Thrombospondin 1 acts as a strong promoter of transforming growth factor beta effects via two distinct mechanisms in hepatic stellate cells

Abstract

Background and aims: Thrombospondin 1 (TSP-1) is an important activator of latent transforming growth factor beta (TGF-beta) but little is known of the expression patterns and functions of TSP-1 in liver cells. We therefore analysed if and how TSP-1 acts on TGF-beta during fibrogenesis.

Methods and results: Using reverse transcription-polymerase chain reaction, we demonstrated that hepatocytes from normal liver expressed no TSP-1 mRNA whereas Kupffer cells and sinusoidal endothelial cells did. TSP-1 mRNA and protein were detected in quiescent and activated cultured hepatic stellate cells (HSC) and TSP-1 expression was highly inducible by platelet derived growth factor BB (PDGF-BB) and, to a lesser extent, by tumour necrosis factor alpha in activated HSC. Furthermore, addition of PDGF-BB directly led to enhanced TGF-beta mRNA expression and a TSP-1 dependent increase in TGF-beta/Smad signalling. Using either a peptide specifically blocking the interaction of TSP-1 with latent TGF-beta or antibodies against TSP-1 not only abrogated activation of latent TGF-beta but also reduced the effects of the active dimer itself.

Conclusions: Our data suggest that TSP-1 expression is important for TGF-beta effects and that it is regulated by the profibrogenic mediator PDGF-BB in HSC. Furthermore, the presence of TSP-1 seems to be a prerequisite for effective signal transduction by active TGF-beta not only in rat HSC but also in other cell types such as human dermal fibroblasts.

Figure 1

Expression of thrombospondin 1 (TSP-1) mRNA in liver cells. (A) Equal amounts of total RNA from hepatic stellate cells (HSC), myofibroblasts (MFB), hepatocytes (HC), sinusoidal endothelial cells (SEC), and Kupffer cells (KC) either freshly isolated from rat livers (0d) or after the indicated periods of culture, were reverse transcribed into cDNA and expression of transforming growth factor β1 (TGF-β1) and TSP-1 was investigated qualitatively by polymerase chain reaction using specific primers, as described in materials and methods. As internal control, amplification of rS6 is shown. (B) TSP-1 mRNA expression during HSC transdifferentiation. Total RNA was isolated from HSC (primary cultures) or MFB (secondary cultures of HSC) at the indicated time points in culture, and TSP-1 mRNA expression was analysed by northern blotting. The graph shows the results of densitometric analyses of five independent cell preparations, and TSP-1 mRNA values were normalised to 18S and 28S rRNA (mean (SD) from 3–4 individual values per time group). The insert shows one of these blots as a representative example.

Figure 2

Analysis of thrombospondin 1 (TSP-1) expression in hepatic stellate cells (HSC) and myofibroblasts (MFB) after cytokine stimulation. (A) Total RNA was prepared after 24 hours of stimulation with platelet derived growth factor BB (PDGF-BB 20 ng/ml), transforming growth factor β1 (TGF-β1 5 ng/ml), interleukin 6 (IL-6 5 ng/ml), or tumour necrosis factor α (TNF-α 10 ng/ml), and by northern blot analysis expression of TSP-1 and TGF-β1 in activated HSC or MFB respectively, was compared with untreated cells. (B) Dose dependency of PDGF-BB or TGF-β1. Total RNA from MFB (3d) was isolated and expression patterns of TSP-1 and TGF-β1 mRNA were investigated by northern blot analysis. Cells were treated with the indicated concentrations of cytokines for four hours. (C) Decreased expression of PDGF-B mRNA during HSC transdifferentiation. Total RNA (1 μg) from one day (1d) or eight day old (8d) HSC and from two day old (2d) MFB was reverse transcribed into cDNA and endogenous expression of PDGF-B mRNA was investigated qualitatively by polymerase chain reaction using specific primers, as described in materials and methods. (D) High TSP-1 expression at early stages of transdifferentiation (day 1) depend on PDGF receptor tyrosin kinase activity. HSC were isolated from healthy rat livers and cultured for one day with or without supplementation of the medium with 250 mM PDGF receptor (PDGFR) tyrosin kinase inhibitor I. Control cultures received only equal amounts of DMSO. Total RNA was isolated and TSP-1 and rS6 mRNA expressions were investigated, as described for fig 1A ▶.

Figure 3

Analysis of thrombospondin 1 (TSP-1) expression in hepatic stellate cells (HSC) after cytokine stimulation. (A) Time course analysis of transforming growth factor β1 (TGF-β1) and TSP-1 mRNA expressions after stimulation with platelet derived growth factor BB (PDGF-BB), TGF-β1, or tumour necrosis factor α (TNF-α). Total RNA from HSC (2d or 3d) was isolated after the indicated periods of stimulation and expression patterns of TSP-1 and TGF-β1 mRNA were investigated by northern blot analysis. (B) PDGF-BB induced TSP-1 and TGF-β1 mRNA expressions more potently than TNF-α. HSC (3d) were stimulated with either PDGF-BB or TNF-α for three hours and total RNA was isolated and analysed by northern blot analysis. Intensities of the TSP-1 and TGF-β1 signals were quantified densitometrically and normalised to the corresponding 18 and 28 S rRNA. The number of experiments per condition (n) is given within each bar. The graph represents mean values (SD) as fold induction compared with the corresponding untreated controls. Concentration of stimulants as in fig 2A ▶.

Figure 4

Determination of thrombospondin 1 (TSP-1) protein levels after platelet derived growth factor BB (PDGF-BB) stimulation. Cell lysates and conditioned medium from transdifferentiating cells with (+) or without (−) stimulation with PDGF-BB (20 ng/ml) for 24 hours or three hours, as indicated, were analysed by western blot under reducing conditions using specific mouse monoclonal antibodies against TSP-1. (A) Total cell lysate (15 μg). (B) Cytosol 15 μl (C) or cell membrane (M) sample. (C) Conditioned medium (30 μl) (containing 0.5% fetal calf serum (FCS)) per lane. For (B) and (C), three day old HSC were used. β-Actin expression is shown to demonstrate equal loading. To accentuate differences in band intensities, the blot under (B) is shown with short and long exposure times.

Figure 5

Sequence alignment of thrombospondin 1 (TSP-1) from rat, human, and mouse: 191 bp of rat TSP-1 cDNA was sequenced using the indicated primers and the resulting fragment was compared with known sequences of human (gi4507484) and mouse (gi202196) TSP-1. The region encoding the transforming growth factor β activating motif KRFK is identical in all three species (box). Single base pairs that differ in the rat and/or human sequence compared with the mouse sequence are underlined.

Figure 6

CAGA₉-MLP-Luc reporter gene studies. (A) Platelet derived growth factor BB (PDGF-BB) treatment increased transforming growth factor β (TGF-β) signalling. One day old hepatic stellate cells (HSC) were infected with adenoviruses encoding a reporter gene (luciferase) whose expression is controlled by multiple copies of a TGF-β response motif (“CAGA₉-MLP-Luc”). Two days later, cells were stimulated with PDGF-BB (20 ng/ml) for the indicated time periods, followed by cell lysis and measurement of luciferase activity. Values are mean (SD) (normalised to the control) of two independent experiments, each performed at least in triplicate, per time point. CPS, counts per second. (B) PDGF-BB and TGF-β effects. As illustrated in the scheme above the graph, HSC were infected with adenoviruses and two days after infection, cells were stimulated with the indicated reagents for three hours. TGF-β was used at 0.1 ng/ml as a positive control. Where recombinant latency associated peptide (LAP) (100 ng/ml), LSKL or KRFK peptides (2000 nM), or antibodies against thrombospondin 1 (TSP-1) (Ab2, Ab3, Ab8; 25 μg/ml each) were added together with PDGF-BB (20 ng/ml) or TGF-β (0.1 ng/ml), cells were preincubated with these substances for one hour. As an internal control for experiments where anti-TSP-1 antibodies were used, cells were also incubated with a mixture of normal IgG antibodies (mouse plus rabbit IgG, 10 μg/ml each). Values are mean (SD) (normalised to the corresponding control) of 3–6 ( = n, as indicated within each bar) independent experiments, each performed at least in triplicate per condition ( = at least nine values per condition). *p<0.05 compared with PDGF-BB (P) alone (at 20 ng/ml) or with untreated control (C). NS (P), not significantly different from PDGF-BB alone (at 20 ng/ml). (C) Effects of tumour necrosis factor α (TNF-α) or purified TSP-1. CAGA-luciferase activities after stimulation (three hours) with increasing amounts of TNF-α or purified human TSP-1 were measured and are presented as described in (B).

Figure 7

CAGA₉-MLP-Luc reporter gene studies. (A) Effects of transforming growth factor β (TGF-β) alone or in combination with platelet derived growth factor BB (PDGF-BB). CAGA-luciferase activities after stimulation (three hours) with either TGF-β alone (0.1 ng/ml) or in combination with PDGF-BB (5 ng/ml) were measured in hepatic stellate cells, and are presented as described in fig 6B ▶. The number of experiments performed per condition (n) is given below the columns. Peptides (2000 nM) or antibodies (25 μg/ml) were added one hour prior to the indicated cytokine(s) and the values in parentheses indicate the amounts used (in ng/ml). Pretreatment of cells with TGF-β neutralising antibodies (20 μg/ml) strongly inhibited reporter gene activation. (B) CAGA-luciferase activities after stimulation (three hours) with TGF-β (0.1 or 0.5 ng/ml) were measured in human dermal fibroblasts, and are presented as described in fig 6B ▶. (C) Western blot of cell lysates (10 μg per lane) from human dermal fibroblasts of three different healthy donors, indicating that these cells express relatively high basal levels of thrombospondin 1 (TSP-1) compared with rat myofibroblasts (MFB). β-actin expression is shown as a loading control. For the CAGA-luciferase assays shown in (B), fibroblasts from donor No 1 were used and the graph shows the average of four independent experiments, each performed in triplicates (SD).