Truncated active matrix metalloproteinase-8 gene expression in HepG2 cellsis active against native type I collagen

Citation
F. Siller-lopez et al., Truncated active matrix metalloproteinase-8 gene expression in HepG2 cellsis active against native type I collagen, J HEPATOL, 33(5), 2000, pp. 758-763
Citations number
30
Categorie Soggetti
Gastroenerology and Hepatology","da verificare
Journal title
JOURNAL OF HEPATOLOGY
ISSN journal
01688278 → ACNP
Volume
33
Issue
5
Year of publication
2000
Pages
758 - 763
Database
ISI
SICI code
0168-8278(200011)33:5<758:TAMMGE>2.0.ZU;2-F
Abstract
Background/Aims: Excess type I collagen accumulation is a major feature of fibrotic diseases such as liver cirrhosis, Reversion of this disease has no t been fully accomplished, Physiologically, collagen is degraded by interst itial collagenases, neutrophil collagenase (MMP-8) being the most active ag ainst 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 eukaryo tic expression vectors and an adenoviral vector, Transfection of MMP-8 in H epG2 was effectuated by CaPO4, polylysine-lactose (P-L) and adenoviral tran sduction, and cells and culture supernatant were harvested 72 h after trans fection for analysis of MMP-8 expression by reverse transcription-polymeras e chain reaction and collagenolytic activity. Results and Conclusions: We show that a truncated neutrophil collagenase (t MMP-8) lacking a portion of the carboxy terminus and with an intact amino-t erminus (latent; l-tMMP-8) or a truncated amino terminus (active; a-tMMP-8) has enzymatic activity against native type I collagen, and the activity wa s inhibited by EDTA, 1,10-phenanthroline and TIMP-1, Both MMP-8 mRNA (laten t and active) were detected by polymerase chain reaction in cells transfect ed with CaPO4, P-L and adenoviral transduction; however, relative expressio n 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 transfect ed with the PEPCK-a-tMMP-8 plasmid DNA up-regulat ed expression of a-tMMP-8 after incubation of the cells with butyryl-cAMP a nd glucagon, while stimulation with insulin slightly down-regulated its exp ression, Recombinant MMP-8 expressed by HepG2-transduced cells can efficien tly degrade soluble type I collagen, which is potentially useful for gene t ransfer therapies.