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Antifibrotic effects of curcumin are associated with overexpression of cathepsins K and L in bleomycin… Zhang, Dongwei; Huang, Chuangfang; Yang, Changfu; Liu, Renzuo J; Wang, Jifeng; Niu, Jianzhao; Brömme, Dieter Nov 29, 2011

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RESEARCH Open AccessAntifibrotic effects of curcumin are associatedwith overexpression of cathepsins K and L inbleomycin treated mice and human fibroblastsDongwei Zhang1, Chuangfang Huang1,2, Changfu Yang2, Renzuo J Liu1, Jifeng Wang2, Jianzhao Niu2 andDieter Brömme1*AbstractBackground: Lung fibrosis is characterized by fibroblast proliferation and the deposition of collagens. Curcumin, apolyphenol antioxidant from the spice tumeric, has been shown to effectively counteract fibroblast proliferationand reducing inflammation and fibrotic progression in animal models of bleomycin-induced lung injury. However,there is little mechanistic insight in the biological activity of curcumin. Here, we study the effects of curcumin onthe expression and activity of cathepsins which have been implicated in the development of fibrotic lung diseases.Methods: We investigated the effects of curcumin administration to bleomycin stimulated C57BL/6 mice andhuman fetal lung fibroblasts (HFL-1) on the expression of cathepsins K and L which have been implicated in matrixdegradation, TGF-b1 modulation, and apoptosis. Lung tissues were evaluated for their contents of cathepsins K andL, collagen, and TGF-b1. HFL-1 cells were used to investigate the effects of curcumin and cathepsin inhibition oncell proliferation, migration, apoptosis, and the expression of cathepsins K and L and TGF-b1.Results: Collagen deposition in lungs was decreased by 17-28% after curcumin treatment which was accompaniedby increased expression levels of cathepsins L (25%-39%) and K (41%-76%) and a 30% decrease in TGF-b1expression. Moreover, Tunel staining of lung tissue revealed a 33-41% increase in apoptotic cells after curcumintreatment. These in vivo data correlated well with data obtained from the human fibroblast line, HFL-1. Here,cathepsin K and L expression increased 190% and 240%, respectively, in the presence of curcumin and theexpression of TGF-b1 decreased by 34%. Furthermore, curcumin significantly decreased cell proliferation andmigration and increased the expression of surrogate markers of apoptosis. In contrast, these curcumin effects werepartly reversed by a potent cathepsin inhibitor.Conclusion: This study demonstrates that curcumin increases the expression of cathepsins K and L in lung whichan effect on lung fibroblast cell behavior such as proliferation, migration and apoptosis rates and on the expressionof TGF-b1 in mouse lung and HFL-1 cells. These results suggest that cathepsin-inducing drugs such as curcuminmay be beneficial in the treatment of lung fibrosis.Keywords: lung fibrosis, curcumin, cathepsins, collagen, TGF-β1, apoptosis, protease inhibitors.BackgroundLung fibrosis is accompanied by fibroblast proliferationand excessive extracellular matrix deposition primarilyin the form of collagens. This leads to a progressive lossof lung function and ultimately death. Besides anti-inflammatory drugs, there is presently no effective andapproved medication available in western countries.However, curcumin, an antioxidant from the spice tur-meric, is used as alternative medicine in India andChina for various inflammatory conditions and lungrelated ailments and the NIH is presently funding sev-eral clinical studies to evaluate the efficacy of curcuminhttp://clinicaltrials.gov/ct2/results?term=curcumin.Smith and coworkers, have recently demonstrated that* Correspondence: dbromme@interchange.ubc.ca1Department of Oral and Biological Science, Faculty of Dentistry, Universityof British Columbia, Vancouver, BC, V6T 1Z3, CanadaFull list of author information is available at the end of the articleZhang et al. Respiratory Research 2011, 12:154http://respiratory-research.com/content/12/1/154© 2011 Zhang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.curcumin administration resulted in a significant reduc-tion of lung inflammation and collagen deposition inbleomycin induced lung fibrosis in mice and relate theseeffects to its anti-proliferative activity on fibroblasts andinterference in TGF-b1 mediated signaling pathways [1].Other laboratories reported similar effects of curcuminin related animal models of lung fibrosis [2-4]. However,the exact mechanism of how curcumin exerts its lungprotective activity remains elusive.Lung fibrosis is also accompanied by abnormal proteo-lytic activity in lungs. Of particular interest are cysteinecathepsins which are potent collagenases and elastasesand which have been implicated in caspase-independentapoptosis pathways [5]. Cathepsins are widely expressedin lung tissue [6,7]. It has been shown that cathepsin K(CatK) deficiency exacerbates lung fibrosis in bleomy-cin-induced lung injury [8] and that it interferes withnormal airway development [9]. On the other hand,increased levels of CatK alleviate excessive extracellularmatrix deposition and consequently protect lungs frombleomycin induced lung fibrosis [10]. Furthermore, over-expression of CatK has been detected in human lungfibrosis derived fibroblasts [8,11]. CatK is a very power-ful collagenase and elastase [12,13]. We have recentlydemonstrated that CatK is also involved in the homeos-tasis of TGF-b1 [9]. On the other hand, TGF-b1 down-regulates CatK expression in fibroblasts and favorsfibrosis [11]. TGF-b1 also reduces the expression ofcathepsin L (CatL) in lung epithelial cell [14]. Accumu-lating evidence suggests that elevated TGF-b1 levels area consistent clinical feature of pulmonary fibrosis [15].Upregulation of TGF-b1 resulted in fibroblast prolifera-tion and differentiation as well as in abnormal collagendeposition [1,16], whereas suppressed activity of TGF-b1afforded a protection from fibrosis [17,18]. We have pre-viously demonstrated that CatK expression suppressesTGF-b1 expression in mouse lung [9]. To what extentcurcumin affects the level of cathepsin expression inlung is unclear. The aim of this study is to investigatethe effect of curcumin on cathepsin expression in bleo-mycin-treated mouse lungs and human lung fibroblasts,and their involvement in fibrosis promoting cell beha-viors such as proliferation, migration and apoptosis.Materials and methodsReagents and AntibodiesBleomycin was obtained from Haizheng Pharmaceuticals(Zhejiang, China). Curcumin, fetal calf serum (FBS),Dulbecco’s Modified Eagle Medium (DMEM), andmouse anti b-actin were purchased from Sigma (SaintLouis, MO, USA), TRIzol Reagent for total RNA isola-tion was from Invitrogen (Paisley, UK), One StepSYBR® PrimeScript® RT-PCR Kit (DRR066A) for realtime RT-PCR from TaKaRa (Dalian, China). TdT-mediated dUTP Nick End-labeling (Tunel) was fromPromega (Madison, WI, USA). Antibodies against Bax,Bcl-2 and cleaved caspase-3 were obtained from CellSignaling Technology (Danvers, MA, USA) and goatpolyclonal anti-CatL from Santa Cruz Biotechnology(Santa Cruz, CA, USA), mouse anti TGF-b1 for immu-nohistochemical staining from sigma, chicken anti TGF-b1 for immunoblot analysis from R&D Systems (Min-neapolis, MN, USA), and rabbit anti CatK from Protein-Tech Group (Chicago, IL, USA). The peptidyl vinylsulfone inhibitor LHVS (Mu-Leu-hPh-VS-Ph) has beensynthesized as described in [19].Bleomycin induced lung fibrosis9-10 C57BL/6 mice per experimental group, age 8-14weeks, were used in the fibrosis model. Mice werehoused in a barrier facility with specific pathogen-freeconditions and all experiments were performed usingprotocols approved by the Beijing University of ChineseMedicine Animal Care Unit. Briefly, mice were anesthe-tized and the trachea was minimally exposed via a ven-tral midline neck incision. Bleomycin sulfate (0.1 ml,0.025 U/mouse, diluted in sterile saline) or equalvolumes of saline as control were slowly instilled intra-tracheally as previously described [20]. The incision wasclosed with surgical adhesive, and mice were allowed torecover with free access to food and water. Mice weretreated (2nd day after instillation) with curcumin (200mg/kg) or saline alone daily in 0.5% carboxymethylcellu-lose by gavage. Mice were euthanized at various timepoints (at 1, 2, and 4 weeks) after bleomycin instillation.Lung tissues were either flash frozen and then stored inliquid nitrogen for further analysis or perfused and fixedin 10% formalin for at least 24 h at room temperaturefor immunohistochemical analysis.Histochemical and immunohistochemical staining5 μm sections were used for Hematoxylin and Eosin(H&E) staining to evaluate the severity of lung inflam-mation [21]. Masson Trichrome staining was performedto assess the degree of fibrosis [22]. Microscopic imageswere acquired with the 20× objective on a Leica micro-scope (Leica Microsystems Inc., Wetzlar, Germany).Immunohistochemical analysis of the lung tissue sam-ples was performed following standard procedure.Briefly, endogenous peroxidase activity was blocked byimmersion of deparaffinized sections in 3% H2O2 inmethanol for 30 min. Antigen retrieval was performedby steaming slides in 0.01 M citrate buffer (pH 6.0) for30 min. Slides were blocked with 1% bovine serum albu-min for 30 min at room temperature and subsequentlyincubated at 4°C overnight with antibodies against CatK(1:100) and CatL (1:100), TGF-b1 (10 μg/ml). To iden-tify and exclude non-specific immunoglobulin bindingZhang et al. Respiratory Research 2011, 12:154http://respiratory-research.com/content/12/1/154Page 2 of 12in tissue sections, non-antigen derived IgGs of the samespecies and at the same concentration as the experimen-tal primary antibodies were used as controls. Subse-quently, the slides were incubated with biotinconjugated horse anti-mouse IgG (Pierce, Rockford, IL,USA), goat anti-rabbit (Pierce) for 30 min at room tem-perature. Then immunoreactions were visualized byusing avidin biotin conjugated horseradish peroxidaseand 3, 3’-diaminiobenzidine (DAB, Vector Labs, Burlin-game, CA, USA) as substrate. Two tissue sections ofeach specimen were assessed at a 40× original magnifi-cation using a Leica microscope and evaluated withOpenlab software (PerkinElmer, Waltham, MA, USA).Tunel assayEnd-labeling of exposed 3’-OH ends of DNA fragmentswas undertaken with the DeadEnd™ FluorometricTunel System as described by the manufacturer. Briefly,the tissue samples on slides were permeabilized withproteinase K, then incubated with fluorescein 12-dUTPand finally stained with propidium iodide. After staining,whole lung was imaged using 5× magnification forexamination. Images were quantified using Openlabsoftware (PerkinElmer).Real-time RT-PCRmRNA levels were evaluated by using the TaKaRa OneStep real-time RT-PCR assay according to the manufac-turer’s protocol. Total cellular RNA was extracted fromlungs using TRIzol reagent (Invitrogen) according to themanufacturer’s instructions. The primer sequences were:b-actin (forward, 5’-AGA GGG AAA TCG TGC GTGAC-3’; reverse, 5’-CAA TAG TGA TGA CCT GGCCGT-3’) and CatK (forward, 5’-ACT TGG GAG ACATGA CCA GTG A -3’; reverse, 5’-TCT TGA CTG GAGTAA CGT ATC CTT TC-3’). To normalize the amountof total RNA present in each reaction, b-actin cDNAwas employed as an internal control.Human lung fibroblast (HFL-1) cultureHFL-1 cells were a gift from Dr. Clive Roberts, Univer-sity of British Columbia (ATCC, CCL153). The cellswere cultured in DMEM containing 10% FBS, penicillin/kanamycin. Cultures were maintained at 37°C in ahumidified atmosphere of 5% CO2. The medium waschanged every other day. Cells were treated with bleo-mycin (0.1 mU/ml; dissolved in Phosphate buffered sal-ine, PBS) and/or various concentrations of curcumin.Drug treatments of cells and their controls were per-formed in DMEM without FBS.MTT assayMTT [thiazolyl blue, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide, Sigma] was used todetermine cell viability and proliferation. At specifiedtime points, the culture medium was removed, and fibro-blasts were incubated with 5 mg/ml MTT solution in anincubator at 37°C for 3 h. After removing the medium,200 μl of DMSO was added to each well of a 96-wellplate to solubilize the blue-colored tetrazolium and theplates were then shaken for 5 min. Absorbance at 570nm was recorded in a Spectramax Plus reader (MolecularDevices, Sunnyvale, CA). Data were expressed in percen-tage with the untreated samples set to 100%.Wound healing assayHFL-1 cells were evenly seeded into a 6-well plate. Cellswere starved for 24 h in serum free DMEM after reachingat least 90% confluence. Three separate parallel scratchwounds were made with a 200 μl pipette tip. After wash-ing twice with PBS, 10% FBS in DMEM medium contain-ing different concentrations of curcumin was added to thecells. Images were taken under phase contrast invertedmicroscopy at 0 and 24 h. The wound area was measuredusing Openlab software and represented as percentage ofwound closure of the time zero wound area.Transmigration assayHFL-1 cells were cultured in DMEM containing 10%FBS. When cells reached 70% confluence they werestarved in DMEM without FBS media for 24 h. Cellswere trypsinized and resuspended in serum free media.Then 104 cells were seeded into the upper level of theinsert membrane (Transwell Permeable Supports, Corn-ing, Lowell, MA) with different concentrations of curcu-min. The bottom of the insert contained DMEM with10% FBS. The invasion assay was carried out for 8 h inthe tissue culture incubator. After 8 h, inserts wererinsed with PBS and stained with 0.5% crystal violetsolution in 25% methanol for 10 min. After washing theinserts by dipping the inserts into distilled water, cells atthe top of the inserts were removed with a cotton swab.The membrane was soaked in 1% SDS in PBS overnight.On the second day, 200 μl of buffer were transferred toa 96-well plate and the optical density (OD) was mea-sured at a wavelength of 570 nm in a Spectramax Plusreader. The percentage of migrated cells was calculatedbased on the ODs of treated and untreated samples.Immunoblot assayProteins in cell lysates were separated by SDS-PAGEand then transferred to nitrocellulose membranes.Membranes were incubated with appropriate primaryantibodies overnight at 4°C. The concentrations of pri-mary antibodies were as follows: Bax (1:10000), BCL-2(1:2000), cleaved caspase-3 (1:1000), b-actin (1:30000),CatL (1:100), TGF-b1 (1:850), CatK (1:2000). Blots werethen washed with 0.05% Tween in PBS and incubatedZhang et al. Respiratory Research 2011, 12:154http://respiratory-research.com/content/12/1/154Page 3 of 12for 1 h at room temperature with horseradish peroxi-dase-conjugated secondary antibodies. Serial images ofthe blots were captured and analyzed using the Chemi-genius bioimaging system (Syngene, Cambridge, UK).All values were normalized to expression of the corre-sponding b-actin in the same membrane. Pixel densitieswere corrected for background staining in the samemembrane.Statistical analysisResults are expressed as means ± SE for all mouserelated experiments. All other results are displayed asmeans ± SD. All image analyses were performed by twoobservers blinded to the group status. The significanceof differences of the mean values was calculated usingparametric ANOVA. A p value of less than 0.05 wasconsidered significant.ResultsExpressions of CatK and L in response to curcumintreatment in lungs with bleomycin induced fibrosis andHFL-1 cellsCathepsins are involved in human lung development [6]and are transiently upregulated in bleomycin inducedlung injury during the regeneration phase [23]. CatK,one of most efficient collagenases [13], has been shownto reduce collagen deposition in lungs of mice withbleomycin induced lung fibrosis when overexpressed[10]. Considering the known anti-fibrotic activity of cur-cumin in lung [24], we evaluated the effect of curcuminon the expression of CatK and L in bleomycin-inducedlung fibrosis. As shown in Figure 1A-C, CatK proteinexpression in curcumin treated mice was increased by63%, 76% and 41% at 7d, 14d and 28d, respectively andthat of CatL by 25%, 39% and 38% when compared tobleomycin treated mice. Further, at 7d, 14d and 28d,CatK mRNA levels (Figure 1D) in the curcumin treatedgroup also increased by 17%, 44% and 20%, respectively.It should be noted that bleomycin treatment causesmaximal fibrosis already after one week.Similar to the expression of CatK and L in the lungtissue, the expression of both enzymes was alsoincreased in human fetal lung fibroblast (HFL-1) cul-tures after treatment with curcumin. As shown in Figure2A and 2B, CatK and L expression increased in the pre-sence of curcumin in a dose-dependent manner aftertreatment of the cells with bleomycin. Treatment with30 μM of curcumin resulted in an about 100% increasein the expression of CatK and L.Effect of curcumin on collagen deposition in the lung andon lung fibroblast proliferation and migrationBleomycin-induced lung fibrosis is strongly associatedwith extracellular matrix production and deposition.When compared to untreated control mice, bleomycin-treated animals showed an almost 100% increase in lungcollagen deposition 7 days after the initial bleomycininstillation (Figure 3A and 3B). The collagen contentfurther increased to 113% at 28d. Oral application ofcurcumin (200 mg/kg/d), beginning two days after thebleomycin treatment, reduced the deposition of collagenby 21% after 7d, 17% after 14d and 29% after 28d.As fibroblasts represent the major cell type in lungfibrotic tissue, we determined the effect of curcumin onthe proliferation and migration rates of HFL-1 cells.HFL-1 cells were exposed to bleomycin (0.1 mU/ml)and stimulated for 24, 48 and 72 h. Curcumin revealeda dose-response related inhibition of cell proliferation(Figure 3C) whereas bleomycin increased the prolifera-tion rate by almost 100%. The anti-proliferative effectbecame significant at curcumin concentrations equal orhigher than 10 μM. At 30 μM, the proliferation rateswere suppressed below the rate observed in the absenceof bleomycin.Fibroblasts migration may accelerate fibroblast fociformation and contribute to the development of lungfibrosis. Thus, we determined the effect of curcumin ontransmigration rates of HFL-1 cells. As shown in Figure3D, curcumin was effective in attenuating dose-depen-dently fibroblast transmigration. Migration rates werereduced up to 45% at 30 μM of curcumin.Effect of curcumin on wound healing and its dependencyon cathepsin activitiesAbnormal wound healing is a main contributor to lungfibrosis. As shown in Figure 4A and 4B, curcumindelayed wound healing in a dose-dependent manner.Wound closure rates were reduced from 75% at 3 μMcurcumin to 17% at 30 μM of curcumin concentration.Further, HFL-1 cells were incubated in the presence orabsence of 10 μM LHVS, a pan-cathepsin inhibitor, priorto the wound making. After wound making, cells wereincubated with curcumin (30 μM) and bleomycin (0.1mU/ml) with or without LHVS for 24 h and subsequentlyallowed to grow in drug-free media (10% FBS in DMEM)for another 48 h. As shown in Figure 4C, we found thatLHVS can significantly increase the rate of wound clo-sure (p < 0.05 or p < 0.01). These results indicate thatcurcumin can inhibit proliferation and migration in thisassay and that this partly depends on cathepsin activity.Effect of curcumin on TGF-b1 expression and itsdependency on cathepsin activitiesTGF-b1 plays a key role in the progression of lungfibrosis and has been shown to be upregulated [25].Inhibition of TGF-b1 attenuates exacerbation of lungfibrosis [26]. First, we determined TGF-b1 expression inbleomycin induced lung fibrosis in the absence orZhang et al. Respiratory Research 2011, 12:154http://respiratory-research.com/content/12/1/154Page 4 of 12presence of curcumin. As shown in Figure 5A, 28 daysafter bleomycin administration, the extent of TGF-b1expression was decreased by 25% in the presence of cur-cumin when compared to the absence of curcumin atsame time point. To reveal the influence of cathepsinactivity and their regulation by curcumin on TGF-b1expression, we treated bleomycin stimulated HFL-1 cellswith curcumin. As shown in Figure 5B, TGF-b1 expres-sion was dose-dependently reduced by curcumin.Further, TGF-b1 expression was increased two-fold bythe addition of the pan-cathepsin inhibitor, LHVS, inthe presence of curcumin (30 μM) and bleomycin (0.1mU/ml) (Figure 5C). To further elucidate cathepsinexpression in response to TGF-b1 stimulation typical inbleomycin-induced lung fibrosis, we treated HFL-1 cellswith a constant amount of TGF-b1 (5 ng/ml) and mea-sured the effect of curcumin on CatK and L expression.The expressions of CatK and L were 2.5-fold lower inthe TGF-b1 only treated cells than in the control cells(absence of TGF-b1 and curcumin) (Figure 5D). Addi-tion of curcumin (20 μM) increased the expression ofCatK and L 7 and 5-fold, respectively.Taken together, this may indicate that curcumin-induced upregulation of cathepsins reduces the TGF-b1content in fibroblasts and lung tissue.Effect of curcumin on apoptosis and its dependency oncathepsin activitiesApoptosis has been associated with both physiologicallung homeostasis and pathological lung remodeling [27].Figure 1 Effect of curcumin on the expression of cathepsins K and L in mouse lungs of bleomycin induced fibrosis. Representativeimages (A) of immunohistochemical staining and quantification (B and C) of the expression of CatK and L in curcumin treated bleomycininduced mice show that expressions of CatK and L were increased at 7d, 14 d and 28 d. Real time PCR results (D) also show RNA levels of CatKwere increased after curcumin and bleomycin treatment at the indicated time points. For each time point 9-10 mice were evaluated. All datarepresent means ± SE (* p < 0.05, all compared to bleomycin treated group only; Con: control, BLM: bleomycin, Cur: curcumin).Zhang et al. Respiratory Research 2011, 12:154http://respiratory-research.com/content/12/1/154Page 5 of 12Tissue sections of mouse lungs revealed a 33-41%increase in Tunel positive (apoptotic) cells in curcumintreated mice when compared to control bleomycin-trea-ted littermates only (Figure 6A). This result suggeststhat apoptosis might contribute to the regeneration offibrotic lung tissues. In order to investigate the effect ofcathepsin expression on promoting fibroblast apoptosisas a response to curcumin administration, we deter-mined the expression of caspase-3 and Bax/Bcl-2 inbleomycin stimulated HFL-1 cells. Figures 6B and 6Cshow that caspase-3 expression and the ratio of Bax/Bcl-2 in HFL-1 cells were dose-dependently increased by 4.5and 2.5-fold after 24 h of 30 μM curcumin treatment.Subsequently, we examined whether the ability of cur-cumin inducing cell apoptosis was cathepsin dependent,LHVS was utilized to evaluate the alterations of caspase-3, Bax, and Bcl-2 in bleomycin treated HFL-1 cells.HFL-1 cells exposed to bleomycin were treated with 30μM curcumin in the presence or absence of LHVS.After 24 h, caspase-3 expression was investigated byimmunoblotting and immunofluorescence (data notshown). Figure 6D reveals that LHVS significantly inhib-ited caspase-3 expression (p < 0.05). Accordingly, theratio of Bax/Bcl-2 was reduced by LHVS as well (p <0.05). These data indicate that curcumin is proapoptoticand that the curcumin-dependent upregulation of cathe-psins is directly contributing to increased apoptosisrates.DiscussionCurcumin has been extensively studied as a potentialdrug for the treatment of lung fibrosis. Most of therecent research indicates that the mechanism of block-ing fibrosis by curcumin is related to decreasing col-lagen accumulation in the lung [1] and to anti-oxidant[2,28-30] and anti-inflammatory activities [24]. In vitro,curcumin has the ability of inducing scleroderma fibro-blast apoptosis [31], protecting rat lung epithelial cellsfrom quartz particle-mediated cytotoxic and inflamma-tory effects [30], inhibiting lung fibroblast proliferation[1], blocking the TGF-b1 signaling cascade [32], andupregulating heme oxygenase-1 [33] in renal cells. Nodata, however, have been reported to demonstrate a cur-cumin-mediated effect on cathepsin expression in bleo-mycin induced lung fibrosis as a pathway to controlextracellular matrix accumulation, cell proliferation, andapoptosis. Here, we show that curcumin increases CatKand L expression in vivo and in vitro and that theobserved effects on lung fibroblast cell proliferation,migration and apoptosis as well as the expression ofTGF-b1 are linked to cysteine cathepsin activities. Itshould be noted that we also observed an increase incathepsin B and S expression (albeit to a lesser degreethan cathepsins K and L) in lung tissues after curcumintreatment (data not shown).Bleomycin causes an increased expression of TGF-b1in activated fibroblasts as shown in Figure 5. IncreasedTGF-b1 levels promote ECM production and depositionby inducing fibroblast differentiation into myofibroblasts[34,35] and decreasing the expression of ECM degradingproteases such as CatK in these cells [11]. CatK is ahighly potent collagenase and elastase and its downregu-lation exerts a further increase in ECM accumulation infibrotic lungs as shown in CatK-deficient mice [8]. Onthe other hand, it has been reported that CatK isFigure 2 Overexpression of cathepsins K and L in curcumintreated bleomycin stimulated human lung fibroblasts (HFL-1cells). Representative images (A and B) of immunoblots andmeasurements show that expressions of CatK and L were dose-dependently increased by curcumin. All data represent means ofthree independent experiments ± SD and represent percentagecathepsin expression compared to b-actin as control (* p < 0.05, allcompared to bleomycin treated group only).Zhang et al. Respiratory Research 2011, 12:154http://respiratory-research.com/content/12/1/154Page 6 of 12overexpressed in human lung fibrosis and silica-inducedfibrosis which has been interpreted as a countermeasureto the increased ECM deposition [8,11]. In support ofthese findings, CatK overexpressing transgenic micerevealed a low degree of fibrosis in response to bleomy-cin challenge when compared with wild type mice [10].Here, we show that the antifibrotic activity of curcuminis accompanied by about 2-fold increases in CatK and Lexpression. An anti-fibrotic effect of cathepsin overex-pression can have multiple causes. First, increased tissuelevels of proteases such as CatK will increase ECMdegradation. This is corroborated by our finding thatincreased cathepsin expression is accompanied with adecrease in collagen deposits in fibrotic lungs of bleo-mycin-challenged mice and supported by several othergroups which reported a decrease in protein type Icollagen and hydroxyproline contents in curcumin trea-ted fibrotic lungs of rats [1-3].Second, increased cathepsin expression may directlycontrol TGF-b1 concentration in tissues by proteolysis.We have recently demonstrated that CatK deficiency inlung is associated with increased expression levels ofTGF-b1 and that CatK is a potent TGF-b1 degradingprotease [9]. Here we show that a cathepsin inhibitorincreased TGF-b1 concentrations in fibroblasts by 100%suggesting a direct regulation of TGF-b1 concentrationby cathepsin activities which will consequently affectproliferation and migration rates of lung cells. It hasbeen demonstrated that abnormal repair and deregu-lated wound healing partly result from abnormal fibro-blast proliferation and migration [11,36] and isstimulated by tissue factors such as collagen [37], PGDFFigure 3 Effects of curcumin on collagen deposition in fibrotic lungs of mice (bleomycin-induced) and HFL-1 cell proliferation andmigration. Representative images of Masson trichrome staining (A) and quantitative image analysis (B) of collagen content in the lungs of 9-10mice/group display a reduction of collagen by curcumin. MTT assay (C) shows curcumin dose-dependent decrease of proliferation of HFL-1 cellsin the presence of bleomycin (0.1 mU/ml) at 24, 48 and 72 h. Cell migration rates (D) of HFL-1 cells were also decreased by curcumin. All thecell-related data represent means of three independent experiments ± SD. The Masson trichrome staining evaluation is based on the imageanalysis of two tissue section per mouse and data represent means ± SE (* p < 0.05, all compared to bleomycin treated group only).Zhang et al. Respiratory Research 2011, 12:154http://respiratory-research.com/content/12/1/154Page 7 of 12[38], and TGF-b1 [39,40]. The degradation of collagenand TGF-b1 by CatK may thus provide stimulation sig-nals to slow down proliferation, migration, and woundhealing. Consequently, the administration of the cysteineprotease inhibitor, LHVS, significantly increased thewound closure rate in a fibroblast scratch assay (Figure4C) which was inhibited by the curcumin-mediatedoverexpression of cathepsins.Interestingly, the increase in cathepsin expression bycurcumin is greater under the condition of adding afixed amount of exogenous TGF-b1 to the culturemedia (Figure 5D) than in the presence of bleomycin(Figure 2A and 2B) which has a TGF stimulatory effect.TGF-b1 itself has a suppressing effect on cathepsinexpression (Figure 5D). This might be explained by thecurcumin-mediated expression of a potent TGF-b1degrading cysteine protease such as CatK [9] which islikely to deplete the amount of exogenously added TGF-b1. In the case of bleomycin stimulation, TGF-b1 willbe continuously produced by the HFL-1 cells and thusits anti-cathepsin expression effect remains stronger.The third effect of increased cathepsin expression islikely related to the apoptosis rates in fibrotic lungs.Wound healing and balancing the deposition of extra-cellular matrix largely depend on decreasing the resis-tance of fibroblasts to apoptosis [41]. Inadequatefibroblast and myofibroblast apoptosis may lead to theformation of fibrotic lesions [42]. Here, we demon-strated that curcumin increases apoptosis rates infibrotic lung tissues and HFL-1 cells. This was evi-denced by the dose-dependent increase of apoptoticmarkers such as of caspase-3 and the Bax/Bcl-2 ratio(Figure 6) and is in line with the finding that curcumindownregulates Bcl-2 in non-small cell lung cancer cells[43]. There are several potential pathways which maybe responsible for curcumin induced fibroblast apopto-sis. One pathway might be again related to TGF-b1.High levels of TGF-b1 have been shown to promotefibroblast resistance to apoptosis [44] whereas lowlevels of TGF-b1 may sensitize fibroblast to apoptosis.As discussed above the upregulation of cathepsinexpression by curcumin leads to an increased degrada-tion of TGF-b1 and thus would increase apoptosisrates. This is corroborated by the finding that curcu-min increased the expression of caspase-3 and Bax/Bcl-2 in our experiments. Furthermore, when LHVSwas employed as an antagonist to cysteine cathepsinsactivities in curcumin treated and bleomycin stimu-lated HFL-1 cells, the expression of proapoptotic mar-kers was reduced. The cleavage of the Bcl-2 familymember Bid by cathepsins has been proposed as a pos-sible mechanism of cathepsin-mediated apoptosis [45].The in vivo efficacy results were obtained after oralapplication of curcumin (200 mg/kg/d). Similar out-comes in terms of collagen deposition and/or TGFexpression were observed by other authors using oralapplications of curcumin in rats [2,3,46], whereas arecent study in mice reported efficacy only after i.p.administration of curcumin [1]. We looked into changesof ECM deposition based on Masson trichrome stainingand immunohistochemical analysis of cathepsin andTGF-b1 expression whereas Smith and coworker [1]studied the inflammatory cell content, airspace, andhydroxyproline content. The hydroxyproline content asa marker of collagen accumulation revealed a trend ofdecrease by approximately 10% though not significantafter 21 days of treatment in the Smith study [1]. Ourdata reflect a reduction in total collagen content by 18%after 14d and 32% after 28d. It is likely that an i.p.Figure 4 Effects of curcumin and a cathepsin inhibitor onwound healing in HFL-1 cell scratch model. Representativeimages of wound healing assay (A) and its quantification (B)demonstrate the dose-dependent reduction of wound closure ratesby curcumin. In contrast, LHVS, a pan-cathepsin inhibitorsignificantly increased wound closure rates (C). All data representmeans of three independent experiments ± SD (* p < 0.05, allcompared to bleomycin treated group only).Zhang et al. Respiratory Research 2011, 12:154http://respiratory-research.com/content/12/1/154Page 8 of 12Figure 5 Effects of curcumin on expressions of TGF-b1, and the effect of TGF-b1 on cathepsins K and L expression. Representativeimmunohistochemical images and image analysis (A) of TGF-b1 expression in bleomycin treated mice (9- 10 mice per group). Curcuminsignificantly decreased TGF-b1 expressions at 14d and 28d. All data besides the TGF-b1 analysis (means ± SE) represent means of threeindependent experiments ± SD (* p < 0.05, all compared to bleomycin treated group only). (B) Representative immunoblot images and theirquantitative analysis display the dose-dependent reduction of TGF-b1 protein content by curcumin in bleomycin treated HFL-1 cells. In contrast,LHVS increased TGF-b1 protein content two-fold (C). Panel D shows a 7 and 5-fold increase in the expressions of CatK and L in TGF-b1 treatedHFL-1 cells when exposed to curcumin (20 μM) whereas TGF-b1 alone significantly decreased cathepsin expression. All data represent means ofthree independent experiments ± SD (* p < 0.05, all compared to bleomycin or TGF-b1 treated group only).Zhang et al. Respiratory Research 2011, 12:154http://respiratory-research.com/content/12/1/154Page 9 of 12administration of curcumin would have resulted to moredramatic differences in our experiment as well.In summary, our data provide evidence that cathepsinexpression is increased in response to curcumin treat-ment in bleomycin challenged mice and HFL-1 cells.The elevated levels of cathepsins may directly promoteECM degradation, apoptosis and a decrease in TGF-b1expression which indirectly affects fibroblast prolifera-tion and thus ECM production, cell migration and apop-tosis. This study adds to the increasing evidence thatcurcumin may represent a potentially effective drug forthe treatment of human lung fibrosis.Figure 6 Effects of curcumin and a cathepsin inhibitor on the expression of apoptosis markers in bleomycin stimulated HFL-1 cells.Quantitative analysis of Tunel-positive staining in lungs of curcumin treated bleomycin-induced lung injury (A). Representative immunoblotimages and their quantitative analysis (B and C) show that curcumin increases caspase-3, Bax/Bcl-2 expression in a dose-dependent manner.LHVS, a pan-cathepsin inhibitor is able to reverse the proapoptotic effect of curcumin as seen by the reduction of caspase-3 expression and theBax/Bcl-2 ratio in HFL-1 cells (D). All data represent means of three independent experiments ± SD. (* p < 0.05, all compared to bleomycintreated group only).Zhang et al. Respiratory Research 2011, 12:154http://respiratory-research.com/content/12/1/154Page 10 of 12AcknowledgementsThis work was supported by the ICSD Canada grant and a Canada ResearchChair Award (D. B.) and a grant from the Ministry of Science and Technologyof the People’s Republic of China (2008DFA31970) (J.W.).Author details1Department of Oral and Biological Science, Faculty of Dentistry, Universityof British Columbia, Vancouver, BC, V6T 1Z3, Canada. 2Preclinical MedicineSchool, Beijing University of Chinese Medicine, Beijing, 100029, China.Authors’ contributionsDWZ designed and carried out experiments, analyzed data and wrote thepaper, CH and CY carried out all mouse experiments and analyzed the lungtissue samples, RJL performed some of the wound healing experiments, JWand JN conceived the mouse experiments and analyzed data, DB conceivedexperiments, analyzed data and wrote the paper. All authors read andapproved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Received: 31 August 2011 Accepted: 29 November 2011Published: 29 November 2011References1. 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Stoka V, Turk B, Schendel SL, Kim TH, Cirman T, Snipas SJ, Ellerby LM,Bredesen D, Freeze H, Abrahamson M, et al: Lysosomal protease pathwaysto apoptosis. Cleavage of bid, not pro-caspases, is the most likely route.J Biol Chem 2001, 276(5):3149-3157.46. Venkatesan N, Ebihara T, Roughley PJ, Ludwig MS: Alterations in large andsmall proteoglycans in bleomycin-induced pulmonary fibrosis in rats. AmJ Respir Crit Care Med 2000, 161(6):2066-2073.doi:10.1186/1465-9921-12-154Cite this article as: Zhang et al.: Antifibrotic effects of curcumin areassociated with overexpression of cathepsins K and L in bleomycintreated mice and human fibroblasts. Respiratory Research 2011 12:154.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitZhang et al. Respiratory Research 2011, 12:154http://respiratory-research.com/content/12/1/154Page 12 of 12


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