Up-regulation of uPARAP/Endo180 during culture activation of rat hepatic stellate cells and its presence in hepatic stellate cell lines from different species

Abstract

Background: The urokinase plasminogen activator receptor associated protein (uPARAP)/Endo180 is a novel endocytic receptor that mediates collagen uptake and is implicated to play a role in physiological and pathological tissue-remodelling processes by mediating intracellular collagen degradation.

Result: This study investigates the expression of uPARAP/Endo180 protein and messenger RNA in primary rat hepatic stellate cell (HSC) cultures. The results show that uPARAP/Endo180 protein is not expressed in freshly isolated HSCs or during the first few days of culture while the cells still display quiescent features. In contrast, uPARAP/Endo180 protein is expressed early during HSC activation when cells are transdifferentiated into myofibroblast-like cells. Very low levels of uPARAP/Endo180 mRNA are detectable during the first days of culture but uPARAP/Endo180 mRNA is strongly up-regulated with increasing time in culture. Moreover, endocytic uptake of denatured collagen increases as transdifferentiation proceeds over time and correlates with increased expression of uPARAP/Endo180. Finally, analysis of uPARAP/Endo180 expression in four hepatic stellate cell lines from three different species showed that all these cell lines express uPARAP/Endo180 and are able to take up denatured collagen efficiently.

Conclusion: These results demonstrate that uPARAP/Endo180 expression by rat HSCs is strongly up-regulated during culture activation and identify this receptor as a feature common to culture-activated HSCs.

Figure 1

Light microscopic analysis of HSCs stained with Oil Red-O at different stages of transdifferentiation in primary culture. (A) Representative micrographs of HSCs cultured for 40 hours (A), 7 days (B) or sub-cultured for 5 days (C) showing changes in the morphology (from stellate shape to myofibroblast-like cells) and contents of lipid droplets. Note that HSCs cultured for 40 h contain variable amounts of lipid droplets. In sub-cultured (first passage) cells, there were no detectable lipid droplets as judged by Oil Red-O staining (data not shown). Original magnification: ×100. The micrographs shown are representative of cells obtained from at least two different preparations.

Figure 2

Time course analysis of the expression of Endo180 and marker proteins in freshly isolated and cultured rat HSCs. (A and B) Western blot analysis of expression of Endo180, desmin, GFAP, and α-SMA during culture activation of rat HSCs. At the time points shown cells were collected and cell lysates were prepared from frozen cell pellets (A) or cells were lysed directly in lysis buffer (B) and the same amount of proteins was analyzed by SDS-PAGE and Western blot using anti-Endo180 antibody. The blots were re-probed with antibodies to β-actin, used as a loading control, and marker proteins as indicated. Total non-parenchymal cells (NPC) were prepared from rat liver by collagenase perfusion as previously described [59]. (C) Densitometric analysis of Western blots showing the time-dependent increase in Endo180 protein expression in cultured rat HSCs. The ratio Endo180/β-actin bands was quantified for each day and expressed as means ± SD of seven different samples (days 1–5, and 7) or four different samples (days 0, 6 and sub-cultured). Note that there is less beta-actin in the three first lanes in Fig. 2A. However, densitometric analysis of bands showed that the amount of beta-actin at day 3 is ~ twice as much as day 7, yet no expression of Endo180 is observed at day 3. (D) RT-PCR analysis of Endo180 mRNA expression during culture activation of rat HSCs. Total RNA was extracted from cells and Endo180 and β-actin, used as an internal control, mRNAs were analyzed by RT-PCR. One representative experiment (out of three independent experiments) is shown. SC, sub-cultured (first passage).

Figure 3

Uptake and degradation of denatured collagen by rat HSCs as a function of cell culture time and the effect of inhibitors of lysosomal function on these processes. (A) At each day, cells cultured in 6-well plates were washed, pre-incubation for 45 min at 37°C, and then incubated for 2 h at 37°C with 10 μg/ml ¹²⁵I-TC-collagen. After washing, cells were lysed to determine total cell-associated radioactivity (= the sum of acid-soluble and acid-precipitable radioactivity), which was taken as a measure of total uptake of ¹²⁵I-TC-collagen. Acid-soluble radioactivity was taken as a measure of degradation of ¹²⁵I-TC-collagen. (B) The calculated percentage of internalized ¹²⁵I-TC-collagen degraded by cells. Results are shown as means ± SD for triplicate wells and represent one of three similar experiments. Data are normalized for cell number. Cells were counted on the indicated days as described in Materials and methods. (C) Sub-cultured (first passage) HSCs were washed, pre-incubation for 45 min at 37°C, and then incubated at 37°C with ¹²⁵I-TC-collagen for either 30 min in the absence or presence of excess unlabeled denatured collagen (100-fold) or for 2 h in the absence or presence of E64d (100 μM) or concanamycin A (Con A, 1.5 μM). Uptake and degradation were determined as described above. Data represent either average of duplicate determinations (unlabeled collagen) or means ± SD for triplicate determinations (E64d and Con A) and are representative for at least two such experiments performed.

Figure 4

Expression of Endo180 protein and mRNA by four established HSC lines from three different species. Detection of Endo180 and β-actin proteins (A) and mRNAs (B) was performed as described in Figure 2. Results are representative of three (A) or two (B) experiments. (C) Uptake and degradation of denatured collagen by different HSC lines. Cells were grown in 6-well plates, washed, pre-incubation for 45 min at 37°C, and then incubated for 2 h at 37°C with 10 μg/ml ¹²⁵I-TC-collagen. Experimental procedure is as in the legend to Figure 3. (D) The calculated percentage of internalized ¹²⁵I-TC-collagen degraded by different HSC lines. Results are shown as means ± SD for triplicate wells and are representative for at least two similar experiments. Cells used from a new passage was treated as a separate experiment. Data are normalized for cell number.