Uptake of denatured collagen into hepatic stellate cells: evidence for the involvement of urokinase plasminogen activator receptor-associated protein/Endo180

Abstract

Tissue remodelling is dependent on the integration of signals that control turnover of ECM (extracellular matrix). Breakdown and endocytosis of collagen, a major component of the ECM, is central to this process. Whereas controlled secretion of matrix-degrading enzymes (such as matrix metalloproteinases) has long been known to mediate ECM breakdown, it is becoming clear that uPARAP/Endo180 (where uPARAP stands for urokinase plasminogen activator receptor-associated protein) serves as a receptor that mediates endocytosis of collagen by several types of cells. In the liver, the stellate cells play a major role in turnover of ECM including collagens. These cells synthesize various collagens and also produce matrix metalloproteinases. In the present study, we investigated the capacity of rat hepatic stellate cells to endocytose and degrade 125I-labelled heat-denatured collagen I. It was found that the collagen is efficiently taken up and degraded by these cells. Degradation was inhibited by inhibitors of lysosomal proteases (leupeptin and E-64d) and the vacuolar proton pump (concanamycin A), indicating that it takes place in lysosomes. Furthermore, endocytosed FITC-labelled collagen was shown to reach late endocytic compartments in which it colocalized with LysoTracker (a marker of late endocytic compartments). Competition experiments showed that uPA and unlabelled collagen are capable of inhibiting binding and uptake of [125I]collagen in a dose-dependent manner. Moreover, Western-blot analysis of cell lysate (using a polyclonal rabbit human-Endo180 antiserum) revealed a single band at 180 kDa. In addition, the antiserum was capable of reducing [125I]collagen binding to the cell surface. Finally, using two primers designed from the human uPARAP/Endo180 mRNA sequence, the expression of uPARAP/Endo180 mRNA was detected by reverse transcriptase-PCR. These results together suggest that uPARAP/Endo180 mediates endocytosis of collagen in rat liver stellate cells.

Figure 1. Uptake and binding of [¹²⁵I]collagen to hepatic stellate cells

(A) Time course of receptor-mediated uptake and degradation of collagen: cells (~10⁶ cells/well) were incubated with [¹²⁵I]collagen (0.2 μg/ml) at 37 °C for the indicated times, washed and then lysed. Cell lysates and medium were treated with TCA to determine acid-soluble and acid-precipitable radioactivities as described in the Experimental section. Values are expressed as percentage of total initial acid precipitable radioactivity in the medium and are means±S.D. results for triplicate samples. Equivalent results were obtained in at least six separate experiments. (B) Concentration-dependent binding and uptake of collagen in stellate cells: cells were preincubated at 4 °C for 30 min with the indicated concentrations of unlabelled collagen. After preincubation, [¹²⁵I]collagen (20 ng/ml) was added and the cells were incubated for 120 min at 4 °C or 30 min at 37 °C. The cells were then washed three times and the cell-associated radioactivity was determined. Values are the means±S.D. results for triplicate samples. Equivalent results were obtained in at least three separate experiments.

Figure 2. Fluorescence microscopy showing co-staining of the internalized FITC-collagen and a marker of late endocytic compartments

Cells were pulse-labelled at 37 °C for 15 min with FITC-collagen (green) in the presence or absence of LysoTracker (red), then washed and either fixed (images on the left) or chased for 90 min, after which the incubation continued for an additional 30 min in the presence of LysoTracker (images on the right). Co-localization of FITC-collagen with LysoTracker (yellow) is shown in the merged images (lowest panels). Results represent two or three experiments.

Figure 3. Effect of leupeptin, E-64d and concanamycin on uptake and degradation of [¹²⁵I]collagen

Cells were preincubated with either leupeptin (2 mM), E-64d (2 μM) (upper panel) or increasing concentrations of concanamycin (lower panel) for 15 min at 37 °C. After preincubation, radio-labelled collagen (40 ng/ml) was added, followed by 90 min incubation at 37 °C and three washes. Cell lysates and medium were treated with TCA to determine acid-soluble and acid-precipitable radioactivities as described in the Experimental section. Values are expressed as percentage of controls and are the means±S.D. results for triplicate samples. Equivalent results were obtained in at least three separate experiments.

Figure 4. Dose-dependent inhibition of binding and uptake of [¹²⁵I]collagen to stellate cells by uPA

Cells were preincubated with increasing concentrations of uPA for 30 min at 4 °C. After preincubation, radiolabelled collagen (20 ng/ml) was added, followed by 120 min incubation at 4 °C and three washes. For uptake, uPA was added just before the addition of [¹²⁵I]collagen and cells were incubated at 37 °C for 30 min. Results are means±S.D. of a representative experiment performed in triplicate.

Figure 5. Endo180 is expressed in rat hepatic stellate cells

The whole lysates (40 μg) of hepatic stellate cells (lane 1), U937 cells (lane 3) and HepG2 cells (lane 4) were analysed by SDS/PAGE on a 6% gel and then transferred on to membrane. The blot was incubated first with a polyclonal antiserum directed against Endo180 and subsequently with mouse anti-rabbit IgG conjugated to horseradish peroxidase followed by detection with ECL®. Lane 2 corresponds to the cell membrane fraction (20 μg) prepared from stellate cells. The membrane was then stripped, blocked and incubated with anti-β-actin. Relative molecular mass standards are shown on the left (masses in kDa). The results shown were reproduced in at least three independent experiments.

Figure 6. Expression analysis of uPARAP/Endo180 mRNA by RT–PCR

Four different types of rat liver cells [liver endothelial cells (rLEC), Kupffer cells (rKC), parenchymal cells (rPC) and stellate cells (rStel)] and J774 mouse macrophages were tested for mRNA expression of Endo180 (lanes 2–6 respectively) with β-actin mRNA as an internal control (lanes 7–11). Three experiments gave equivalent results. Sizes are indicated in bp.

Figure 7. Uptake and degradation of [¹²⁵I]TC-OVA by hepatic stellate cells

Cells (~10⁶ cells/well) were incubated at 37 °C with 2 nM [¹²⁵I]TC-OVA in the presence and absence of a 50-fold excess of unlabelled OVA or 2.2 mM EGTA. At timed intervals the cells were washed and cell-associated radioactivity (acid-soluble and acid-precipitable) was measured as described in the Experimental section. Inset: the effect of unlabelled OVA and EGTA on the uptake.