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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 12, 2006 as doi:10.1096/fj.05-4870fje. |
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* Immunology and Allergy, Department of Internal Medicine,
Department of Pathology and Immunology,
Rheumatology, Department of Internal Medicine, Geneva University Hospital, Geneva School of Medicine, Geneva, Switzerland
1Correspondence: Immunology and Allergy, Geneva University Hospital, Geneva 14-1211, Switzerland. E-mail: chizzolini{at}medecine.unige.ch
SPECIFIC AIMS
Fibrosis ensues when the balance between extracellular matrix (ECM) deposition and degradation is disrupted in favor of deposition. The aim of this study was to investigate the effects of proteasome inhibition (PI) on the biosynthetic machinery of human dermal fibroblasts in the context of fibrosis. We assessed the effect of PI in resting fibroblasts and fibroblasts activated by TGF-ß, a master cytokine leading to fibrosis. In some experiments we used fibroblasts generated from individuals affected by systemic sclerosis (SSc), a human disease model of fibrotic conditions. Our read-out was the production of type I collagen, the main constituent of dermal ECM, and of metalloproteinase-1 (MMP-1), which has the distinctive capacity to cleave the triple helix of type I collagen. We also assessed levels of the tissue inhibitor of metalloproteinase-1 (TIMP-1).
PRINCIPAL FINDINGS
1. Proteasome inhibitors reduce type I collagen and TIMP-1, while increasing MMP-1 protein production in dermal fibroblasts
Type I collagen production was strongly and dose-dependently reduced in normal dermal fibroblasts incubated with PSI. In the micromolar range, PSI consistently inhibited the constitutive production of type I collagen by >80%, while at nanomolar concentration it still had some inhibitory activity. In the same cultures, PSI induced a dose-dependent increase of MMP-1 and decrease of TIMP-1 levels. Three additional PI (MG-132, synthetic lactacystin, and bortezomib) were able to reduce type I collagen and TIMP-1 and increase MMP-1 production. The relative potency of these compounds differed: 0.01 µM bortezomib reduced collagen production by 50%; PSI and MG-132 achieved similar levels of reduction at a concentration higher by one order of magnitude (0.1–0.2 µM); lactacystin at the highest concentration tested (20 µM) did not reach 50% of inhibition.
2. Proteasome inhibitors reduce COL1A1 and TIMP-1, and increase steady-state mRNA levels of MMP-1 in dermal fibroblasts
The changes observed at the protein level were matched by changes at the mRNA level, as assessed by quantitative real time RT-PCR and Northern blot. In fibroblasts cultured for 24 h PSI, MG-132 and lactacystin reduced COL1A1 steady-state mRNA levels by 87%, 96%, and 20% of control cultures, respectively. In the same experimental conditions, MMP-1 mRNA levels increased by 370%, 820%, and 160%, while TIMP-1 mRNA levels were 80%, 50%, and 60% of controls. These effects were dose and time dependent.
3. Proteasome blockade results in decreased COL1A1 gene transcription
To assess whether the decreased levels of COL1A1 mRNA were due to changes in mRNA transcription rate or mRNA stability, fibroblasts were treated with the transcription inhibitor DRB in the presence or absence of PSI (4 µM), and the decay of COL1A1 mRNA was monitored as a function of time by quantitative RT-PCR. The half-life of COL1A1 mRNA in the presence of DRB was 
11 h, and it was the same when DRB and PSI were used together. These results strongly indicate that the effect of PI on type I collagen synthesis was essentially due to decreased synthesis rather than stability of mRNA.
4. Effect of PSI in normal and SSc skin fibroblasts
In SSc fibroblasts, the transcriptional machinery is set in a profibrotic mode, a characteristic maintained in culture. Doses of PSI needed to reduce 50% of type I collagen protein levels did not significantly differ between SSc and control fibroblasts, but those required to enhance MMP-1 production by 50% tended to be higher in SSc than in control fibroblasts (P=0.066). Significantly higher doses of PSI were required to inhibit type I collagen than to enhance the production of MMP-1 both in control and SSc fibroblasts (P=0.01). The IC50 was 0.36 ± 0.22 µM for type I collagen, 0.10 ± 0.05 µM for MMP-1, and 1.14 ± 1.00 µM for TIMP-1.
5. Proteasome blockade inhibits the profibrotic activity of TGF-ß
TGF-ß exerts in vitro and in vivo potent profibrotic effects. In our experiments, type I collagen protein production doubled in the presence of TGF-ß compared with medium. In TGF-ß-stimulated fibroblasts, PSI reduced 5-fold type I collagen levels, well below the levels of spontaneous production, and doubled the amount of MMP-1 (Fig. 1
A). TGF-ß increased 3-fold the production of TIMP-1, the effect of which was abrogated by PSI (Fig. 1A
). Similar results were obtained when steady-state mRNA levels were assessed by quantitative RT-PCR (Fig. 1B
). In addition, PI strongly reduced total soluble collagens as detected by a Sirius red-based assay in supernatants of control and stimulated (TGF-ß) cultures (Fig. 1C
) and increased by 10-fold the collagenolyic activity exerted on type I collagen in either the absence or presence of TGF-ß. (Fig. 1D
). Thus, our data indicate that proteasome blockade abolishes the profibrotic activity of TGF-ß.
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6. Proteasome blockade results in c-Jun phosphorylation and c-Jun nuclear accumulation
To unravel the mechanisms involved in the phenomena observed, we tested whether PI could affect the levels of various MAP kinases and of NF-
B family members. Whereas TNF-
used as positive control induced prompt phosphorylation of c-Jun, ERK, p38, and IB- (accompanied by IB degradation), MG-132 specifically induced in a time-dependent manner c-Jun phosphorylation and c-Jun accumulation, which peaked at 12 h. Consistent with these findings, fluorescence intensity of nuclear c-Jun was considerably increased in a time-dependent manner upon PI treatment but unstimulated NF-B levels were not affected. In control cultures, PI suppressed NF-B nuclear translocation induced by TNF-. To ascertain whether the PI effect on c-Jun accumulation was due to c-Jun phosphorylation, we used SP-600125, a specific inhibitor of c-Jun N-terminal kinase (JNK). SP-600125 abrogated c-Jun phosphorylation and accumulation. Thus, in dermal fibroblasts, proteasome inhibition induces c-Jun accumulation in the nucleus, an event favored by enhanced JNK activity.
7. Inhibition of JNK abrogates the enhanced MMP-1 production induced by PI
The promoter region of the MMP-1 gene contains an AP-1 binding site. Since c-Jun is a member of the proteins forming AP-1, we tested whether inhibition of JNK would reverse the enhanced MMP-1 production induced by PI. In fibroblasts cultured in the presence of bortezomib, the addition of SP-600125 reduced the production of MMP-1 in a dose-dependent manner with no substantial modification in type I collagen or TIMP-1 levels. In parallel control experiments, we tested whether the inhibition of NF-B nuclear translocation by SN50 cell permeable inhibitor peptide would reverse the enhanced MMP-1 production induced by PI. No such effect was found; on the contrary, a 2-fold increase was observed at high inhibitor concentrations. These data stress the specificity of the effect of PI on c-Jun.
8. Effect of PI on fibroblast viability
Compared with fibroblasts cultured in medium alone, cells exposed to increasing doses of PSI did not show impaired growth except at very high concentrations (40 µM), and only after 72 h. The viability of fibroblasts cultured for 48 h in the presence of bortezomib (0.1 µM) and JNK inhibitor SP-600125 (20 µM) was 85.1 ± 5.9% that of fibroblasts cultured in medium alone. Thus, PI did not substantially affect dermal fibroblast viability.
CONCLUSIONS AND SIGNIFICANCE
An increase in collagen synthesis unmet by adequate degradation facilitates ECM deposition leading to fibrosis and pathology (Fig. 2
). The down-regulation of type I collagen and TIMP-1 and the up-regulation of MMP-1 protein and collagenolytic activity induced by PI (described here in dermal fibroblasts) has the potential to reverse excessive ECM deposition and fibrosis.
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Metalloproteinases are induced in many cell types by proinflammatory cytokines, an effect blocked by PI. In contrast, we report that in resting and TGF-ß-stimulated fibroblasts, PI leads to increased levels of MMP-1. Similarly, PI was reported to increase MMP-3 mRNA levels in rat mesangial cells. Our data point to a role for c-Jun in PI-induced MMP-1 production. Indeed, PI induced c-Jun phosphorylation and c-Jun accumulation whereas pharmacological inhibition of JNK reversed this effect and abrogated the enhanced production of MMP-1. Such effects were specific insofar as 1) we did not detect significant modifications induced by PI in the phosphorylation and protein levels of other MAP kinases, of IB- and NF-B; 2) inhibition of nuclear translocation of NF-B by SN-50 did not reverse the effect of PI; and 3) JNK inhibition did not reverse the effect of PI on collagen and TIMP-1 reduction. These findings are consistent with previous data indicating that PI results in: 1) c-Jun accumulation in various cell types; 2) the presence of AP-1 sites on the promoter of MMP-1 gene; and 3) the capacity of AP-1 to increase MMP-1 production.
Despite the expected differences in potency of the four PI used (bortezomib>MG-132
PSI>lactacystin), their capacity to reduce type-I collagen and TIMP-1 and increase MMP-1 strongly suggests that the observed phenomena were due to a class effect and points to proteasome blockade as a strategy worth exploring for treatment of diseases characterized by excessive ECM deposition.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4870fje;
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