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Updated: Jul 10, 2026
Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels
Published on: January 22, 2019
Truncated active matrix metalloproteinase-8 gene expression in HepG2 cells is active against native type I collagen
F Siller-López1, J García-Bañuelos, K A Hasty
1Instituto de Biología Molecular en Medicina, CUCS, U. de G., Guadalajara, Jal., México.
Background/Aims:
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.
Results And 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.
