Expression of exogenous rat collagenase in vitro and in a rat model of liver fibrosis

Abstract

Aim: The present study was conducted to test the hypothesis that the introduction of the collagenase gene into tissue culture cells and into a rat model of liver fibrosis would result in the expression of enzymatically active product.

Methods: FLAG-tagged full-length rat collagenase cDNA was PCR amplified and cloned into a mammalian expression vector. NIH3T3 cells were then transiently transfected with this construct. Expression of exogenous collagenase mRNA was assessed by RT-PCR, and the exogenous collagenase detected by Western blotting using anti-FLAG monoclonal antibody. Enzymatic activity was detected by gelatin zymography. To determine the effects of exogenous collagenase production in vivo, the construct was bound to glycosyl-poly-L-lysine and then transduced into rats that had developed liver fibrosis as a result of CCl(4) plus ethanol treatment. The hepatic expression of the construct and its effect on the formation of liver fibrosis were demonstrated using RT-PCR and immunohistochemistry.

Results: It was found that exogenously expressed rat collagenase mRNA could be detected in NIH3T3 cells following transfection. Enzymatically active collagenase could also be detected in the culture medium. The recombinant plasmid was also expressed in rat liver after in vivo gene transfer. Expression of exogenous rat collagenase correlated with decreased deposition of collagen types I and III in the livers of rats with experimentally induced liver fibrosis.

Conclusion: The expression of active exogenous rat collagenase could be achieved in vitro and in vivo. It was suggested that in vivo expression of active exogenous collagenase may have therapeutic effects on the formation of liver fibrosis.

Figure 1

Map of plasmid pCMV-RC-F. The FLAG tagged full length rat collagenase cDNA was inserted into a mammalian expression pasmid vector Ptarge^TM which carries the human cytomegalovirus (CMV) immediate-early enhancer/promoter region.

Figure 2

RT-PCR assay of the mRNA expression of recombinant collagenase in NIH 3T3 cells. The PCR-amplified 652 bp fragment of recombinant collagenase cDNA derived from their respective mRNA was found in NIH 3T3 cells which were transfected with pCMV-RC-F (lane 1), while no signal was detected in cells which transfected with control plasmid pTarge^TM (lane 2). Lane M is the molecular weight marker (DNA/Hind III, BamH I).

Figure 3

Westernblot analysis of the FLAG-tagged rat collagenase expression using mouse anti-FLAG M2 McAb. The immunoreactive band with anti-FLAG M2 McAb at about M_r 55000 was observed in the culture supernatant of cells transfected with PCMV-RC-F (lane 1) and the supernatant of 1:2 dilution (lane 2). No immunoreactive was found in the supernatant of cells transfected with pTarge^TM (lane 3, 4) or in culture medium alone (lane 5, 6).

Figure 4

Gelatin zymography analysis for the activity of collagenase. The enhanced gelatin degradation activity was found in the culture supernatant of pcmv-RC-F transfected cells (lane 1-7 represent the zymograph of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 diluted solutions of the supernatant) when compared with pTarge^TM transfected cells (lane 10, 11 represent the zymograph of 1:2 and 1:1 diluted solutions of the surpernatant). No activity was found at about M_r 55000 in the culture medium (lane 8, 9).

Figure 5

RT-PCR assay for the expression of recombinant interstitial collagenase in rat livers. The 652 bp PCR amplified fragment of recombinant collagenase cDNA which was derived from their respective mRNA could be detected in liver samples from pCMV-RIC-F plasmid transduced rat groups C1 (lane 1, 2) and C2 (lane 3, 4) but not in the normal control group A (lane 5, 6) and disease control group B (lane 7). 335 bp fragment was PCR amplified β-actin cDNA. Lane M was 100 bp-1200 bp DNA marker ladders.

Figure 6

RT-PCR assay for the endogenous expression of collagenase in rat livers. The 753 bp PCR-amplified fragment of endogenous collagenase cDNA which was derived from their respective mRNA could be detected in all liver samples from the normal control groups A (lane 1) and disease control group B (lane 4), as well as pCMV-RIC-F plasmid transduced rat groups C1 (lane 3) and C2 (lane 4).335 bp fragment was PCR amplified β-actin cDNA. Lane M was 100 bp-1200 bp DNA marker ladders.

Figure 7

Immunohistochemical analysis of the expression of recombinant plasmid with anti-FLAG antibody in pCMV-RIC-F plasmid transduced group C1 (× 400). Nearly about 30 percent of total cells presented positive signals. The distributions of positive signals were found in both the hepatocytes and the perisinusoidal cells.

Figure 8

Immunohistochemical analysis of collagen types I (A, B) and III (C, D) in livers (× 100). The accumulations of collagen in the disease control group (A, C) were more severe than that in the pCMV-RIC-F plasmid transduced C1 group (B, D).