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Published ahead of print on May 5, 2008, doi:10.1165/rcmb.2007-0320OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 458-465, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0320OC

In Vivo Investigations on Anti-Fibrotic Potential of Proteasome Inhibition in Lung and Skin Fibrosis

Serena Fineschi1, Massimo Bongiovanni2, Yves Donati3, Souad Djaafar1, Filippo Naso4, Laurence Goffin1, Constance Barazzone Argiroffo3, Jean-Claude Pache2, Jean-Michel Dayer1, Sylvie Ferrari-Lacraz1 and Carlo Chizzolini1

1 Immunology and Allergy, Department of Internal Medicine, School of Medicine and University Hospital; 2 Department of Genetics and Laboratory Medicine, University Hospital; 3 Department of Pediatrics and Pathology-Immunology, School of Medicine and University Hospital, Geneva, Switzerland; and 4 Department of Experimental Biomedical Sciences, Padua University, Padua, Italy

Correspondence and requests for reprints should be addressed to Carlo Chizzolini, M.D., Immunology and Allergy, Geneva University Hospital, 1211 Geneva 14, Switzerland. E-mail: chizzolini{at}medecine.unige.ch


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
In systemic sclerosis (SSc), a disease characterized by fibrosis of the skin and internal organs, the occurrence of interstitial lung disease is responsible for high morbidity and mortality. We previously demonstrated that proteasome inhibitors (PI) show anti-fibrotic properties in vitro by reducing collagen production and favoring collagen degradation in a c-jun N-terminal kinase (JNK)-dependent manner in human fibroblasts. Therefore, we tested whether PI could control fibrosis development in bleomycin-induced lung injury, which is preceded by massive inflammation. We extended the study to test PI in TSK-1/+ mice, where skin fibrosis develops in the absence of overt inflammation. C57Bl/6 mice received bleomycin intratracheally and were treated or not with PI. Lung inflammation and fibrosis were assessed by histology and quantification of hydroxyproline content, type I collagen mRNA, and TGF-β at Days 7, 15, and 21, respectively. Histology was used to detect skin fibrosis in TSK-1/+mice. The chymotryptic activity of 20S proteasome was assessed in mice blood. JNK and Smad2 phosphorylation were evaluated by Western blot on lung protein extracts. PI reduced collagen mRNA levels in murine lung fibroblasts, without affecting their viability in vitro. In addition, PI inhibited the chymotryptic activity of proteasome and enhanced JNK and TGF-β signaling in vivo. PI failed to prevent bleomycin-induced lung inflammation and fibrosis and to attenuate skin fibrosis in TSK-1/+mice. In conclusion, our results provide direct evidence that, despite promising in vitro results, proteasome blockade may not be a strategy easily applicable to control fibrosis development in diseases such as lung fibrosis and scleroderma.

Key Words: proteasome inhibitors • bleomycin-induced pulmonary fibrosis • TSK-1/+mice • TGF-β



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
In the quest for novel anti-fibrotic strategies, we explored the potential of proteasome inhibition in models of lung and skin fibrosis driven by distinct pathogenetic mechanisms. In both, proteasome inhibition failed to control fibrosis development.

 
Fibrosis is a complex tissue response to various pathologic insults predominantly characterized by excessive deposition of extracellular matrix (ECM), which results in altered tissue and organ architecture leading to dysfunction and ultimately pathology. Fibrosis may be seen as a process not appropriately controlled and terminated aimed at repairing tissue damage (1, 2). Inflammatory cells profoundly affect ECM turnover by releasing soluble products or by direct cell-to-cell interactions that modify fibroblast metabolism during the early phases of fibrosis development (3). However, some authors suggest that cells of noninflammatory origin may be directly responsible for fibrosis development. For instance, in idiopathic pulmonary fibrosis, it has been proposed that fibrosis involves abnormal wound healing in response to multiple microscopic sites of ongoing alveolar epithelial injury. In this perspective, epithelial damage directly drives fibroblast migration and proliferation and leads to the formation of myofibroblast foci with excess of ECM deposition (4).

We recently reported that proteasome inhibitors (PI) in vitro profoundly alter human fibroblast metabolism, favoring ECM degradation over production (5). Both in resting and TGF-β–stimulated fibroblasts, proteasome inhibition results in increased metalloproteinase-1 (MMP-1) production, enhanced collagenolytic activity, and simultaneous reduction in collagen synthesis. Further, we showed that MMP-1–enhanced production was dependent on c-jun N-terminal kinase (JNK) activation leading to c-Jun phosphorylation and nuclear accumulation (5). Proteasomes are present in all mammalian cells, and possess trypsin, chymotrypsin, and caspase proteolytic activities, enabling the digestion of endogenous proteins including transcription factors and cell cycle cyclins, in addition to improperly or damaged cytosolic proteins as well as virus coded proteins (6, 7). PI have been developed and have been tested in clinical trials with the aim of reducing inflammation and neoplastic cell proliferation (8, 9). PS-341 (bortezomib), a potent, stable, reversible, and selective inhibitor of the chymothryptic threonine protease activity of proteasome, has been approved for the treatment of refractory multiple myeloma (10).

Systemic sclerosis (SSc) or scleroderma is a connective tissue disease of presumed autoimmune origin characterized by vascular abnormalities and widespread fibrosis affecting the skin and internal organs, including the lung (11). Indeed, interstitial lung disease in SSc is a primary reason of morbidity and mortality, for which there is a need of improved therapeutic approaches (12).

The aim of the present work was to assess the potential PI as anti-fibrotic agents in two distinct animal models of lung fibrosis and scleroderma (13, 14). The first, dependent on the intratracheal injection of bleomycin, is characterized by an early and dramatic inflammatory response that precedes lung fibrosis (13). It has however been questioned whether inflammation is directly responsible for the development of fibrosis in this model, since mice deficient in β6-integrin develop exaggerated inflammation but are protected from fibrosis (15) and fibrosis also develops in mice lacking T cells (16). The second, dependent on a partial duplication within the fibrillin-1 gene, which codes for a nonstructural ECM protein, results in collagen accumulation leading to a tight skin phenotype (TSK-1/+) (17). Interestingly, the TSK-1/+ phenotype develops in the absence of a detectable inflammatory cell response (18). Thus, while both models are characterized by fibrosis, they allow testing of the potential of PI to control fibrosis in substantially different circumstances, one preceded by exuberant inflammation and the other not.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Cultures
Primary C57Bl/6 lung fibroblasts were grown in 100-mm culture dishes at 37°C in 5% CO2-air, in Dulbecco's modified Eagle's medium/10% FCS supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 1% nonessential amino acids, 1% sodium pyruvate (Gibco, Invitrogen AG, Basel, Switzerland), until confluence (19). For COL1A1 mRNA determination and protein phosphorylation analysis, fibroblasts were serum-starved overnight, then cultured for 16 hours or the indicated time in the presence of PI or TGF-β (R&D Systems, Minneapolis, MN), or both. Their viability was assessed using the tetrazolium salts reduction test (EZ4U reagent; Biomedica, Vienna, Austria) (5).

Mice Treatments
The institutional animal care and use committee of the University of Geneva approved all experimental procedures involving mice. Twelve-week-old C57Bl/6 male mice received food and water ad libitum. Bleomycin (Baxter AG, Volketswil, Switzerland) (4 U/kg body weight in 100 µl of saline), or saline was administered by a single intratracheal injection under anesthesia. Twenty-four hours after the intratracheal injection, mice were randomly assigned to treatment with PI or vehicle. Every experiment involved 24 mice, divided into four groups of 6 mice each. PI were PS-341 (bortezomib; Millennium Pharmaceuticals, Cambrige, UK) and MG-132 (Z-Leu-Leu-Leu-CHO; Calbiochem, Darmstadt, Germany). In experiments with MG-132 the vehicle was DMSO, with PS-341 saline. Two sets of experiments were performed using PS-341 given intraperitoneally (0.1 mg/kg daily), and one using PS-341 given intravenously (0.8 mg/kg twice in week). MG-132 was given daily at 0.1 mg/kg intraperitoneally. Different schedules were used in two additional experiments. In one, mice were assigned to 15 days of treatment with PS-341 intraperitoneally or vehicle 1 week after being injected with bleomycin. In the other, mice were pre-treated with PS-341 or vehicle intraperitoneally for 1 week before being injected with bleomycin and continued their treatment for 2 additional weeks.

Lung Preparations
Mice were killed at Day 7 or Day 15 to evaluate inflammation, and at Day 21 to evaluate fibrosis. The right lung was removed and weighed, lobes were separated, one was snap-frozen in liquid nitrogen for hydroxyproline determination, and the other was put in 500 µl of RNAlater (Qiagen, Hilden, Germany) for COL1A1 mRNA. Then the trachea was cannulated, left lung was instilled with 4% paraformaldehyde in PBS, removed, kept for 6 hours in 4% paraformaldehyde, and subsequently subjected to paraffin embedding for histologic examination.

Determination of Hydroxyproline
The hydroxyproline content was determined according to Stegemann's procedure as modified by Woessner (20). After rinsing with PBS, the upper left lung lobe was lyophilized, further dried under vacuum over P2O5 at 37°C for 36 hours, weighed, and hydrolyzed for 22 hours at 110°C in 6 N HCl under N2 atmosphere. Aliquots were then assayed by adding chloramine T solution for 20 minutes, 3.15 M perchloric acid for 5 minutes, and Erlich's reagent at 60°C for 20 minutes. Absorbance was measured at 561 nm, and the amount of hydroxyproline was determined against a standard curve. Total lung collagen was calculated assuming that lung collagen contains 12.2% (wt/wt) hydroxyproline and expressed as milligrams of collagen per lung.

Real-Time RT-PCR Quantification of COL1A1 mRNA
Total RNA was isolated with TRIzol reagent (Gibco). One µg of total RNA was reverse-transcribed with random primers and superscript II (Invitrogen). The cDNA obtained was diluted 1:10 in water as template for PCR and amplified. The TaqMan assay reagents (Universal PCR Master Mix Buffer) and primers for mouse COLA1A, GADPH, and 18S RNA, were purchased by Applied Biosystems (Foster City, CA). The results were normalized to the geometric mean of GAPDH and 18S RNA. PCR was performed in triplicate using ABI PRIS 7900 Sequence Detection System (Applied Biosystems) in a final volume of 10 µl. The thermal cycler conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, then 50 cycles of 95°C for 15 seconds and 60°C for 1 minute. cDNA synthesis reaction without RNA or cDNA synthesis reaction without reverse transcriptase were used as controls and were negative for amplification products.

Analysis of Bronchoalveolar Lavage Fluid
The trachea was cannulated and bronchoalveolar lavage (BAL) performed with 700 µl of saline twice (1.4 ml total). Recovery of BAL fluid (BALF) ranged from 70 to 80% and did not significantly differ among the groups. BALF was collected and centrifuged at 500 x g for 10 minutes at 4°C, cell viability was assessed by trypan blue and differential counts by Papanicolau stain, total protein content by Bradford's method, and total TGF-β1 by enzyme-linked immunosorbent assay upon heating the samples at 80°C for 5 minutes (DuoSet mouse TGF-β1; R&D Systems) (21).

Histologic Analysis
For histologic examination, the paraffin sections were stained with either hematoxylin and eosin (H&E) or Masson's trichrome. Two different methods were used to quantify interstitial fibrosis. With software specifically created to identify sections stained by Masson's trichrome, a threshold of positivity for the blue color, representative of collagen, was established and used to compute the percentage of fibrosis of lung parenchyma excluding airways. At least 40 fields were analyzed for each lung. In addition, two observers, blinded to the treatment groups, assigned the Ashcroft score independently (22). This score ranges from 0 (normal lung) to 8 (complete fibrosis) and was averaged upon examination of five microscopic fields. To assess inflammation on slides stained with H&E, we established a score based on leukocyte counts (0 = scattered, 1 = < 15 leukocytes per field, 2 = 15–50 leukocytes per field, 3 = >50 leukocytes per field) in the interstitial, perivascular, and peribronchial areas, separately. The mean score of all fields was taken as the inflammation score of that lung section.

TSK-1/+Mice
TSK-1/+ and pa/pa mutant mice on a C57Bl/6 background were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred in our facility by mating TSK-1/+ male mice with pa/pa female mice. The TSK-1/+ progeny was identified by the black coat and eye color as well as by increased skin tethering. Twenty-eight male, 4-week-old, TSK-1/+ mice and 12 pa/pa littermates were randomly assigned to treatment with PS-341 0.1 mg/kg body weight intraperitoneally daily or saline for 1 month. Mice were killed and the skin was removed from the interscapular region as full-thickness section extending to the body wall musculature and the vertebral column. Tissues were fixed in 10% formaldehyde for 48 hours; decalcified in 2% formalin, 8% formic acid; then embedded in paraffin, cut into sections, stained with H&E, and independently examined by two investigators blind to treatment. Hypodermal thickness, defined by depth of the subcutaneous loose connective tissue below the panniculus carnosus, was measured in transverse perpendicular sections, at five positions along the length of each section.

Western Blotting
Cultured primary C57Bl/6 lung fibroblasts were washed in ice-cold PBS, scraped off with a rubber policeman, and incubated on ice in lysis buffer (50 mM Hepes pH 7.5; 150 mM NaCl; 5 mM EDTA; 1 mM orthovanadate; 50 mM NaF; 0.25% Na-DOC; 0.15 vol:vol complete protease inhibitor mix from Roche [Mannheim, Germany]; 0.2 mM PMSF; 100 nM okadaic acid, and 1% NP-40) for 15 minutes. Total proteins were recovered by 2-minute centrifugation at 10,000 rpm. Lungs were gently homogenized with Dounce homogenizer in 400 µl of lysis. After 30 minutes of incubation in ice, they were centrifuged at 13,000 rpm for 15 minutes at 4°C and supernatants were collected. Concentration of proteins obtained from lungs or cultured lung fibroblasts was determined using Bradford technique (protein assay reagent from Bio-Rad, Hercules, CA). Fifty (lung) or twenty (cell culture) micrograms of total protein extracts were separated by 10% SDS-PAGE under reducing conditions and electroblotted onto nitrocellulose membrane (Amersham Hybond ECL, GE Healthcare Europe, Otelfingen, Switzerland). Blots were incubated with antibodies against phospho c-jun (ser 37), phospho-Smad2 (ser 465/467) (Cell Signaling, Bervely, MA), phospho-JNK (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and β-actin (Sigma, St. Louis, MO). Horseradish peroxidase–conjugated antisera were used to reveal primary binding and detected by chemiluminescence using the ECL system (Amersham). The densitometric data from each experimental band was normalized to β-actin.

Assessment of Proteasome Inhibition in Mice Treated with PS-341
20S proteasome activity in whole blood of mice, collected by retro-orbital venous plexus, was determined in triplicate, as described (23). Whole blood samples were lysated and tested for the chymotryptic activity of proteasome by measuring the increase in fluorescence emission at 440 nm ({lambda}ex = 380 nm) that accompanies cleavage of AMC from peptide-AMC substrates (Succinyl-Leu-Leu-Val-Tyr-AMC) (SLLVY). The activity was normalized to the amount of proteins present in the lysate, and expressed as pmol AMC/second/mg prot.

Statistical Analysis
Student's t test was used to analyze differences between two groups. A one-way ANOVA test (Dunnett method) was used to assess the differences among groups. The survival data were analyzed using the log-rank test. A P value ≤ 0.05 was considered statistically significant.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Proteasome Inhibitors Reduce Collagen mRNA Levels in Murine Lung Fibroblasts
We previously showed that PI reduce collagens production in human dermal and lung fibroblasts (5). We therefore tested whether PI could affect collagen synthesis also in murine fibroblasts. PS-341 (1 µM) reduced type I collagen mRNA expression in unstimulated and TGF-β–stimulated mouse fibroblasts by 75.3% and 55.3%, respectively (Figure 1A). Importantly, PS-341 did not impaired fibroblast viability (Figure 1B).


Figure 1
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  		Figure 1. PS-341 reduces type I collagen mRNA levels but not cell viability in murine lung fibroblasts. (A) Quantification by real-time RT-PCR in C57Bl/6 lung fibroblasts after 24 hours of culture. Results are expressed as fold compared with medium and are normalized to GADPH expression. The relative mean quantity of GADPH was medium (solid bars) = 0.502, PS-341 (open bars) = 0.490, TGF-β (shaded bars) = 0.984, TGF-β+PS-341 (diagonally hatched bars) = 0.777. Bars represent the mean ± SEM of three independent experiments. *P < 0.05 compared with medium. (B) Cell viability was assessed by reduction of tetrazolium salts after 24 hours of culture. PS-341 = 1 µM, TGF-β = 10 ng/ml, NP-40 (horizontally hatched bar) = 1%. Data presented are the mean ± SD of triplicate cultures.

 
Proteasome Inhibitors Do Not Improve Survival and Weight Loss in Bleomycin-Injured Mice
We assessed the capacity of PI to improve survival in C57Bl/6 mice receiving bleomycin (4 U/kg body weight). At Day 21 after bleomycin, the survival rate of mice treated with PI (PS-341 or MG-132, 0.1 mg/kg intraperitoneally, daily) was 60.7% versus 73.5% in controls (Figure 2). Further, the intravenous administration of 0.8 mg/kg body weight every 3.5 days of PS-341 induced early death of all mice previously injected with bleomycin. However, PI did not affect mice survival when given alone. This was observed both in C57Bl/6 and in TSK-1/+ (Figure 2). Of interest, bleomycin-injured mice lost weight to a similar extent whether receiving PI or vehicle; however, PI per se did not affect body weight (Table 1).


Figure 2
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  		Figure 2. Effect of proteasome inhibitors on C57Bl/6 mice survival. Intratracheal injection of bleomycin or saline performed at Day 0. *P < 0.001 compared with saline treated mice. #P < 0.001 compared with bleomycin-injected mice. Squares, bleomycin + vehicle (n = 34); open diamonds, bleomycin + intraperitoneal proteasome inhibitors (PI) (0.1 mg/kg body weight daily) (n = 28); open circles, bleomycin + intravenous PI (0.8 mg/kg body weight every 3.5 d) (n = 6); triangles, saline + vehicle (n = 40); solid circles, saline + intraperitoneal PI (0.1 mg/kg body weight daily) (n = 40); solid diamonds, saline + intravenous PI (0.8 mg/kg body weight every 3.5 d) (n = 19).

 

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  		TABLE 1. BODY WEIGHT OF MICE TREATED WITH PS-341

 
Proteasome Inhibitors Do Not Modify Bleomycin-Induced Lung Inflammation
The early response to bleomycin lung injury is characterized by a dramatic increase in microvascular leak and inflammatory cell recruitment, which precedes fibrosis development (13). At Days 7 and 15, bleomycin induced a strong cellular infiltrate in perivascular, interstitial, and peribronchial areas (Figures 3A and 3B), and increased protein content (Figure 3C) and cell counts in BALF (Table 2) in all experiments. PI did not modify the inflammatory response to bleomycin (Figures 3A and 3B, Table 2). Of interest and unexpectedly, in mice injected intratracheally with saline, PI treatment resulted in an increase in inflammatory cells recruitment, particularly in peribronchial areas examined at Day 15 (Figure 3B).


Figure 3
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  		Figure 3. Effect of proteasome inhibitors on lung inflammation 7 and 15 days after intratracheal injection of bleomycin. (A) Histologic semiquantitative score (from 0–3) of global lung inflammation 7 days after intratracheal bleomycin or saline. Bars represent the mean ± SD of an experiment with six mice per group. (B) Histologic semiquantitative score (from 0–3) of lung inflammation in perivascular, interstitial, and peribronchial areas at Day 15. (C) Total protein concentration in bronchoalveolar lavage fluid (BALF) at Day 15. In B and C the bars represent the mean ± SEM of three independent experiments with six mice per group. Statistics were obtained by applying the ANOVA test. Solid bars, bleomycin + vehicle; open bars, bleomycin + PI; shaded bars, saline + vehicle; hatched bars, saline + PI.

 

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  		TABLE 2. CELL COUNTS IN BRONCHOALVEOLAR LAVAGE FLUID OF MICE INJECTED INTRATRACHEALLY WITH BLEOMYCIN AND TREATED WITH PROTEASOME INHIBITORS

 
Proteasome Inhibitors Do Not Modify Bleomycin-Induced Lung Fibrosis
At Day 21, bleomycin increased COL1A1 mRNA levels (Figure 4E), total lung collagen (Figure 4A), and fibrosis assessed by quantitative automated image analysis (Figure 4B) or by observer examination (Ashcroft score) (Figure 4C) in all experiments. However, PI did not attenuate the fibrotic response to bleomycin (Figures 4A–4E). In contrast, PI tended to increase peribronchial collagen deposition in controls (saline), as shown in Figure 4D4 and quantified by the Ashcroft score (P < 0.05 versus saline + vehicle) (Figure 4C). We further assessed whether different schedules of treatment could attenuate fibrosis. PI given for a week before and 2 weeks after bleomycin, or given for a fortnight starting a week after bleomycin, did not control fibrosis (not shown).


Figure 4
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  		Figure 4. Effect of proteasome inhibitors on lung fibrosis 21 days after intracheal injection of bleomycin. (A) Total lung collagen as hydroxyproline content. (B) Histologic assessment of lung fibrosis by quantitative image analysis. Percentage = (fibrosis/total tissue) x 100. (D) Representative lung histology of Masson's trichrome. 1 = bleomycin + vehicle, 2 = bleomycin + PS-341, 3 = saline + vehicle, 4 = saline + PS-341. Original magnification: x200. Trait = 50 µm. (E) Quantification of COL1A1 mRNA by real-time RT-PCR in total lung extract. Values are expressed as fold of the mean value of saline + vehicle group. (F) Total TGF-β was assessed by enzyme-lined immunosorbent assay in BALF obtained at Day 15. All experiments included six mice per group. Bars represent the mean ± SEM of three independent experiments in B, E, and F, and the mean ± SEM of two experiments in A and C. Solid bars, bleomycin + vehicle; open bars, bleomycin + PI; shaded bars, saline + vehicle; hatched bars, saline + PI.

 
Since TGF-β has been shown to be a mediator involved in the fibrotic response induced by bleomycin (24), we were interested to assess whether PI could affect TGF-β levels. In our experiments, total TGF-β levels were 3-fold higher in bleomycin-injected mice than in controls, but PI did not significantly modify these levels (Figure 4F). Overall, these data strongly indicate that PI do not attenuate bleomycin-induced lung fibrosis.

Proteasome Inhibitors Do Not Modify TSK-1/+ Mice Hypodermal Fibrosis and Skin Tethering
We then asked the question whether PI could prevent fibrosis development in mice in which skin fibrosis develops in the absence of an inflammatory cell infiltrate. To test the capacity of PI to prevent the establishment of fibrosis, TSK-1/+ mice were treated daily for a month from Days 30 to 60 of age, when they develop maximal skin thickness and hypodermal fibrosis. Hypodermal thickness was 6-fold greater in TSK-1/+ compared with pa/pa littermates, and similar in PS-341 and vehicle-treated TSK-1/+ mice (Figures 5A and 5B). Therefore, PI were unable to modify the establishment of skin fibrosis in this animal model of SSc.


Figure 5
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  		Figure 5. Effect of proteasome inhibitors on TSK-1/+ skin fibrosis. (A) Representative H&E-stained skin sections from 2-month-old male TSK-1/+ mice (A1, A2) and pa/pa littermates (A3, A4). A1 and A3 received vehicle, and A2 and A4 received PS-341 0.1 mg/kg intraperitoneally daily for a month. d = dermis. Arrowhead, panniculus carnosus. h, hypodermis. Original magnification: x25. Trait = 200 µm. (B) The bars represent the mean ± SD of 14 mice per group. Hypodermal thickness was the average of measurements at five distinct positions along the length of each section. Solid bar: TSK-1/+ mice, PS-341; open bar: TSK-1/+ mice, vehicle; shaded bar: pa/pa mice, PS-341; hatched bar: pa/pa mice, vehicle.

 
Proteasome Inhibitors Block Proteasome Activity and Induce JNK Activation In Vivo
In view of the above negative results, it was important to check whether the PI we used had a detectable pharmacologic activity in vivo. This was the case, as the chymotryptic activity of 20S proteasome was reduced by more than 70 percent in mice receiving PS-341 compared with controls, both in the bleomycin-induced lung fibrosis and in TSK-1/+ mice (Table 3). Since in our in vitro experiments we previously demonstrated that PI results in JNK and c-Jun phosphorylation and nuclear accumulation, and that c-Jun signaling pathway is required for the enhanced collagenolytic activity induced by PI (5), we tested whether PI could activate JNK in vivo in lung extracts. PS-341 induced JNK activation, as demonstrated by enhanced JNK and c-Jun phosphorylation both in TSK-1/+ and in pa/pa littermates (Figure 6A). Similar data were obtained also in C57Bl/6 mice, where PI treatment induced c-Jun phosphorylation in the absence of previous exposure to bleomycin (not shown). These data strongly argue in favor of a substantial pharmacologic activity of PI in vivo, which results in clear proteasome blockade.


Figure 6
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  		Figure 6. In vivo activation of JNK and enhanced Smad-2 phosphorylation by PS-341. (A) Western blot using proteins extracted from whole lung of TSK-1/+ and pa/pas mice treated or not with PS-341 for a month. Each lane represents an individual mouse. (B) Western blot using proteins extracted from C57Bl/6 lung fibroblasts cultured in vitro with TGF-β (10 ng/ml), with PS-341 (1 µM), or with both for the indicated time. (D) Western blot using proteins extracted from whole lung of C57Bl/6 intratracheally injected with bleomycin or saline and then treated with PS-341 or saline for 7 days. Each lane represents an individual mouse. Specific phospho-protein levels were quantified, normalized to β-actin, and reported either (C) as protein increase or (E) as protein expression. In E, the bars represent the mean ± SD of P-Smad2 levels of five mice per group shown in D. Statistically significant differences are reported (Student's t test). In C, solid bars indicate TGF-β, hatched bars indicate PI, and shaded bars indicate TGF-β + PI.

 

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  		TABLE 3. PROTEASOME ACTIVITY IN THE BLOOD OF MICE TREATED OR NOT WITH PS-341

 
To further investigate the paradoxical increase of peribronchial fibrosis in mice receiving intratracheal saline and then treated with PI, we explored whether in vitro and in vivo PI could modify TGF-β signals. As expected, in primary mouse fibroblasts TGF-β induced Smad2 phosphorylation, which peaked at 1 hour and vanished by 3 hours (Figures 6B and 6C). The joint presence of PI resulted in sustained Smad2 phosphorylation, which lasted for at least 3 hours and was still faintly present at 6 hours (Figures 6B and 6C). Of interest, PI by itself did not induce Smad 2 phosphorylation, thus indicating that PI in vitro modifies signals initiated by TGF-β and mediated by the canonical TGF-β signaling pathway. To verify the in vivo relevance of such a finding, we assessed the levels of phospho-Smad2 in the lungs of mice that received intratracheal saline and were then treated or not for 7 days with PI. Additional controls included mice that received intratracheal bleomycin and then were treated or not for 7 days with PI. As shown in Figure 6D, where each lane represents an individual mouse, mice that received bleomycin had very high (probably maximal) levels of phospho-Smad2 in the lung extracts. The PI treatment did not substantially modify these levels (Figures 6D and 6E). Of major interest, mice that did not receive bleomycin had some phospho-Smad2 basal expression that was significantly increased by PI (Figures 6D and 6E). These data indicate that PI indeed modifies TGF-β signaling in a direction that may enhance rather than inhibiting signals leading to fibrosis in C57Bl/6 mice.


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The primary goal of this study was to investigate whether pharmacologic proteasome inhibition could have a potential in preventing or reversing fibrosis development in two distinct animal models in which the fibrotic response is driven by two substantially different processes. In bleomycin-induced lung injury, an exuberant inflammatory response precedes fibrosis (13). Inflammatory mediators thought to be important in alveolar damage and subsequent widespread fibrosis include TNF-{alpha}, IFN-{gamma}, reactive oxygen species, IL-4, TGF-β, CTGF, and MCP-1, as well as the transcription factors Smad and NF-{kappa}B (24–28). Increased epithelial cell apoptosis takes part to this process (29). It should, however, be noted that uncoupling between inflammation and fibrosis has been reported, indicating that the inflammatory mediators here listed may not all have redundant roles (15, 16). At variance with this, immuno-inflammatory cells are absent from TSK-1/+ skin and TGF-β has been implicated, since TGF-β gene targeting attenuates the phenotype (30). Indeed, the mutation of the fibrillin-1 gene in TSK-1/+ mice results in duplication of TGF-β binding domains, thus allowing the recruitment of a greater number of TGF-β molecules, leading to excessive collagen production (17). Unfortunately, the results we obtained strongly indicate that PI do not have the potential to control fibrosis in these two models. This is in contrast with the reported efficacy of PI in preventing heart fibrosis in hypertensive rats (31), renal fibrosis in rat obstructive nephropathy (32), liver fibrosis in bile-duct ligated mice (33), and myelofibrosis induced by high levels of thrombopoietin in mice (34). Although some of the mediators are in common, it is worth stressing that fibrosis is the outcome of complex and redundant (though not overlapping) processes. Thus, the discrepancy between our results and those reported in the literature is consistent with substantial differences in molecular mechanisms leading to fibrosis.

In several in vivo models, including the bile-duct ligation liver fibrosis, obstructive nephropathy, and high thrombopoietin myelofibrosis, the administration of PI was associated with high levels of I{kappa}B{alpha} in target organs compared with untreated animals, consistent with NF-{kappa}B blockade under proteasome inhibition (35). NF-{kappa}B was reported to be involved in the bleomycin-induced lung fibrosis, since antisense oligonucleotides to NF-{kappa}B improved survival and a selective I{kappa}B kinase inhibitor was shown to ameliorate bleomycin-induced pulmonary fibrosis (26, 36). However, in our experimental conditions, baseline levels of I{kappa}B{alpha} in the lung were similar when controls and bleomycin-treated mice were compared, and obviously PI could not modify normal levels of I{kappa}B{alpha} (not shown). Since our experimental protocol powerfully induced extensive fibrosis, the discrepancy between ours and other results in term of NF-{kappa}B signaling involvement remains to be explained, although slight differences in the timing of organ sampling could be considered. In the other hand, we have shown that the JNK pathway was activated in PI-treated mice, consistently with results we and others have obtained in vitro (5, 37). c-Jun has been implicated in lung fibrosis induced by amiodarone, radiation, and bleomycin, and in idiopathic pulmonary fibrosis (38–40). There is also evidence of c-Jun involvement in renal and hepatic fibrosis (41, 42). It can be hypothesized that c-Jun activation induced by PI could trigger cell type–dependent responses, the final effect of which is pro-fibrotic, thus counterbalancing the possible anti-fibrotic effect of NF-{kappa}B blockade.

TGF-β is thought to be involved in fibrosis development both in bleomycin-induced lung injury and in skin fibrosis in TSK-1/+ mice. Consistent with previous reports, we observed high levels of TGF-β in the BALF of bleomycin mice (43). It is also known that signaling generated by TGF-β upon binding to its receptor complex, including both positive and negative transducers, is tightly regulated by proteasome (44). Inhibition of the proteasome activity causes accumulation of these components in the cells and modulates TGF-β signaling in a time-dependent and gene-specific manner (45). Consistent with these observations, our data show prolonged Smad-2 phosphorylation in lung fibroblasts when activated by TGF-β in conjunction with PI and enhanced levels of phospho-Smad2 in the lungs of mice treated with PI (Figure 6). Through a physical interaction with different ubiquitin E3 ligases, Smads participate in ubiquitination and proteasome degradation of several key regulators, such as the oncoprotein SnoN and the multi-domain docking protein HEF1, linking TGF-β signaling to multiple signaling pathways (46). Our data indicate that PI indeed modifies TGF-β signaling in a direction that may enhance rather than inhibit signals leading to fibrosis. However, PI reduced type I collagen transcription in vitro, both in resting and in TGF-β–stimulated conditions. This indicates that the fibrotic responses observed in vivo in PI-treated mice may be due to additional mechanisms synergistic with TGF-β.

It is unlikely that the lack of antifibrotic effects in our two experimental models was due to trivial explanations such as suboptimal or inefficacious treatment schedules. First, we tested two different PI (PS-341 and MG-132) at doses validated in other in vivo studies (31, 33). Second, we observed inhibition of the proteasome chymotryptic activity in blood of treated mice. Third, we demonstrated in vivo activation of JNK and TGF-β signaling pathway in lungs of treated mice. In an attempt to improve PI efficacy, we varied timing of administration and doses with no changes in outcome except when PS-341 was given at high doses (0.8 mg/kg body weight intravenously every 3.5 d), which resulted in excess and rapid mortality in bleomycin-injured mice. The autopsy revealed pulmonary edema and pneumonia, while the liver, kidney, and heart were histologically normal (data not shown). Since PI by themselves did not significantly affect mice survival in controls, it can be hypothesized that PI at high dose inhibited some rescue mechanism from acute bleomycin injury. Of interest, the neutral cysteine protease named bleomycin hydrolase bears structural similarity to 20S proteasome and is essential for bleomycin resistance (47). It is possible that at high doses PI reduces the activity of such an enzymatic pathway. In addition, proteasome controls the levels of several transcription factors or enzymes involved in apoptosis, cell cycle, and signal transduction, which may in multiple ways affect the capacity of the organism to resist an acute insult (7). Finally, off-target effects of PI may not be excluded as important to explain excess mortality.

In C57Bl/6 receiving PI only, we noticed an accentuation of collagen fibers and inflammatory cells localized in peribronchial areas. This unexpected observation may be in keeping with the reported severe pulmonary complications in some patients suffering from multiple myeloma under therapy with PS-341 (48–50). However, neither an inflammatory infiltrate nor collagen accumulation has been observed in the lung of TSK-1/+ mice (not shown). Thus, we speculate that an initial lung stress (intratracheal instillation of PBS) is required for allowing PI to induce such a reaction.

Overall, the results here reported indicate that PI, while resulting in proteasome blockade in vivo, cannot reduce fibrotic responses in the two models we used, possibly in relationship with the complexity of inflammatory events and intracellular signals involved, which bypass signals controlled by proteasome.


   Acknowledgments
 
The authors thank Mrs. M. Alvarez (Immunology and Allergy) for her precious help with Western blotting and Mr. T. Le Minh (Division of Clinical Pathology, Geneva School of Medicine) for providing valuable technical assistance with histology. The authors are indebted to Mrs. P. Vinken and Dr. R. De Coster (Johnson & Johnson Pharmaceutical Research & Development, Beerse, Belgium) for measurements of proteasome activity in blood lysate.


   Footnotes
 
This work was supported in part by grant 310000–112180/1 (C.C.) from the Swiss National Science Foundation, and from the Association des Sclérodermiques de France. S.F. was a recipient of an AMPLI fellowship.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0320OC on May 5, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 31, 2007

Accepted in final form March 22, 2008


   References
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 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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