4, P-L and adenoviral transduction; however, relative expression of MMP-8 was enhanced when the plasmid was delivered as a P-L complex and increased by adenoviral infection. Finally, a-tMMP-8 cDNA was cloned in a vector under transcriptional control of a regulated promoter (PEPCK-a-tMMP-8). HepG2 cells transfected with the PEPCK-a-tMMP-8 plasmid DNA up-regulated expression of a-tMMP-8 after incubation of the cells with butyryl-cAMP and glucagon, while stimulation with insulin slightly down-regulated its expression. Recombinant MMP-8 expressed by HepG2-transduced cells can efficiently degrade soluble type I collagen, which is potentially useful for gene transfer therapies.
Siller-López
F
F
Instituto de Biología Molecular en Medicina, CUCS, U. de G., Guadalajara, Jal., México.
García-Bañuelos
J
J
Hasty
K A
KA
Segura
J
J
Ramos-Márquez
M
M
Qoronfleh
M W
MW
Aguilar-Cordova
E
E
Armendáriz-Borunda
J
J
D003094:Collagen / Q000378:metabolism*; D015316:Genetic Therapy; D006801:Humans; D008099:Liver / Q000378:metabolism*; D020784:Matrix Metalloproteinase 8 / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D003094:Collagen; D020784:Matrix Metalloproteinase 8