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ELQGLQJ GRPDLQ ).%' RI ).%3V LV FRQVHUYHG IURP DUFKDHD WR SULPDWHV .DQJHWDO Figure 3. PPIase activity. PPIases catalyze the cis/trans isomerization of proline residues. (Figure adapted from Lu, et al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igure 4. Examples of FKBP members of each subgroup. FKBP1 and FKBP1B belong to the cytoplasmatic FKBPs. FKBP3 and 15 are nuclear FKBPs. FKBP4, 5, 6, 8 contain a tetratricopeptide (TRP) domain and are therefore grouped as TRP-domain containing FKBPs. FKBP7, 9, 10, 11, 14 belong to the secretory pathway FKBPs. 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Staab-Weijnitz1, Isis E. Fernandez1, Larissa Knuppel ¨ 1, Julia Maul1, Katharina Heinzelmann1, Brenda M. Juan-Guardela2, Elisabeth Hennen1, Gerhard Preissler3, Hauke Winter3, Claus Neurohr4, Rudolf Hatz3,5, Michael Lindner5,J¨urgen Behr4,5, Naftali Kaminski2, and Oliver Eickelberg1 1Comprehensive Pneumology Center, Helmholtz Zentrum Munchen, ¨ Member of the German Center of Lung Research (DZL), Munich, Germany; 2Pulmonary, Critical Care and Sleep Medicine, Yale School of Medicine, New Haven, Connecticut; 3Thoraxchirurgisches Zentrum, Klinik fur ¨ Allgemeine, Viszeral, Transplantations, Gefaß- ¨ und Thoraxchirurgie, Klinikum Großhadern, Ludwig-Maximilians- Universitat, ¨ Munich, Germany; 4Medizinische Klinik und Poliklinik V, Klinikum der Ludwig-Maximilians-Universitat, ¨ Member of the German Center of Lung Research (DZL), Munich, Germany; and 5Asklepios Fachkliniken Munchen-Gauting, ¨ Munich, Germany Abstract transcriptase–polymerase chain reaction, Western blot, and quantification of secreted collagens in the lung and in primary human Rationale: Increased abundance and stiffness of the extracellular lung fibroblasts (phLF). Effects on collagen secretion were compared matrix, in particular collagens, is a hallmark of idiopathic pulmonary with those of the drugs nintedanib and pirfenidone, recently fibrosis (IPF). FK506-binding protein 10 (FKBP10) is a collagen approved for IPF. chaperone, mutations of which have been indicated in the reduction of extracellular matrix stiffness (e.g., in osteogenesis imperfecta). Measurements and Main Results: FKBP10 expression was up-regulated in bleomycin-induced lung fibrosis and IPF. Objectives: To assess the expression and function of FKBP10 in IPF. Immunofluorescent stainings demonstrated localization to 1 interstitial (myo)fibroblasts and CD68 macrophages. Transforming Methods: We assessed FKBP10 expression in bleomycin- growth factor-b , but not endoplasmic reticulum stress, induced induced lung fibrosis (using quantitative reverse 1 FKBP10 expression in phLF. The small interfering RNA–mediated transcriptase–polymerase chain reaction, Western blot, and knockdown of FKBP10 attenuated expression of profibrotic immunofluorescence), analyzed microarray data from 99 mediators and effectors, including collagens I and V and a-smooth patients with IPF and 43 control subjects from a U.S. cohort, muscle actin, on the transcript and protein level. Importantly, loss of and performed Western blot analysis from 6 patients with IPF FKBP10 expression significantly suppressed collagen secretion by and 5 control subjects from a German cohort. Subcellular phLF. localization of FKBP10 was assessed by immunofluorescent stainings. The expression and function of FKBP10, as well as its Conclusions: FKBP10 might be a novel drug target for IPF. regulation by endoplasmic reticulum stress or transforming b fi growth factor- 1, was analyzed by small interfering Keywords: FKBP65; peptidyl-prolyl isomerase; lung brosis; RNA–mediated loss-of-function experiments, quantitative reverse collagen cross-linking; extracellular matrix (Received in original form December 15, 2014; accepted in final form May 18, 2015 ) Supported by the Helmholtz Association, the German Center for Lung Research, and National Institutes of Health grants RO1HL108642 and RC2HL101715 (N.K.). Author Contributions: Conception and design: C.A.S.-W., I.E.F., K.H., J.B., N.K., and O.E. Experimental work, analysis, and interpretation: C.A.S.-W., I.E.F., L.K., J.M., K.H., B.M.J.-G., E.H., G.P., H.W., C.N., R.H., M.L., N.K., and O.E. Drafting the manuscript and intellectual content: C.A.S.-W., I.E.F., L.K., K.H., N.K., and O.E. Correspondence and requests for reprints should be addressed to Oliver Eickelberg, M.D., Comprehensive Pneumology Center, Ludwig-Maximilians-Universitat ¨ and Helmholtz Zentrum Munchen, ¨ Max-Lebsche-Platz 31, 81377 Munchen, ¨ Germany. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 192, Iss 4, pp 455–467, Aug 15, 2015 Copyright © 2015 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201412-2233OC on June 3, 2015 Internet address: www.atsjournals.org Staab-Weijnitz, Fernandez, Knuppel, ¨ et al.: FKBP10 in Lung Fibrosis 455 ORIGINAL ARTICLE such, the need to identify novel drug Taufkirchen, Germany) dissolved in sterile At a Glance Commentary targets for IPF remains imperative, as saline and applied using the MicroSprayer we require alternative treatment options Aerosolizer, Model IA-1C (Penn-Century, Scientific Knowledge on the for nonresponders, cell-type–specific Wyndmoor, PA). Control mice were Subject: Accumulation of targeted therapy, and combination instilled with 50 ml of saline. After extracellular matrix plays an important therapy options. instillation, mice were kept for 14, 28, and role in idiopathic pulmonary fibrosis The intracellular chaperone 56 days. Before death, mice were (IPF) disease progression. Deficiency of FK506-binding protein 10 (FKBP10, anesthetized with ketamine/xylazine the chaperone FK506-binding protein also termed FKBP65) has been reported followed by lung function measurement 10 (FKBP10) has been reported to to directly interact with collagen I (11). and tissue harvesting as previously attenuate collagen secretion and FKBP10 belongs to the FKBP subfamily described (20). Fibrosis was further assessed decrease extracellular collagen of immunophilins, molecular chaperones by bronchoalveolar lavage cell counts cross-linking in, for example, with peptidyl-prolyl isomerase activity, and histology evaluation. All animal osteogenesis imperfecta. which bind the immunosuppressive experiments were conducted under strict drug FK506 (tacrolimus) (12, 13). governmental and international guidelines What This Study Adds to the Collagen triple helix formation heavily and were approved by the local government Field: FKBP10 is specifically relies on proline isomerization to for the administrative region of Upper up-regulated in interstitial fibroblasts trans-proline as a prerequisite of linear Bavaria, Germany. in IPF. Inhibition of FKBP10 in primary chain assembly (14). Mutations in IPF fibroblasts attenuates gene FKBP10 lead to collagen-related disorders Gene Expression Data expression of various profibrotic genes such as osteogenesis imperfecta, and Data for FKBP10 in IPF lungs (n = 99) and and decreases collagen secretion. studies in the last 4 years have suggested normal histology control lungs (n = 43) was an association of these mutations with extracted from the gene expression attenuated collagen secretion and microarray data generated by us on lung Idiopathic pulmonary fibrosis (IPF) is diminished collagen I cross-linking in samples obtained from the National Lung, the most fatal interstitial lung disease, dermal fibroblasts and bone (15–18). A Heart, and Blood Institute–funded Tissue 2 2 with a 5-year survival rate of 30 to 50% recent study showed that Fkbp10 / mouse Resource Consortium, as described previously and few treatment options (1). The embryos are postnatally lethal and display (21, 22). Gene expression microarray data etiology of IPF is poorly understood. reduced collagen cross-linking in calvarial (Agilent Technologies, Santa Clara, CA), and The current concept involves repeated bone (19). associated clinical data are available on the alveolar injuries of unclear nature, Given that FKBP10 is a collagen- Lung Genomics Research Consortium providing signals for fibroblast activation, processing enzyme with potential impact website (https://www.lung-genomics.org/ proliferation, and differentiation to on ECM protein secretion and cross-linking, research/) as well as on accession number myofibroblasts (2–6). The latter overgrow we sought to assess its role in IPF. We GSE47460 or the Lung Tissue Research the delicate alveolar lung tissue and analyzed FKBP10 expression in lungs of Consortium website (http://www.ltrcpublic. secrete increased amounts of extracellular mice subjected to bleomycin and patients com). Significance was calculated using t matrix (ECM) proteins. According to with IPF. Loss-of-function studies were statistics, and multiple testing was controlled a recently published study, the ECM performed in primary human lung by the false discovery rate method at 5% (23). appears to play a major role not only fibroblasts (phLF) to investigate the effect in the composition but also in the of FKBP10 on ECM protein synthesis and Human Material maintenance of the fibrotic phenotype secretion. Resected human lung tissue and lung in IPF, contributing to the irreversibility explant material were obtained from the of the disease (7). Therefore, matrix Asklepios biobank for lung diseases at the and matrix-processing enzymes will Methods Comprehensive Pneumology Center. provide promising novel drug targets Biopsies were obtained from six patients for IPF. For instance, inhibition of the For more details on methods, see the online with IPF (usual interstitial pneumonia collagen cross-linking enzyme lysyl supplement. Statistical analysis was pattern, mean age 54 6 9 yr, five men, one oxidase-like 2 (LOXL2) is currently performed in GraphPadPrism 5 (GraphPad woman). All participants gave written investigated as an IPF treatment option Software, San Diego, CA). Results are given informed consent, and the study was in a phase II trial (8). as mean 6 SEM, and paired t test was used approved by the local ethics committee of Pirfenidone and nintedanib, two for statistical analysis, if not mentioned Ludwig-Maximilians University of Munich, drugs recently approved by the U.S. otherwise. Germany. Food and Drug Administration for IPF therapy, decelerate but do not attenuate Induction and Measurement of Isolation and Culture of phLF disease progression in patients with IPF, Murine Pulmonary Fibrosis For FKBP10 knockdown, phLF were by decreasing lung function decline. Pulmonary fibrosis was induced in female isolated from IPF biopsies (n = 4), the They also show considerable side effects, C57BL/6 mice (10–12 wk old) by a single adjacent normal region of a lung tumor and their mechanism of action is intratracheal instillation of 50 mlof resection (n = 2), and donor tissue (n = 3). incompletely understood (9, 10). As bleomycin (3 U/kg; Sigma Aldrich, For more details, see the online supplement. 456 American Journal of Respiratory and Critical Care Medicine Volume 192 Number 4 | August 15 2015 ORIGINAL ARTICLE Treatment of phLF with Transforming Dulbecco’s modified Eagle medium/F12 (R&D Systems, Minneapolis, MN), Growth Factor-b1, Nintedanib, or with 0.5% fetal bovine serum and 0.1 mM nintedanib, and pirfenidone (both Selleck, Pirfenidone 2-phospho-L-ascorbic acid, followed by Houston, TX) in starvation medium for the Cells were seeded at a density of 20,000 to treatment with the indicated concentrations indicated time points. For more details, see 2 b 25,000 cells/cm , starved for 24 hours in of transforming growth factor (TGF)- 1 the online supplement. A PBS Bleo d14 d28 d56 B D 0.06 O] ** 2 ** 0.04 HeLa PBS d14 Bleo d14 Fkbp10 0.02 Actb Compliance [ml/cmH 0.00 PBS d14 Bleo d14 PBS d28 Bleo d28 PBS d56 Bleo d56 C E –4 ** 8 *** *** –5 *** 6 –6 –7 4 –8 (Fkbp10/Actb) 2 Ct (Fkbp10-Gapdh) Ct Δ –9 relative band intensity – –10 0 PBS d14 Bleo d14 PBS d28 Bleo d28 PBS d56 Bleo d56 PBS d14 Bleo d14 Figure 1. FK506-binding protein 10 (FKBP10) is up-regulated in a mouse model of bleomycin-induced lung fibrosis. (A) Representative Masson trichrome staining demonstrating that increased collagen deposition peaked at Day 14 and was gradually lost afterward. Bottom row shows magnification of the boxed areas in the top row. Upper scale bar =1,000mm; lower scale bar =100mm. (B) Bleomycin instillation led to a significant decrease in lung compliance at Day 14, which was completely restored after 56 days. (C) FKBP10 gene expression was up-regulated at Day 14 after bleomycin instillation. (D) Representative Western blot showing that FKBP10 levels in total lung homogenate were significantly increased at Day 14 after bleomycin instillation; HeLa lysate was used as positive control. (E) Quantification by densitometric analysis demonstrating that FKBP10 levels returned to baseline at Day 28 and Day 56. Data shown are mean 6 SEM, and a two-tailed Mann-Whitney test was used for statistical analysis for comparison between bleomycin groups (Bleo) at different time points and phosphate-buffered saline (PBS) control vs. bleomycin for each time point. Actb = b-actin as loading control. Gapdh = glyceraldehyde phosphate dehydrogenase. **P , 0.01, ***P , 0.001. Staab-Weijnitz, Fernandez, Knuppel, ¨ et al.: FKBP10 in Lung Fibrosis 457 ORIGINAL ARTICLE Transfection of phLF performed in three different cell lines with (GAPDH) and hypoxanthine-guanine Cells were reverse transfected either with pGL3-CAGA(9)-luc (24) or pGL3 control phosphoribosyltransferase (HPRT) were used human FKBP10 small interfering RNA vector (Promega, Madison, WI). After as endogenous controls for standardization of (siRNA) (s34171; Life Technologies, incubation for 6 hours, cells were starved relative mRNA expression in mice and phLF, Carlsbad, CA) or negative control siRNA for 18 hours, followed by treatment with respectively. For more technical details on b No. 1 (Life Technologies). Twenty-four hours 2 ng/ml TGF- 1. Luminescence was RNA isolation and quantitative reverse after transfection, cells were starved for recorded in a TriStar LB 941 Multimode transcriptase polymerase chain reaction, see another 24 hours in Dulbecco’s modified Reader (Berthold Technologies, Bad the online supplement. Eagle medium/F-12 including 0.5% fetal Wildbad, Germany), and results were bovine serum and 0.1 mM 2-phospho-L- normalized to pGL3 control luciferase activity. Protein Isolation and Western Blot ascorbic acid, followed by treatment with For more details, see the online Analysis 2 ng/ml TGF-b1 in starvation medium. supplement. See online supplement. Twenty-four and 48 hours after beginning b the TGF- 1 treatment, cells and cell culture Real-Time Quantitative Reverse Quantification of Secreted Collagen supernatants were harvested for RNA and Transcriptase Polymerase Chain For quantification of total secreted collagen, protein analysis. In total, eight completely Reaction Analysis the Sircol assay was performed according to independent knockdowns were performed in Relative transcript abundance of a gene manufacturer’s instructions (Biocolor, fi target eight different human primary broblast is expressed as 2DCt values (DCt =Ct 2 Carrickfergus, UK). reference fi fi lines. Ct ) or as fold change derived from the For speci c quanti cation of secreted DD 2(DDCt) After 24 hours of siRNA transfection, relevant Ct values, using 2 .For collagen I, collagens were precipitated from forward transfection with the mothers specific gene amplification, primers listed in cell culture supernatant as follows: Proteins against decapentaplegic homolog (SMAD) Table E1 in the online supplement were used. were precipitated with 0.2 g/ml ammonium signaling luciferase reporter plasmid was Glyceraldehyde phosphate dehydrogenase sulfate and incubated on ice for 30 minutes, A Control IPF FKBP10 B *** C 5000 Control IPF 4000 FKBP10 3000 α-SMA 2000 ACTB Relative FKBP10 1000 transcript abundance 0 8 IPF D ** Control 6 -actin) β 4 2 (FKBP10/ relative band intensity 0 IPF Control Figure 2. FK506-binding protein 10 (FKBP10) is up-regulated in idiopathic pulmonary fibrosis (IPF). (A) Heat map of FKBP10 gene expression, extracted from microarray data of normal histology control (n = 43) and samples from patients with IPF (n = 99). Every column corresponds to a patient. Increases and decreases are denoted in increasing shades of yellow and purple, respectively, and gray is unchanged (false discovery rate , 5%). (B) Box-and- whisker plot for FKBP10 gene expression data. The difference is highly significant (***P , 1 3 1027). (C) Western blot analysis of total lung tissue homogenate showed up-regulation of FKBP10 in patients with IPF relative to donor samples in an independent cohort. (D) Densitometric analysis of the Western blot showed that FKBP10 up-regulation in IPF is highly significant. Data shown are mean 6 SEM, and a two-tailed Mann-Whitney test was used for statistical analysis (**P , 0.01). a-SMA = a-smooth muscle actin; ACTB = b-actin as loading control. 458 American Journal of Respiratory and Critical Care Medicine Volume 192 Number 4 | August 15 2015 ORIGINAL ARTICLE followed by centrifugation at 20,000 3 g at Day 14 after instillation of bleomycin appeared to correlate with levels of at 48C for 30 minutes. The pellet was (Figure 1D). After resolution of fibrosis, a-smooth muscle actin (a-SMA), a marker dissolved in 0.1 M acetic acid containing at Day 56, FKBP10 expression had returned of myofibroblasts (Figure 2C). 0.1 mg/ml pepsin (Thermo Fisher to baseline levels (cf. Figures 1A, 1B, Scientific, Waltham, MA) and incubated and 1E). With a fold change of 3.6, FKBP10 Is Expressed in Interstitial overnight at 48C. Then, 5 M NaCl was up-regulation of FKBP10 at Day 14 Fibroblasts, Including Myofibroblasts added to yield a final concentration of occurred in part on the transcriptional Immunofluorescent stainings of lung 0.7 M, followed by incubation on ice level (Figure 1C). tissue sections from the bleomycin-treated for 30 minutes and centrifugation at Next, we studied FKBP10 gene mice and patients with IPF confirmed 20,000 3 g at 48C for 30 minutes. The expression in IPF. Analysis of microarray increased expression of FKBP10 in pellet containing collagen I was data of 99 IPF samples and 43 normal fibrotic lungs, with little staining of FKBP10 resuspended in 0.1 M acetic acid and histology control samples revealed in phosphate-buffered saline–instilled analyzed by immunoblotting. significant up-regulation of FKBP10 (fold control mice or donor lungs. FKBP10 change, 1.7; false discovery rate , 5%; expression was predominantly localized 27 Immunofluorescent and Masson P , 1 3 10 ) (Figures 2A and 2B). Up- to interstitial fibroblasts, as evidenced by Trichrome Stainings regulation of FKBP10 was confirmed on the colocalization with a-SMA (Figures 3 Cultured phLF were seeded on coverslips, protein level using lung homogenates from and 4A) or desmin (Figure 4A), both and immunostaining was performed as patients with IPF of an independent markers of myofibroblasts. In addition, described previously (25). For staining of cohort: FKBP10 was highly increased in a fraction of FKBP10-expressing cells comparison with donor samples (Figures were found to be interstitial macrophages tissue sections, human and murine lung 1 tissue was fixed in 10% formalin before 2C and 2D). Moreover, FKBP10 levels (evidenced by CD68 staining; Figure 4B). paraffin embedding. Three-micron sections were prepared and mounted on slides, followed by deparaffinization and PBS Bleo immunofluorescent staining or Masson 100 μm FKBP10 α-SMA trichrome staining according to a standard DAPI protocol. For more details, see the online supplement. Subcellular Fractionation Protein fractionation was performed with a Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Fisher Scientific) according to the manufacturer’s FKBP10 instructions. Purity of the fractions was T1α DAPI assessed using the following marker proteins for Western blot analysis of the obtained fractions: calreticulin and protein disulfide isomerase 3 (PDIA3) for the membrane extract, glyceraldehyde phosphate dehydrogenase (GAPDH) for the cytosolic extract, and lamin A/C for the nuclear and chromatin-bound extract. FKBP10 TTF1 DAPI Results FKBP10 Expression Is Increased in Lung Fibrosis We first studied FKBP10 expression in the mouse model of bleomycin-induced lung fibrosis. Bleomycin instillation led to increased ECM deposition, as demonstrated Figure 3. FK506-binding protein 10 (FKBP10) is expressed in interstitial fibroblasts, including myofibroblasts, in a mouse model of bleomycin-induced lung fibrosis. Immunofluorescent stainings of by Masson trichrome staining, and fi paraffin sections from control (phosphate-buffered saline [PBS], left panels) and bleomycin-treated a signi cant decrease in lung compliance (Bleo, right panels) mice at Day 14 after bleomycin instillation. Representative Fkbp10 immunostaining at Day 14, which was completely restored is shown in red, a-smooth muscle actin (a-SMA), podoplanin (T1a), or thyroid transcription factor 1 after 56 days (Figures 1A and 1B). Western (TTF1) in green, and 49,6-diamidino-2-phenylindole (DAPI) in blue, as indicated on the right side. blot analysis showed a clear increase of White squares in the Bleo stainings are shown enlarged to the right. Sections shown are FKBP10 protein levels in total lung lysates representative stainings from three PBS- and three bleomycin-treated mice. Staab-Weijnitz, Fernandez, Knuppel, ¨ et al.: FKBP10 in Lung Fibrosis 459 ORIGINAL ARTICLE A Donor IPF In contrast, FKBP10 did not colocalize with 100 μm FKBP10 a α-SMA T1 (AT1 cells) or TTF1 (AT2 cells) DAPI (Figures 3 and 4A). FKBP10 Is an Endoplasmic Reticulum Resident Protein but Not Up-regulated by ER Stress in Primary Human Lung Fibroblasts In immunofluorescent stainings of phLF, FKBP10 localized mainly to the cytoplasm FKBP10 Desmin and showed colocalization with PDIA3, an DAPI endoplasmic reticulum (ER)-resident protein (Figure 5A). In some cells, positive FKBP10 staining was also observed in the nucleus. In contrast, costaining with a marker for the Golgi apparatus, Golgin subfamily A member 1 (GOLGA1) showed no colocalization (Figure 5B). Subcellular FKBP10 fractionation of phLF resulted in a clear T1α DAPI enrichment of FKBP10 in the ER/ membrane extract (Figure 5C). Additionally, FKBP10 was also weakly detected in the cytosolic and the nuclear extract. Although tunicamycin and thapsigargin up-regulated the ER stress markers BiP, ATF4, and CHOP, FKBP10 FKBP10 levels were drastically decreased TTF1 DAPI by both (see Figure E1 in the online supplement). FKBP10 usually migrated at approximately 70 kD, but in the presence of tunicamycin an additional FKBP10 band appeared at approximately 65 kD, in agreement with the theoretical size of the unmodified protein (cf. Figure E1A). FKBP10 TGF-b Up-regulates FKBP10 B 50 μm 1 CD68 Expression in phLF DAPI b TGF- 1 induced FKBP10 expression in a dose-dependent manner, with a clearly visible up-regulation for concentrations of 1 ng/ml and higher after 48 hours of fi b treatment (Figure 6A). Ef cacy of TGF- 1 treatment was confirmed by Western blot analysis of phosphorylated and total SMAD3 (Figure 6A). From these initial experiments, an effective concentration of 2 ng/ml TGF-b1 was derived and used in Figure 4. FK506-binding protein 10 (FKBP10) is expressed in interstitial fibroblasts, the following FKBP10 knockdown including myofibroblasts, and interstitial CD681 macrophages in human idiopathic pulmonary b experiments, where the TGF- 1 effect on fibrosis (IPF). Representative immunofluorescent stainings of paraffin sections from donor FKBP10 expression was also quantified (left panels) and IPF tissue (right panels). Representative FKBP10 immunostaining is always shown in red 9 (Figure 6B). Results in Figure 6B are based and 4 ,6-diamidino-2-phenylindole (DAPI) in blue.(A) Representative immunofluorescent costainings with on results with control siRNA from the a-smooth muscle actin (a-SMA), desmin, podoplanin (T1a), and thyroid transcription factor 1 (TTF1) in green show FKBP10 expression in interstitial fibroblasts but not in alveolar epithelial cells. loss-of-function experiments (cf. Figure 6C White squares in the IPF stainings are shown enlarged to the right. Scale bar =100 mm. (B) for a representative Western blot). Representative immunofluorescent costainings with CD68 show FKBP10 expression in interstitial Induction of FKBP10 expression by b macrophages (indicated by white arrows). Sections shown are representative stainings from three TGF- 1 was already observed at transcript donors and three patients with IPF. Scale bar =50 mm. level (Figure E2). 460 American Journal of Respiratory and Critical Care Medicine Volume 192 Number 4 | August 15 2015 ORIGINAL ARTICLE Loss of FKBP10 in IPF Fibroblasts this effect for collagen I, but not for (Figure 7A). However, the corresponding Attenuates ECM Protein Synthesis collagen V or fibronectin (Figure 6D). TGF-b–induced transcript fold changes and Decreases Levels of Secreted Moreover, levels of the myofibroblast did not indicate attenuation of TGF-b Collagen Independent of TGF-b marker a-SMA were also significantly signaling in the FKBP10 knockdown Signaling decreased by knockdown of FKBP10 in the (Figure 7B), and neither a Smad signaling The siRNA-mediated knockdown of b luciferase reporter assay (Figure 7C) nor presence of TGF- 1 (Figure 6D, bottom FKBP10 in IPF fibroblasts showed right panel). The effects observed for analysis of SMAD 3 phosphorylation or significant down-regulation of collagen I, collagen I and V manifested at transcript protein levels (Figure 7D and Figure E3) collagen V, and fibronectin protein levels level already and also transcription of the showed an appreciable effect of FKBP10 knockdown on canonical TGF-b signaling. in crude cell lysates after 48 hours of TGF-b target gene PAI1 were decreased by b Finally, we assessed levels of secreted TGF- 1 treatment (Figures 6C and 6D). knockdown of FKBP10, suggestive of an b b collagen in these experiments by two Interestingly, TGF- 1 partly counteracted effect on overall TGF- signaling approaches. First, specifically collagen type I levels were semiquantified using Western blot analysis followed by densitometry A (Figures 8A and 8B); second, total secreted collagen was quantified using the Sircol assay (Figure 8C). Both approaches showed significant decreases of secreted collagen in the FKBP10 knockdown in absence and b presence of TGF- 1. In our efforts to interpret the relevance of FKBP10 knockdown in IPF fibroblasts in the context of currently approved IPF therapeutics, we used the same experimental setup to address inhibition of total collagen secretion in IPF fibroblasts by nintedanib and pirfenidone. Importantly, FKBP10 knockdown significantly decreased total collagen secretion by about 20%, in an amount similar to the effects observed with B C CE ME NE CB nintedanib, whereas we did not observe any effect of pirfenidone on total collagen FKBP10 secretion (Figure 9). CALR Discussion PDIA3 In the present study, we show that FKBP10 is up-regulated in bleomycin-induced lung fi Lamin A/C brosis and IPF. FKBP10 is predominantly expressed in interstitial (myo)fibroblasts 1 b and CD68 macrophages. TGF- 1, but not GAPDH 20 μm ER stress, induced FKBP10 expression in phLF. Loss of FKBP10 in phLF led to Figure 5. FK506-binding protein 10 (FKBP10) is mainly an endoplasmic reticulum (ER)-resident attenuation of collagen synthesis and protein and does not localize to the Golgi apparatus. (A) Immunostaining of FKBP10 and the secretion and reduced the expression of ER-marker protein disulfide isomerase A3 (PDIA3) is shown in red and green, respectively. 49,6- a-SMA and PAI1 independent of the diamidino-2-phenylindole (DAPI) staining is shown in blue. The white square was chosen as the TGF-b signaling pathway. Finally, FKBP10 region of interest and enlarged to the right. Arrows in the region of interest depict examples for knockdown inhibited collagen secretion with colocalization of FKBP10 and PDIA3. While the staining shown to the left is an extended-focus picture an efficiency similar to that of nintedanib. generated from a z-stack, the enlarged figure to the right shows one focal plane from the z-stack only. FKBP10 was up-regulated in the mouse (B) Immunostaining of FKBP10 and the Golgi-marker protein Golgin subfamily A member 1 is shown model of bleomycin-induced lung fibrosis in red and green, respectively. DAPI staining is shown in blue. The presented staining is an and returned to baseline with resolution of extended-focus picture generated from a z-stack. (C) Western blot analysis of subcellular fractionation of primary human lung fibroblasts. FKBP10 is enriched in the membrane extract the disease (Figure 1). In agreement, (ME), similar to the ER-resident proteins calreticulin (CALR) and PDIA3. FKBP10 was additionally a previous study has indicated found in the cytosolic extract (CE) and the nuclear extract (NE) but not in the chromatin-bound up-regulation of FKBP10 mRNA during fraction (CB). Lamin A/C and glyceraldehyde phosphate dehydrogenase (GAPDH) were used as bleomycin-induced lung injury (26). In this marker proteins for NE/CB and CE, respectively. study, however, analysis was restricted to Staab-Weijnitz, Fernandez, Knuppel, ¨ et al.: FKBP10 in Lung Fibrosis 461 ORIGINAL ARTICLE A B * 24h 48h ** ctrl siRNA 2.5 FKBP10 FKBP10 siRNA TGF-β TGF-β ctrl ctrl 2.0 -actin) β 1.5 FKBP10 1.0 pSMAD3 0.5 SMAD3 (FKBP10/ relative band intensity 0.0 β ACTB TGF- – – + + – – + + FKBP10 siRNA – + – + – + – + 24h 48h C 24h 48h D 8 Collagen I * 10 Collagen V ** β 6 8 TGF- – – + + – – + + * -actin) 6 FKBP10 siRNA – + – + – + – + -actin) β β 4 * 4 * FKBP10 2 (Coll I/ 2 (Coll V/ relative band intensity Collagen I 0 relative band intensity 0 8 * 150 Collagen V Fibronectin α-SMA * 6 100 Fibronectin -actin) β -actin) β 4 α-SMA 50 2 -SMA/ (FN1/ α ( ACTB relative band intensity relative band intensity 0 0 TGF-β – – + + – – + + TGF-β – – + + – – + + FKBP10 siRNA – + – + – + – + FKBP10 siRNA – + – + – + – + 24h 48h 24h 48h Figure 6. FK506-binding protein 10 (FKBP10) is up-regulated by transforming growth factor (TGF)-b1 in primary human lung fibroblasts (phLF), and knockdown of FKBP10 attenuates synthesis of collagen I, collagen V, and a-smooth muscle actin (a-SMA). (A) Western blot analysis of phLF treated with increasing concentrations of TGF-b1 (0.1, 0.2, 1.0, 2.0, and 5.0 ng/ml) shows up-regulation of FKBP10 at 48 hours in the presence of 1.0 ng/ml and higher TGF-b1 concentrations. Efficacy of TGF-b1 treatment was confirmed by monitoring phosphorylation of mothers against decapentaplegic homolog 3 (SMAD3, depicted as pSMAD3) in comparison with total SMAD3 levels (SMAD3). (B) 2.0 ng/ml TGF-b1 significantly increased FKBP10 in phLF after 48 h, as quantified by densitometric analysis from Western blot analysis (cf. Figure 7C for representative Western blot, lanes with scrambled [2] small interfering RNA [siRNA] only). The effect of the knockdown was always significant (P , 0.01 or P , 0.001) but is not specified in the interest of clarity. (C) FKBP10 knockdown effects in phLF in combination with 24 and 48 hours of TGF-b1 treatment (2.0 ng/ml). A representative Western blot analysis for detection of FKBP10, collagen I, collagen V, fibronectin, and a-SMA is shown. (D) Densitometric quantification of collagen I, collagen V, fibronectin, and a-SMA protein levels. b-actin (ACTB) was used as loading control. Scrambled siRNA was used as control. Data are based on eight completely independent experiments and are given as mean 6 SEM. Statistical analysis for comparison of FKBP10 siRNA vs. scrambled siRNA control was performed by paired two-tailed t test. *P , 0.05, **P , 0.01. The well-known effect of TGF-b1 on these proteins was mostly significant but is not specified in the interest of clarity. ctrl = control. Day 10 after bleomycin administration, and this in two completely independent cohorts, in the mouse model as well as in the IPF up-regulation was merely assessed at one in the United States and one in Europe, sections, were FKBP10-negative, transcript level. We clarify that FKBP10 strongly arguing for an important function highlighting the phenotypic diversity of overexpression occurs on the protein level of FKBP10 in IPF. Immunofluorescent interstitial mesenchymal cell types in and correlated with the fibrotic phase and stainings of fibrotic mouse and IPF tissue fibrosis. lung function, both aspects of importance sections revealed strong FKBP10 expression The subcellular localization provided for the translation of these results to IPF, by interstitial fibroblasts, including an important clue for FKBP10 function in a state of presumably irreversible fibrosis. myofibroblasts, the major ECM-producing fibroblasts, as FKBP10 substrates proposed Most importantly, we show for the first cell type in fibrotic lung disease (Figures 3 in the literature include not only ECM time that FKBP10 expression is increased and 4), indicating that FKBP10 might have protein precursors, such as procollagen I or in IPF (Figure 2). This appears to be a function in ECM protein synthesis in tropoelastin (11, 27), but also cytosolic a universal phenomenon, as we discovered phLF. Notably, many myofibroblasts, both proteins, such as HSP90 and c-Raf-1 (28). 462 American Journal of Respiratory and Critical Care Medicine Volume 192 Number 4 | August 15 2015 ORIGINAL ARTICLE A ctrl siRNA B 25 FKBP10 siRNA 24h ctrl siRNA 20 24h FKBP10 siRNA 48h ctrl siRNA 14 COL1A1 ** 15 * 48h FKBP10 siRNA ** 12 *** *** 10 10 5 8 Transcript Fold Change 0 (COL1A1-HPRT) t C 6 Δ PAI1 – COL1A1 COL5A1 8 COL5A1 * 6 *** C 0.5 ctrl siRNA 4 * 0.4 FKBP10 siRNA 2 0.3 (COL5A1-HPRT) t 0 C 0.2 Δ – –2 0.1 0.0 10 *** relative luciferase activity PAI1 ** TGF-β – – + + – – + + 8 ** FKBP10 siRNA – + – + – + – + 6 * 24h 48h (PAI1-HPRT) t 4 C Δ – D 2 P-SMAD3 TGF-β – – + + – – + + – + – + – + – + FKBP10 siRNA tSMAD3 24h 48h ACTB TGF-β – – + + – – + + FKBP10 siRNA – + – + – + – + 2h 24h Figure 7. Transforming growth factor (TGF)-b1 induces FK506-binding protein 10 (FKBP10) expression, and knockdown of FKBP10 in primary human lung fibroblasts (phLF) significantly decreases expression of collagen a-1(I) chain (COL1A1), collagen a-1(V) chain (COL5A1), and plasminogen activator inhibitor 1 (PAI1) on the transcriptional level independent of the canonical TGF-b signaling pathway. (A) Quantitative reverse transcriptase–polymerase chain reaction analysis of transcript levels of COL1A1 and COL5A1 (encoding the a1-chain of collagen I and V, respectively) and PAI1 as a representative TGF-b1–transduced gene after FKBP10 knockdown in combination with 24 and 48 hours of TGF-b1 treatment (2.0 ng/ml). Data are depicted as mean 6 SEM from eight independent experiments, and a paired two-tailed t test was used for statistical analysis for comparison of FKBP10 small interfering RNA (siRNA) vs. scrambled siRNA control. The well-known effect of TGF-b1 on these transcripts was always highly significant (P , 0.01 or P , 0.001) but is not specified in the interest of clarity. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as endogenous control. (B)TGF-b–induced transcript fold changes obtained from the same data do not show attenuation of TGF-b signaling. (C) Transfection of phLF with a mothers against decapentaplegic homolog (SMAD) signaling luciferase reporter plasmid during FKBP10 knockdown showed no effect on canonical TGF-b signaling (n = 3). (D) Western blot analysis (n = 3) demonstrated that FKBP10 knockdown did not affect levels of total and phosphorylated SMAD3. b-actin (ACTB) was used as loading control. For quantification of these data, see Figure E3 in the online supplement. *P , 0.05, **P , 0.01, ***P , 0.001. ctrl = control. Here, we demonstrate that FKBP10 associated with transport processes across previous studies in chondroblasts and localized mainly to the ER in phLF the trans-Golgi network to the extracellular embryonic fibroblasts (19, 27). (Figure 5). No colocalization was observed space. This suggests that FKBP10 mainly As there is evidence that ER stress with the Golgi-specific protein GOLGA1 functions as an ER-resident chaperone contributes to IPF pathology (30, 31), we (29), indicating that FKBP10 is not and/or foldase in phLF, which agrees with assessed whether FKBP10 is induced by ER Staab-Weijnitz, Fernandez, Knuppel, ¨ et al.: FKBP10 in Lung Fibrosis 463 ORIGINAL ARTICLE A and implies that adult IPF phLF have secreted retained the capacity to up-regulate fi collagen I FKBP10 under brotic conditions, supporting the use of this cell culture system for the functional analysis of β TGF- – – + + – – + + FKBP10 in vitro. In agreement with FKBP10 siRNA – + – + – + – + a collagen chaperone function, inhibition of FKBP10 by an siRNA-mediated 24h 48h approach in this model resulted in 4 a consistent decrease of collagen I and B ctrl siRNA * collagen V protein levels (cf. Figure 6C and FKBP10 siRNA 3 6D) as well as in decreased secreted * collagen (Figure 8). In line with our results, deficiency of FKBP10 in osteogenesis 2 imperfecta has been shown to attenuate collagen secretion and, moreover, band intensity 1 decrease the extent of lysyl hydroxylation (secreted collagen I) and extracellular collagen cross-linking and 0 increase protease sensitivity of extracellular collagen I in dermal fibroblasts from patients C 25 ctrl siRNA * with osteogenesis imperfecta (15, 17, 18). 20 FKBP10 siRNA Surprisingly, loss of FKBP10 affected collagen levels not only on protein but also on 15 transcript levels (Figure 7A). This is ** — g/ml] unexpected, as FKBP10 as a chaperone and μ [ 10 peptidyl-prolyl isomerase—is mainly ascribed a post-transcriptional role in procollagen I 5 processing (11, 14, 15, 17, 18). Interestingly, total secreted collagen 0 loss of FKBP10 also attenuated the expression b– β of the TGF- responsive genes PAI1 and TGF- – – + + – – + + a-SMA (Figures 6D and 7A). However, this FKBP10 siRNA – + – + – + – + was not due to inhibition of the canonical b – 24h 48h TGF- signaling pathway (Figure 7B 7D). Hence, it appears that FKBP10 exerts Figure 8. Knockdown of FK506-binding protein 10 (FKBP10) attenuates collagen secretion. (A) a previously unappreciated function in signal Western blot analysis of secreted collagen I. Collagen I was precipitated from cell culture supernatant after FKBP10 knockdown in combination with 24 and 48 hours of transforming growth factor transduction processes for the transcriptional regulation of profibrotic genes. Nevertheless, (TGF)-b1 treatment (2.0 ng/ml) and analyzed by Western blot analysis. (B) Densitometric analysis of secreted collagen I as detected in A. Data shown are based on seven independent experiments and the data also show an increase in TGF- given as mean 6 SEM. (C) Results of a Sircol assay for assessment of total secreted collagen in cell b–induced collagen expression and secretion, culture supernatant after FKBP10 knockdown in combination with 24 and 48 hours of TGF-b1 even in the context of FKBP10 knockdown, treatment. Data shown are based on eight independent experiments and given as mean 6 SEM. indicating that alternative, FKBP10- Statistical analysis was performed using paired two-tailed t test for comparison of FKBP10 siRNA vs. independent, profibrotic mechanisms scrambled siRNA control. ctrl = control; siRNA = small interfering RNA. *P , 0.05, **P , 0.01. contribute to overall ECM deposition. Importantly, the effects of FKBP10 knockdown on TGF-b–induced collagen stress as a possible explanation for its ER stress (34). As the observed decrease of secretion in phLF were comparable to increased expression in IPF. Interestingly, FKBP10 might reflect deglycosylation of the those of submicromolar concentrations of we observed a drastic decrease in FKBP10 protein rather than an overall decrease in nintedanib (Figures 9A and 9B). In levels (band at 70 kD; cf. Figure E1A) in expression levels, a mechanistically contrast, we did not observe any effect of response to tunicamycin, an inhibitor of independent ER stress inducer, thapsigargin, pirfenidone in this context. Previous N-protein glycosylation (32). This decrease was used to confirm FKBP10 down- studies in human lung fibroblasts and A549 was accompanied by the appearance of regulation in ER stress (Figure E1B). Hence, it cells have shown moderate reduction of a lower band at the height of the predicted is unlikely that the unfolded protein response collagen I expression only at concentrations size of the unmodified protein (65 kD; cf. contributes to FKBP10 up-regulation in IPF. higher than those used in this study (1.0 Figure E1A), in line with the previous In contrast, TGF-b1 significantly mM vs. 1.6 and 2.7 mM pirfenidone, identificationofFKBP10asanN-glycosylated induced FKBP10 expression in phLF on the respectively [35, 36]). The effect of protein (33). This is in agreement with mRNA and protein level (cf. Figure E2, pirfenidone on TGF-b–induced collagen a previous study showing that FKBP10 is Figure 6B). This effect has previously only secretion in vitro has, to our knowledge, degraded by the proteasome in response to been observed in fetal lung fibroblasts (26) only been studied in trabecular meshwork 464 American Journal of Respiratory and Critical Care Medicine Volume 192 Number 4 | August 15 2015 ORIGINAL ARTICLE A * in underlying mechanisms or involve 100 ctrl siRNA metabolic activation. FKBP10 siRNA The ability to bind the 80 immunosuppressive drug FK506 (tacrolimus) via at least one peptidyl-prolyl 60 isomerase domain is characteristic for all (% of ctrl) 40 FKBPs, including FKBP10 (11, 39). Interestingly, immunosuppressive 20 peptidyl-prolyl isomerase inhibitors like total secreted collagen 0 FK506 have been ascribed potent antifibrotic effects in lung fibrosis – B ** (40 43). FK506 suppresses collagen synthesis and expression of the TGF-b ** 1 Nintedanib receptor in dermal and lung fibroblasts, 100 ctrl but the underlying mechanisms are 80 μ largely unclear (41, 44, 45). Our 0.01 M fi 0.1 μM ndings put forward the possibility that 60 inhibition of FKBP10 might contribute 1 μM * to the antifibrotic effects of FK506. (% of ctrl) 40 In conclusion, we show that 20 FKBP10 is overexpressed in bleomycin- total secreted collagen induced lung fibrosis and IPF. Up- 0 regulation of FKBP10 expression is at least b partly mediated by TGF- 1.Lossof C Pirfenidone FKBP10 in phLF derived from patients 140 with IPF or control patients attenuated ctrl ECM protein expression and secretion 120 100 μM and suppressed fibroblast-to- 100 500 μM myofibroblast differentiation/activation. 80 1000 μM Although FKBP10 has been suggested to 60 be important for normal lung (% of ctrl) development, with little expression in 40 normal adult tissues, its expression 20 total secreted collagen appears to be reactivated during lung 0 fibrosis (26, 27). Considering the impact – TGF-β + TGF-β of FKBP10 on collagen cross-linking (15, 17, 18) and the effects described herein Figure 9. In idiopathic pulmonary fibrosis fibroblasts, FK506-binding protein 10 (FKBP10) knockdown on collagen secretion, FKBP10 might inhibits collagen secretion with similar efficiency as nintedanib, whereas pirfenidone shows no effect. (A–C) provide a very specificandeffectivedrug Normalized levels of secreted collagen in response to (A)FKBP10knockdown,(B) nintedanib, and (C) n pirfenidone treatment at varying concentrations. In contrast to pirfenidone, nintedanib shows a dose- target for treatment of IPF. dependent effect. In comparison, FKBP10 knockdown performs with similar efficiency as submicromolar concentrations of nintedanib. For FKBP10 knockdown, scrambled small interfering RNA (siRNA) was Author disclosures are available with the text 6 used as control. Data shown are based on four independent experiments and given as mean SEM. of this article at www.atsjournals.org. Statistical analysis was performed using paired two-tailed t test. *P , 0.05, **P , 0.01. ctrl = control; TGF-b = transforming growth factor-b. Acknowledgment: The authors thank Daniela Dietel for excellent technical assistance, Mona cells, where collagen secretion was hardly physiologically relevant, indicating Dotzler for initial contributions to this project, and significantly reduced at a concentration of that the well-known antifibrotic effects of Melanie Konigshoff ¨ and Kathrin Mutze for 2.5 mM (37). These concentrations are pirfenidone in vivo (38) differ substantially valuable help and discussion. References 3. Renzoni E, Srihari V, Sestini P. Pathogenesis of idiopathic pulmonary fibrosis: review of recent findings. F1000Prime Rep 2014;6:69. 1. Kim DS, Collard HR, King TE Jr. Classification and natural history of the 4. Wolters PJ, Collard HR, Jones KD. Pathogenesis of idiopathic idiopathic interstitial pneumonias. Proc Am Thorac Soc 2006;3:285–292. pulmonary fibrosis. 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Respiratory Research (2018) 19:67 https://doi.org/10.1186/s12931-018-0768-1 RESEARCH Open Access FK506-binding protein 10 (FKBP10) regulates lung fibroblast migration via collagen VI synthesis Larissa Knüppel1,2, Katharina Heinzelmann1,2, Michael Lindner3, Rudolf Hatz3,4, Jürgen Behr3,5, Oliver Eickelberg1,2,6 and Claudia A. Staab-Weijnitz1,2* Abstract Background: In idiopathic pulmonary fibrosis (IPF), fibroblasts gain a more migratory phenotype and excessively secrete extracellular matrix (ECM), ultimately leading to alveolar scarring and progressive dyspnea. Here, we analyzed the effects of deficiency of FK506-binding protein 10 (FKBP10), a potential IPF drug target, on primary human lung fibroblast (phLF) adhesion and migration. Methods: Using siRNA, FKBP10 expression was inhibited in phLF in absence or presence of 2ng/ml transforming growth factor-β1 (TGF-β1) and 0.1mM 2-phosphoascorbate. Effects on cell adhesion and migration were monitored by an immunofluorescence (IF)-based attachment assay, a conventional scratch assay, and single cell tracking by time-lapse microscopy. Effects on expression of key players in adhesion dynamics and migration were analyzed by qPCR and Western Blot. Colocalization was evaluated by IF microscopy and by proximity ligation assays. Results: FKBP10 knockdown significantly attenuated adhesion and migration of phLF. Expression of collagen VI was decreased, while expression of key components of the focal adhesion complex was mostly upregulated. The effects on migration were 2-phosphoascorbate-dependent, suggesting collagen synthesis as the underlying mechanism. FKBP10 colocalized with collagen VI and coating culture dishes with collagen VI, and to a lesser extent with collagen I, abolished the effect of FKBP10 deficiency on migration. Conclusions: These findings show, to our knowledge for the first time, that FKBP10 interacts with collagen VI and that deficiency of FKBP10 reduces phLF migration mainly by downregulation of collagen VI synthesis. The results strengthen FKBP10 as an important intracellular regulator of ECM remodeling and support the concept of FKBP10 as drug target in IPF. Keywords: FKBP10, FKBP65, migration, focal adhesion, collagen VI, lung fibrosis, fibroblast, fibulin Background transforming growth factor β1(TGF-β1), followed by myo- Patients suffering from idiopathic pulmonary fibrosis (IPF), fibroblast differentiation, increased fibroblast migration, ahighlyprogressiveinterstitiallungdisease,haveamedian and, ultimately, excessive deposition of extracellular matrix survival prognosis of 2-5 years after diagnosis [1]. The (ECM) in the alveolar region [3–6]. More recent evidence pathogenic processes are not completely understood. It is suggests that the composition of the ECM strongly affects currently believed that the fibrotic response is caused by re- fibroblast phenotypes and therefore plays a crucial role in peated micro-injuries of the respiratory epithelium [2] disease progression [4, 7, 8]. which leads to the release of profibrotic mediators like Aberrant fibroblast adhesion and migration are com- mon features of fibrosis [9–11] and targeting fibroblast migration, e.g. by inhibition of focal adhesion kinase * Correspondence: [email protected] (FAK) or of integrins, has been proposed as a treatment 1Comprehensive Pneumology Center, Ludwig-Maximilians-Universität and Helmholtz Zentrum Munich, Max-Lebsche-Platz 31, 81377 Munich, Germany strategy [12, 13]. For instance, myofibroblasts possess an 2Member of the German Center of Lung Research (DZL), Munich, Germany increased ability to adhere to the ECM, which is Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Knüppel et al. Respiratory Research (2018) 19:67 Page 2 of 14 mediated by focal adhesions (FA) attaching the actin elucidate the underlying mechanisms, we analyzed the cytoskeleton to the matrix [14]. Cell attachment to the effect of siRNA-mediated knockdown of FKBP10 on ECM via clustering of integrins leads to the recruitment intracellular and membrane-spanning components of of numerous FA proteins with adapter, structural, and the FA complex, on regulatory events of FA turnover, on enzymatic functions [15, 16]. For instance, structural proteins involved in actin dynamics, and, finally, on a se- proteins like talin, vinculin and α-actinin facilitate the lection of ECM proteins with important emerging func- connection between integrins and actin fibers and pro- tions in migration. vide the basis for the transmission of mechanical forces between cell and ECM [17]. To enable cell migration, Methods turnover of FA is necessary. Several factors are involved Material in FA disassembly, like actin dynamics, FAK and Src Primers were obtained from MWG Eurofins (Ebersberg, phosphorylation, and ERK/MAP kinase-mediated activa- Germany) and are shown in Table 1. Table 2 contains tion of calpain-2, a calcium-dependent protease [15]. used primary antibodies. HRP-linked and fluorescent la- Finally, migration is strongly influenced by topology beled secondary antibodies were purchased from GE and composition of the ECM including integrin ligands Healthcare Life Sciences (Freiburg, Germany). like collagen, fibronectin (FN), and laminin [7]. Collagen type VI appears to play a particularly important role in Statistical Analysis this context, as several studies indicate a central, albeit Statistical analysis was performed in GraphPadPrism 5 context-dependent and tissue-specific role of collagen VI (GraphPad Software, San Diego, CA, USA). Results are for migration and adhesion [18–20]. shown as mean ± SEM. Paired t-test was used for statis- FK506-binding protein 10 (FKBP10, also termed tical analysis. Notably, analysis using a Wilcoxon signed FKBP65), a member of the family of immunophilins, is rank test yielded very similar results except for the an endoplasmic reticulum (ER) -resident peptidyl prolyl scratch assays shown in Fig. 5a where results just failed isomerase and a collagen I chaperone [21]. We have pre- significance (not shown). Significance is indicated as fol- viously reported upregulation of FKBP10 in experimen- lows: *p<0.05, **p<0.01, ***p<0.001. tal lung fibrosis and IPF, where it is mainly expressed by (myo)fibroblasts [22]. Deficiency of FKBP10 by siRNA- Human Lung Material, Isolation and Culture of phLF mediated knockdown in primary human lung fibroblasts Primary human lung fibroblasts (phLF) were isolated (phLF) reduced the expression of profibrotic markers from human lung tissue and derived from in total eight like α-smooth muscle actin (α-SMA), FN and collagen I, different patients. The tissue derived from human lung and suppressed collagen secretion [22]. explant material of IPF patients or histologically normal As properties of the ECM play an important role in regions adjacent to resected lung tumors was obtained adhesion dynamics and FKBP10 has been identified pre- from the BioArchive CPC-M for lung diseases at the viously as a regulator of collagen biosynthesis in phLF, Comprehensive Pneumology Center (CPC Munich, the aim of this study was to assess the effect of FKBP10 Germany). The study was approved by the local ethics deficiency on adhesion and migration in phLF. To committee of the Ludwig-Maximilians University of Table 1 Primer table for Real-Time Quantitative Reverse-Transcriptase PCR (qRT-PCR). Primers were synthesized by MWG Eurofins (Ebersberg, Germany). Target Species Forward primer (5′-3′) Reverse primer (5′-3′) CAPNS1 human GACGCTACTCAGATGAAAGT TCTTTGTCAAGAGATTTGAAG CAV1 human TCACTGTGACGAAATACTG CGTAGATGGAATAGACACG COL6A1 human GACGCACTCAAAAGCA ATCAGGTACTTATTCTCCTTCA COL6A2 human AGAAAGGAGAGCCTGCGGAT AGGTCTCCCTCACGTAGGTC COL6A3 human CTCTACCGAGCCCAGGTGTT ATGAGGGTGCGAACGTACTG CORO1C human GTTAACAAATGTGAGATTGC TGGAAAAGGTCAGACTTC DHX8 human TGACCCAGAGAAGTGGGAGA ATCTCAAGGTCCTCATCTTCTTCA ERK1 human TTCGAACATCAGACCTACT AGGTCCTGCACAATGTAG FBLN1C human GCCCTGAGAACTACCG GAGAGGTGGTAGTAGGTTATTC FKBP10 human CGACACCAGCTACAGTAAG TAATCTTCCTTCTCTCTCCA ITGB1 human TTACAAGGAGCTGAAAAACT AAAATGACTTCTGAGGAAAG TLN1 human GCTCTTTCTGTCAGATGAT CATAGTGTCCCCATTTC Knüppel et al. Respiratory Research (2018) 19:67 Page 3 of 14 Table 2 Primary antibodies used in Western Blot analysis, Immunofluorescence and Proximity Ligation Assays Target Abbreviation Antibody Provider Application β-actin ACTB HRP-conjugated anti-ACTB antibody Sigma Aldrich, St. Louis, USA WB Calpain-4 CAPNS1 mouse monoclonal anti-Calpain-4 Abnova, Taipei City, Taiwan WB, IF, PLA Caveolin-1 CAV1 rabbit monoclonal anti-Caveolin-1 antibody Cell Signaling, Boston, USA WB Collagen VI α1 COL6A1 mouse monoclonal anti-Collagen VI A1 antibody Santa Cruz, Dallas, USA WB, IF, PLA Collagen VI α3 COL6A3 mouse monoclonal anti-Collagen VI A3 antibody Santa Cruz, Dallas, USA IF, PLA Coronin 1C CORO1C mouse monoclonal anti-CORO1C antibody Santa Cruz, Dallas, USA WB, IF, PLA Extracellular Signaling Related ERK1 mouse monoclonal anti-ERK1 antibody BD Biosciences, New Jersey, USA WB Kinase 1 ER protein 57 (Protein ERp57 mouse monoclonal anti-ERp57 Abcam, Cambridge, UK PLA disulfide-isomerase A3) Fibulin-1 FBLN1 mouse monoclonal anti-FBLN1 antibody Santa Cruz, Dallas, USA WB, IF, PLA FK506-binding protein 10 FKBP10 rabbit polyclonal anti-FKBP10 antibody ATLAS, Stockholm, Sweden WB, IF, PLA Focal Adhesion Kinase FAK rabbit polyclonal anti-FAK antibody Santa Cruz, Dallas, USA WB Golgin97 CDF4 mouse monoclonal anti-Golgin97 antibody Invitrogen, Carlsbad, USA PLA mouse IgG (neg. ctrl) mouse IgG mouse IgG1κ isotype control eBioscience, San Diego, USA PLA Integrin-β1 ITGB1 mouse monoclonal anti-ITGB1 antibody Abcam, Cambridge, UK WB Phosphorylated Extracellular p-ERK1/2 rabbit monoclonal anti-pERK1/2 (Thr202/Tyr204) Cell Signaling, Boston, USA WB Signaling Related Kinase 1/2 Phosphorylated Focal p-FAK Y397 rabbit monoclonal anti-pFAK (Tyr397) Cell Signaling, Boston, USA WB Adhesion Kinase Phosphorylated Focal p-FAK Y566/577 rabbit monoclonal anti-pFAK (Tyr576/Tyr577) Biomol, Hamburg, Germany WB Adhesion Kinase Phosphorylated SRC p-Src rabbit polyclonal anti-pSrc (Tyr416) Cell Signaling, Boston, USA WB Proto-Oncogene, Non- Receptor Tyrosine Kinase SRC Proto-Oncogene, Non- Src mouse monoclonal anti-Src Cell Signaling, Boston, USA WB Receptor Tyrosine Kinase Talin 1 TLN1 mouse monoclonal anti-TLN1 Sigma Aldrich, Dt. Louis, USA WB, IF, PLA Secondary HRP-linked antibodies and secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 568 goat anti-rabbit IgG) for IF were purchased from GE Healthcare Life Sciences (Freiburg, Germany). 4',6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining (Sigma-Aldrich, St. Louis, USA) Munich, Germany, and all participants gave written in- harvesting for RNA and protein analysis. Unless stated formed consent. Isolation and culture of phLF was per- otherwise, all data is derived from eight independent formed as described previously [22, 23]. Notably, in knockdown experiments in at least three and maximally previous studies, we have never seen consistent expres- in eight different human primary fibroblast lines. For sion differences between control and IPF fibroblasts, nei- fibroblast lines that had been used for more than one ther in terms of basal and TGF-β1-induced gene knockdown experiment in different passages, typically a expression of collagens and collagen biosynthetic en- mean was formed for these experiments prior to statistical zymes, nor in terms of collagen secretion [22, 23]. analysis to avoid overrepresentation of one biological replicate in the data. Transfection of phLF and TGF-β1 Treatment Cells were seeded at a density of 20.000–25.000 cells/ cm2. Reverse transfection was carried out with human RNA Isolation and Real-Time Quantitative Reverse- small interfering RNA of FKBP10 (siRNA) (s34171; Life Transcriptase PCR (qRT-PCR) Analysis Technologies, Carlsbad, CA) or negative control siRNA. RNA isolation and qRT-PCR analysis were performed as Twenty-four hours later starvation for another 24 hours described previously [22, 23]. in Dulbecco’s modified Eagle medium/F-12 including 0.5% fetal bovine serum and 0.1 mM 2-phospho-L-as- corbic acid was performed. Then, cells were treated with Protein Isolation and Western Blot analysis 2 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN) in Protein isolation and Western Blot analysis were per- starvation medium for another 24 - 48 h, followed by formed as described previously [22, 23]. Knüppel et al. Respiratory Research (2018) 19:67 Page 4 of 14 Cell Fixation and Immunofluorescent Stainings Germany) and the section was marked by a black dot. Cells were seeded on FN-coated coverslips (6 μg/mL, After 24h, additional images were taken to compare Sigma-Aldrich, St. Louis, USA), followed by serum star- wound closure between control and FKBP10 deficient vation and TGF-β1 treatment for 48h as described cells. Results are given in percent of wound closure nor- above. The fixation method was chosen accordingly to malized to untreated control. the used antibodies. For methanol fixation, cells were washed once in 1x phosphate-buffered saline (PBS), Single Cell Migration Assay Using Time Lapse Microscopy followed by fixation with 100% methanol for 2 minutes Cells were seeded at a density of 5.500 cells/cm2 on on ice and three additional washing steps with 1x PBS to uncoated, collagen I-coated (~50 μg/mL, Sigma-Aldrich, remove the residual methanol. St. Louis, USA), or collagen VI-coated (10 μg/mL, For para-formaldehyde (PFA) fixation, cells were Abcam, Cambridge, UK) wells. Knockdown by siRNA of washed once with 1x PBS and 4% PFA was added to the FKBP10 was performed as described above. After 24h, cells followed by incubation at room temperature for 20 cells were serum-starved for another 24h followed by minutes. Residual PFA was removed by three washing TGF-β1 treatment (2 ng/mL) for 24h. Movies were gen- steps with 1x PBS. Staining of the cover slips was per- erated over a period of 12h - 24h using Axio Observer formed as described before [22]. Immunofluorescence Z1 microscope equipped with an AxioCam camera (Carl (IF) was examined by acquiring z-stack images with an Zeiss, Jena, Germany) and images were taken in 20 min Axio Imager M2 Microscope (Carl Zeiss, Jena, Germany) intervals. Single cell movement was analyzed using the and analysed by AxioVision 4.8 software. cell tracking tool of the AxioVision 4.8 software (Carl Zeiss, Jena, Germany). Proximity Ligation Assay (PLA) Cells were seeded, treated with TGF-β1 for 48h, and Results methanol-fixed as described above. The Duolink® PLA Kit Deficiency of FKBP10 attenuates migration and adhesion (Sigma Aldrich, St. Louis, USA) was used and carried out of primary human lung fibroblasts according to the manufacturer’s protocol. Interaction of SiRNA-mediated knockdown of FKBP10 expression in FKBP10 with target proteins was visualized using an Axio phLF was highly efficient on both protein and mRNA Imager M2 Microscope (Carl Zeiss, Jena, Germany). level (Fig. 1a, b, knockdown efficiency 86% ± 5%). Wound closure in a conventional scratch assay was sig- Cell Adhesion Assay nificantly reduced in FKBP10-deficient cells compared For analysis of cell attachment in FKBP10-deficient to control in absence of TGF-β1. A similar trend was phLF, cells were seeded and treated as described above. observed in presence of TGF-β1, which just failed sig- After 48h of TGF-β1 treatment and 96h of siRNA nificance (Fig. 1c, d). These results were confirmed with knockdown, cells were trypsinized and counted. Per con- an independent and more accurate method, namely a dition, four replicates with 100.000 cells per 48-well single cell migration assay using time-lapse microscopy were seeded and incubated for 1 hour at 37°C, 5% CO2. to track individual cells. In this assay, FKBP10-deficient Wells were carefully washed once with 1 x PBS to re- cells showed a highly significant reduction of mean vel- move non-adherent cells and attached cells were fixed ocity both in absence and presence of TGF-β1 (Fig. 1e). with 4% PFA as described before. Cells were stained with Next, we investigated the effect of siRNA-mediated 4′,6-Diamidin-2-phenylindol (DAPI) and Phalloidin, la- knockdown of FKBP10 on adhesion of phLF using an beled with Alexa Fluor 568 (Invitrogen) followed by im- IF-based attachment assay. In presence of TGF-β1, aging using an LSM T-PMT microscope (Carl Zeiss, FKBP10 deficiency significantly reduced the ability of Jena, Germany). Attached cells were counted using phLF to adhere to the cell culture dish surface (Fig. 1f). IMARIS Software 9.0. Results were normalized to non- While TGF-β1 had no significant effect on migration, treated control and visualized as percentage of attached neither in the scratch nor in the single cell tracking cells. assay, TGF-β1 significantly increased fibroblast adhesion. Scratch Assay Deficiency of FKBP10 upregulates expression of key Cells were seeded at a density of 35.000 cells/cm2 and molecules of adhesion and migration in primary human siRNA-mediated knockdown of FKBP10 was performed lung fibroblasts, but does not alter the activation of focal as described above. To reach 100% confluency, cells adhesion kinase and downstream pathways were grown for 48h followed by starvation for 24h. A To gain a better understanding of the effect of FKBP10 scratch was executed using a 1000 μL pipette tip and deficiency on adhesion and migration, we analyzed sev- TGF-β1 was added. Images were taken at time point 0h eral selected proteins with key functions in these pro- using an inverse microscope (Primovert, Carl Zeiss, Jena, cesses. In terms of altered gene expression of focal Knüppel et al. Respiratory Research (2018) 19:67 Page 5 of 14 a b c d e f Fig. 1 Knockdown of FKBP10 reduces migration and adhesion of phLF. a Western Blot analysis of phLF treated with scrambled siRNA as control (sc) or FKBP10 siRNA (kd) and 2-phosphoascorbate (0.1 mM) in absence and presence of TGF-β1 (2 ng/mL) for 48h. Densitometric analysis and representative blots show the effect of FKBP10 knockdown on the expression of FKBP10 relative to β-actin as loading control (ACTB). b Quantitative reverse transcriptase-polymerase chain reaction analysis of phLF treated with sc siRNA as control or FKBP10 siRNA (kd) and 2- phosphoascorbate (0.1 mM) in absence and presence of TGF-β1 (2 ng/mL) for 48h. Transcript levels are shown as -ΔCt values. DEAH (Asp-Glu-Ala-His) Box Polypeptide 8 (DHX8) was used as endogenous control. Data (a, b) is based on eight independent experiments. c Representative images of a scratch assay of phLF treated with sc siRNA as control or FKBP10 and 2- phosphoascorbate (0.1 mM) in absence or presence of TGF- β1 (2 ng/mL). Images were taken at 0h and after 24h. d Analysis of open wound areas as shown in (c) normalized to controls at 0h (100%), given in % of the remaining wound area. Data is based on four independent experiments. e SCM assay of phLF treated with sc siRNA as control or FKBP10 siRNA and 2- phosphoascorbate (0.1 mM) in absence and presence of TGF-β1(2ng/mL).Cellsweretracked over a period of 12h - 24h. Results of five independent experiments are shown as mean velocity of around 80 tracked cells per condition. f Cell attachment assay of phLF treated with sc siRNA as control or FKBP10 siRNA in absence or presence of TGF-β1(2ng/mL)for48h. Results originate from six independent experiments and are visualized as percentage of cell adhesion normalized to non TGF-β1- treated cells. Statistical significance between control and FKBP10 kd is indicated by horizontal brackets and asterisks adhesion and ECM components, we only observed mar- calpain-4 (CAPNS1), the small regulatory subunit of ginal differences between fibroblasts isolated from nor- calpain-1 and calpain-2, which is indispensable for for- mal histology control or from IPF lung tissue and mation and strengthening of adhesions and for mechan- therefore decided to pool the data. Deficiency of FKBP10 osensing during fibroblast migration [24]. Similar to in phLF led to induction of the focal adhesion compo- TLN1, CAPNS1 expression was significantly upregulated nent talin-1 (TLN1) on protein and mRNA level in ab- in absence and presence of TGF-β1 on transcript level sence and presence of TGF- β1 (Fig. 2a, b). Next, we (Fig. 2c, d); this effect, however, translated to the protein investigated the effect of FKBP10 knockdown on level only in absence of TGF-β1. Moreover, FKBP10 Knüppel et al. Respiratory Research (2018) 19:67 Page 6 of 14 a c e b d f g i hj Fig. 2 FKBP10 deficiency alters the expression of molecules implicated in adhesion and migration. a, c, e, g, i Western Blot analysis of phLF treated with scrambled siRNA as control (sc) or FKBP10 siRNA (kd) and 2- phosphoascorbate (0.1 mM) in absence and presence of TGF-β1(2ng/mL) for 48h. Densitometric analysis and representative blots show the effect of FKBP10 kd on the expression of talin-1 (TLN1) (a), calpain-4 (CAPNS1) (c), integrin β1(ITGB1)(e), caveolin-1 (CAV1) (g) and coronin 1C (CORO1C) (i)relativetoβ-actin as loading control (ACTB). b, d, f, h, j Quantitative reverse transcriptase-polymerase chain reaction analysis of phLF treated with scrambled siRNA as control (sc) or FKBP10 siRNA (kd) and 2- phosphoascorbate (0.1 mM) in absence and presence of TGF-β1 (2 ng/mL) for 48h. Transcript levels are presented as -ΔCt values showing the effect of siRNA mediated kd of FKBP10 on talin-1 (TLN1) (b), calpain-4 (CAPNS1) (d), integrin β1 (ITGB1) (f), caveolin-1 (CAV1) (h) and coronin 1C (CORO1C) (j). DEAH (Asp-Glu- Ala-His) Box Polypeptide 8 (DHX8) was used as endogenous control. All data is based on eight (protein) or seven (mRNA) independent experiments. Statistical significance between control and FKBP10 kd is indicated by horizontal brackets and asterisks knockdown also increased protein levels of the trans- creating a motif which results in binding and conform- membrane collagen receptor integrin-β1 (ITGB1), par- ational activation of proto-oncogene tyrosine-protein ticularly in presence of TGF-β1 (Fig. 2e), while kinase Src. Active Src triggers further FAK phosphor- transcript levels were not significantly changed (Fig. 2f). ylation on Tyr sites like Tyr 576/577, starting the ras- Finally, two modulators of cytoskeleton dynamics, raf-MEK-ERK signal transduction cascade which is caveolin-1 (CAV1) and coronin-1C (CORO1C), were implicated in many processes including cell adhesion regulated in opposite directions: Whereas CAV1 expres- and migration. MEK1/2 catalyzes ERK1/2 phosphoryl- sion (protein and mRNA) was downregulated upon ation on specific Tyr and Thr residues essential for FKBP10 knockdown in presence of TGF-β1 (Fig. 2g, h), enzyme activity [25, 26]. CORO1C protein levels were upregulated (Fig. 2i, j). Deficiency of FKBP10 slightly upregulated total FAK FAK activation by either integrins or growth factors levels (Fig. 3a); however, there was no significant effect leads to autophosphorylation of Tyr 397 in FAK, on the phosphorylation on Tyr 397 or Tyr 576/577 of Knüppel et al. Respiratory Research (2018) 19:67 Page 7 of 14 a d f b e g c Fig. 3 Neither FAK, Src, nor ERK1/2 activation is affected by FKBP10 kd. Western Blot analysis of phLF treated with scrambled siRNA as control (sc) or FKBP10 siRNA (kd) and 2- phosphoascorbate (0.1 mM) in absence and presence of TGF-β1 (2 ng/mL) for 48h. Densitometric analysis and representative blots show the effect of FKBP10 kd on the expression of FAK (a), Src (d)andERK1(f)relativetoβ-actin as loading control (ACTB). b, c Densitometric analysis and representative blots showing the effect of FKBP10 kd on the phosphorylation of FAK (Y397) (b) and FAK (Y576/577) (c)relativetoFAK.e, g Densitometric analysis and representative blots showing the effect of FKBP10 kd on the phosphorylation of Src (Y416) (e)relativetoSrc and pERK1/2 (T202/Y204) (g) relative to ERK1. All data is based on eight independent experiments. Statistical significance between control and FKBP10 kd is indicated by horizontal brackets and asterisks FAK (Fig. 3b, c). Similarly, none of the assessed down- the process of cell migration [18, 27]. Here, we found stream signaling pathways appeared affected: Neither that collagen VI and fibulin-1 expression were regulated levels of total Src and phospho-Src nor levels of total in different directions in response to FKBP10 knock- ERK1 and phosphorylated ERK1/2 changed upon down. Collagen 6A1 was significantly reduced on protein FKBP10 knockdown (Fig. 3d-g). level in FKBP10 deficient phLF with and without TGF- β1, whereas COL6A1 transcript was only reduced in FKBP10 interacts with type VI collagen and fibulin-1 and presence of TGF-β1 (Fig. 4a, b). Interestingly, mRNA regulates their expression levels of COL6A2 and COL6A3 chains were not influ- As changes in expression of focal adhesion complex enced by FKBP10 deficiency (Fig. 4c, d). In contrast, components and in events during FAK signaling did not fibulin-1 protein expression was increased in response to explain the observed attenuated migration upon FKBP10 FKBP10 knockdown in absence and presence of TGF- knockdown, we reasoned that changes in ECM compos- β1; transcript levels of FBLN1C were significantly upreg- ition could be the main cause of reduced migration vel- ulated under basal conditions upon FKBP10 knockdown ocity. Besides the ECM proteins collagen I and FN, (Fig. 4e, f). which we reported to be downregulated upon FKBP10 Co-localization of FKBP10 with both, collagen VI knockdown previously [22], collagen VI and fibulin-1 (COL6A1 and COL6A3) (Fig. 4g, h) and fibulin-1 (Fig. 4i) also have been described to maintain important roles in was examined by IF stainings and PLA (Fig. 4j). Negative Knüppel et al. Respiratory Research (2018) 19:67 Page 8 of 14 abc def g hi j Fig. 4 FKBP10 interacts with COL6A1 and FBLN1 and regulates their expression. a, e Western Blot analysis of phLF treated with scrambled siRNA as control (sc) or FKBP10 siRNA (kd) and 2- phosphoascorbate (0.1 mM) in absence and presence of TGF-β1 (2 ng/mL) for 48h. Densitometric analysis and representative blots showing the effect of FKBP10 kd on the expression of Collagen VI alpha1 (COL6A1) (a) and fibulin-1 (FBLN1) (e)relativetoβ- actin as loading control (ACTB). b, c, d, f Quantitative reverse transcriptase-polymerase chain reaction analysis of phLF treated with sc siRNA as control or FKBP10 siRNA and 2-phosphoascorbate (0.1 mM) in absence and presence of TGF-β1 (2 ng/mL) for 48h. Transcript levels are shown as -ΔCt values of showing the effect of FKBP10 kd on collagen VI alpha 1 (COL6A1) (b), collagen VI alpha 2 (COL6A2) (c), collagen VI alpha 3 (COL6A3) (d)and fibulin-1C (FBLN1C) (f). DEAH (Asp-Glu-Ala-His) Box Polypeptide 8 (DHX8) was used as endogenous control. All data is based on eight (protein) or seven (mRNA) independent experiments. Statistical significance is indicated by horizontal brackets and asterisks. g-i Immunofluorescence staining of FKBP10 (green) and COL6A1 (red) (g), COL6A3 (red) (h) and FBLN1 (red) (i). 4`, 6-diamidino-2-phenylindole (DAPI) staining is shown in blue. The region of interest is indicated by a white square and enlarged in the picture below. White arrows specify examples for co-localization of FKBP10 with COL6A1, COL6A3 or FBLN1. Stainings were taken as z-stack and the enlarged pictures show one focal plane from this z-stack. Representative images were selected from three independent experiments. j Representative images of in situ localization of the interaction of FKBP10 and COL6A1, COL6A3,FBLN1,mouseIgG1κ (negative control), Golgin97 (negative control) and ERp57 (positive control), assessed by proximity ligation assay. Representative images were selected from three independent experiments, except for IgG negative control (n=1) Knüppel et al. Respiratory Research (2018) 19:67 Page 9 of 14 controls (mouse IgG and Golgin97) did not show any that exclusion of ascorbate from the culture medium interaction with FKBP10. Endoplasmic Reticulum Protein would give us a first indication whether decreased 57 (ERp57), previously reported by IF to co-localize with biosynthesis of collagen or increased biosynthesis of FKBP10 [22] was used as positive control and showed fibulin may underlie the observed effects of FKBP10 proximity to FKBP10. Positive signals were also observed deficiency on migration and adhesion. Notably, in ab- for COL6A1, COL6A3 and FBLN1, indicating interaction sence of 2-phosphoascorbate the effect of FKBP10 de- with or proximity to FKBP10. ficiency in phLF on adhesion and migration was lost, in comparison to the results in presence of 2- The effects of FKBP10 deficiency on migration and phosphoascorbate (Fig. 5), pointing towards a adhesion depend on 2-phosphoascorbate and are collagen-dependent mechanism. abolished by coating cell culture dishes with collagen VI As both collagen I and collagen VI are downregulated Post-translational modifications (PTM) like proline and upon FKBP10 deficiency [22], we coated dishes with ei- lysine hydroxylation of collagens (including collagen VI) ther collagen I or collagen VI and analyzed migration in are crucial steps in collagen biosynthesis and catalyzed a single-cell migration approach. Notably, the effect of by ascorbate-dependent prolyl and lysyl hydroxylases FKBP10 deficiency in phLFs on migration was com- [28–30]. In contrast, FBLN1 biosynthesis is not pletely lost when culture dishes were coated with dependent on ascorbate [31]. Therefore, we reasoned collagen VI (Fig. 6b) when compared to uncoated dishes a b Fig. 5 The inhibitory effect of FKBP10 deficiency on migration and adhesion is 2-phosphoascorbate dependent. a Analysis of open wound areas in scratch assays of phLF treated with scrambled siRNA (sc) as control or FKBP10 siRNA (kd) in absence and presence of TGF-β1 (2 ng/mL) and 0.1 mM 2-phosphoascorbate. Images were taken at 0h and after 24h. Data is normalized to controls at 0h (100%), given in % of the remaining wound area and based on four independent experiments. b Cell attachment assay of phLF treated with scrambled siRNA (sc) as control or FKBP10 siRNA (kd) in absence or presence of TGF-β1 (2 ng/mL) and 2-phosphoascorbate (0.1 mM) for 48h. Results originate from six independent experiments and are visualized as percentage of cell adhesion normalized to non TGF-β1 treated. Experiments without and with 2- phosphoascorbate were performed in parallel Knüppel et al. Respiratory Research (2018) 19:67 Page 10 of 14 a b c Fig. 6 Coating of dishes with collagen VI abolishes the inhibitory effect of FKBP10 knockdown on migration. SCM assay of phLF treated with scrambled siRNA as control (sc) or FKBP10 siRNA (kd) and 2- phosphoascorbate (0.1 mM) in absence and presence of TGF-β1 (2 ng/mL). Cells were tracked over a period of 12h -24h. Results of three independent experiments are shown as mean velocity of around 80 tracked cells per condition. Effect of collagen VI coated dishes on mean velocity of phLF is shown in (b) and of collagen I coated dishes in (c)comparedto uncoated dishes for control (a). Statistical significance between control and FKBP10 kd is indicated by horizontal brackets and asterisks (Fig. 6a). Some compensation was also visible in collagen deficiency, even if the scratch assay results in presence I-coated dishes, albeit not that pronounced (Fig. 6c). of TGF-β1 failed significance (p=0.06). We believe that this minor discrepancy reflects the well-known disadvan- Discussion tages of the scratch assay, most importantly variations in In this study we demonstrated that FKBP10 deficiency gap width due to the manually applied and therefore inhibited phLF adhesion and migration. This effect could often uneven scratch, the fact that ECM substrate is neither be explained by changes in expression or activa- equally scraped off together with the cells, and also the tion of components of the FA complex, nor by changes mechanical cell damage which may introduce artifacts. in FAK downstream signaling events, nor by changes in These technical drawbacks may explain the greater vari- regulators of actin dynamics. Instead, we found that the ations observed in this assay, leading to results that just effect of FKBP10 deficiency on migration and adhesion fail significance. depended on 2-phosphoascorbate, pointing towards a Our finding that TGF-β1 did not affect fibroblast mi- central role of collagen biosynthesis in this context. Loss gration is in agreement with previous studies by others of FKBP10 downregulated the expression of collagen VI, [33–35]. Increased fibroblast adhesion in presence of a collagen type increasingly recognized as a central TGF-β1 may in part reflect increased expression of β1- player in migration, and coating culture dishes with col- integrin (this work, cf. Fig. 2e, f, scr ctrl vs. scr TGF-β1, lagen VI completely abolished the effect of FKBP10 defi- p=0.0642) and/or β3-integrins [36]. ciency on phLF migration. Initially, we sought to assess the effect of FKBP10 defi- Next to excessive ECM deposition by interstitial fibro- ciency on several regulatory levels of FA turnover to elu- blasts, aberrant fibroblast adhesion and increased migra- cidate the mechanism underlying attenuated migration. tion are also important features of IPF [6, 9–11]. Here Cell migration is a complex cyclic process starting with we show that loss of the collagen chaperone FKBP10, the extension of membrane protusions (lamellipodia)at which we previously identified as potential IPF drug tar- the leading edge followed by their adhesion to the ECM get due to its role in ECM synthesis and secretion [22], and retraction of the cell tail [37]. During cell migration inhibited wound closure and reduced the mean velocity assembly and disassembly of FA is dynamically regu- and adhesion capacity of phLF (Fig. 1). This observation lated. The process of cell attachment to the ECM is initi- is in line with the very recent report by Liang et al., ated by clustering of integrins on the cell surface, showing that siRNA-mediated knockdown of FKBP10 heterodimeric transmembrane receptors consisting of α led to reduced migration in human hypertrophic scar fi- and β subunits. The intracellular domains of the clus- broblasts [32]. Collectively, these studies are confirma- tered integrins serve as a platform for FA protein re- tive of our concept of FKBP10 as potential drug target in cruitment and ultimately link the ECM via FA to actin fibrotic disease. stress fibers [38]. In this context, TLN1 mediates the ini- We assessed fibroblast migration both in a conven- tiation of FA assembly by interacting with the cytoplas- tional scratch assay as well as by tracking individual cells mic domain of the integrin β subunit on the one hand with videomicroscopy in time-lapse experiments, a more and with actin and actin-binding proteins on the other accurate approach. Overall, both assays consistently [15]. Another initial event upon integrin clustering is ac- showed reduced migration under conditions of FKBP10 tivation of FAK including autophosphorylation of Tyr Knüppel et al. Respiratory Research (2018) 19:67 Page 11 of 14 397 and Src-mediated phosphorylation of additional At first, observing inhibition of adhesion and migra- tyrosine residues within FAK (e.g. Y 566 and 577), which tion in the absence of changes in activation of FAK and are essential for full FAK activity. Active FAK interacts related signaling pathways seemed contradictory to us. with multiple signalling molecules including Grb7, PI3K, However, similarly, Asano and colleagues have reported paxillin, MLCK and ERK, activating signalling pathways that siRNA-mediated knockdown of α-smooth muscle which result in protrusion extension, increased FA turn- actin (α-SMA) in phLF led to inhibition of migration over, and therefore, ultimately, in increased cell motility without affecting the FAK signaling pathway [59]. This [15, 39–42]. The protease calpain 2, a heterodimer con- observation suggested that changes in actin dynamics sisting of a catalytic subunit and a regulatory subunit may underlie the observed inhibition of migration and, (calpain-4, CAPNS1) plays a central role in this context indeed, from our previous studies, we know that FKBP10 as it, when recruited by FAK and activated by ERK/MAP deficiency reduces α-SMA expression in phLF [22]. Also, kinase, mediates amongst others proteolysis of TLN1 deficiency of the actin binding protein CORO1C typic- and FAK, considered the rate-limiting step in FA turn- ally results in inhibition of migration; however, here, we over [15, 38, 39, 43, 44]. Finally, directional cell migra- observed a moderate increase of CORO1C protein ra- tion is strongly dependent on polarized actin dynamics. ther than downregulation (Fig. 2i)[60, 61]. Expression of Rho-like GTPases like RhoA and Rac1 control cytoskel- CAV1, deficiency of which typically results in decreased eton contractility, polymerization, and protrusion. The migratory speed in variable cell types [62–64], was only integral membrane protein caveolin-1 (Cav-1), activated moderately downregulated in presence of TGF-β1 by small kinases like Src, is a central regulator of Rho- (Fig. 2g, h). Collectively, these observations do not like GTPase signaling in this context [45, 46]. Another argue for altered actin dynamics as a major mechan- regulator of actin filament turnover in the lamellipodia ism underlying inhibition of migration in response to during cell migration is the actin binding protein coro- FKBP10 deficiency. nin 1C (CORO1C) [47]. Importantly, cell migration is influenced by properties FKBP10 deficiency led to several significant changes in of the ECM, like density of adhesion ligands (collagen, expression of FA components on all levels assessed, i.e. FN), ECM composition, and stiffness [7, 65]. Previously, upregulation of ITGB1, TLN1, CAPNS1, FAK, CORO1C we have observed downregulation of expression and se- and downregulation of CAV1 (Fig. 2). Upregulation of cretion of type I collagen and FN, both major compo- ITGB1 may associate with reduced FA turnover and nents of the fibroblast ECM, in response to FKBP10 slow migration [48–50] but impaired migration and ad- knockdown [22]. Here we extended this analysis and hesion has also been reported as a result of ITGB1 defi- assessed additional ECM components with important ciency [51]. Collectively, these findings suggest that roles in cell migration, namely type VI collagen and manipulation of ITGB1 protein levels in general is un- FBLN1. Both proteins colocalized with FKBP10 in phLF, favorable to cell motility, regardless of direction of regu- as assessed by both immunofluorescence colocalization lation. As ITGB1 functions as an adapter protein analysis and proximity ligation assay (Fig. 4g-j), indicat- between the ECM and intracellular FA complexes, it is ing direct interaction with FKBP10 in the ER. Interest- conceivable that its expression levels must be tightly reg- ingly, we found that loss of FKBP10 significantly ulated to allow for functional intermolecular interactions increased FBLN1 expression (Fig. 4e, f), but decreased between different interacting partners. protein levels of COL6A1 (Fig. 4a, b). Notably, COL6A1 The same may apply to TLN1, an adapter protein link- deficiency is sufficient to inhibit collagen VI deposition ing the cytoskeleton to ITGB1, where reports in the lit- in the ECM, as no triple helical molecules can be formed erature are also seemingly controversial: Upregulation of without the α1(VI)-chain [66]. These results suggest op- TLN1, but also suppression of TLN1 has been reported posing functions of FKBP10 in FBLN1 and collagen VI to reduce migration and adhesion [52–54]. Interestingly, biosynthesis in phLF. It is tempting to speculate, for in- downregulation of TLN1 has also been associated with stance, that FKBP10 acts as a FBLN1 chaperone, seques- increased migration [52, 55], which is in support of its tering FBLN1 in the ER, prohibiting packing in vesicles function as a regulator of FA turnover together with its for secretion or targeting FBLN1 for ER-associated pro- protease calpain-2 [15, 38, 39, 43, 44]. In our system, tein degradation, while at the same time FKBP10 is likely however, the simultaneous increase of the regulatory cal- required for efficient collagen VI triple helix formation, pain 2 subunit CAPNS1, argues for overall little change similar to collagen I and III [21, 67, 68]. These aspects in FA turnover, at least in absence of TGF-β1 (Fig. 2c, will be interesting to explore in future studies. d). This is consistent with our observation that phos- As to function of these proteins in migration, reduced phorylation levels of FAK, Src, and ERK (Fig. 3), central attachment and decreased migratory speed has been re- signaling events in the process of FA turnover [39, 56–58] ported for a human breast cancer cell line (MDA remained unchanged. MB231) in response to FBLN1 overexpression [27] and Knüppel et al. Respiratory Research (2018) 19:67 Page 12 of 14 siRNA mediated knockdown of FBLN1 in corneal fibro- of ITGB1, TLN1, CAPNS1, total FAK, and CORO1C blasts upregulated cell migration [69]. Therefore, taken may ultimately reflect an attempt of the cells to over- together, it was plausible that increased FBLN1 levels come decreased migration by overcompensation, in- may underlie the observed inhibition of migration under creasing expression of ECM receptors or components of conditions of FKBP10 deficiency. the FA turnover machinery. While collagen VI begins to emerge as an important Interestingly, FKBP10 mediates dimerization with col- regulator of cell migration, reports on its direction of ef- lagen lysyl hydroxylase 2 and thus contributes to the fect, inhibiting or promoting migration, are controversial generation of collagen hydroxylysines, which act as sub- [18–20]. For instance, collagen VI-deficient tendon fi- strates for extracellular lysyl-oxidase-mediated collagen broblasts show delayed wound closure, i.e. lower migra- crosslinking [74–76]. Extracellular collagen lacking tion speed, in a scratch assay [18], while human dermal proper crosslinks is prone to proteolytic degradation and fibroblasts displayed higher migration speed on matrices may not be able to contribute to the higher ordered derived from collagen VI-deficient cells [20]. These dis- organization of the ECM anymore [77–80]. Therefore, crepancies may be a result of the divergent approaches downregulation of FKBP10 may also contribute to in the mentioned studies (assessment of newly formed disorganization of the ECM by providing less crosslink- ECM versus assessment of ECM deposited within 10 ing sites in type I and type VI collagen. days, respectively), different collagen VI chains assessed In summary, we found that loss of FKBP10 in phLF re- (COL6A1 versus COL6A2), but also of the different cell sults in decreased adhesion and migration. We found origins, thus possibly reflecting time-, chain-, and cell- that the main underlying mechanism was reduced colla- specific effects of collagen VI. gen VI biosynthesis, as both ascorbate deficiency and Ascorbic acid is a cofactor necessary for proline and coating of cell culture dishes with collagen VI abolished lysine hydroxylation during collagen synthesis [68] in- the effect of FKBP10 knockdown on migration. As in- cluding collagen VI [30, 70], but not required for FBLN1 creased fibroblast migration is a characteristic of IPF, the synthesis. Therefore, to differentiate between increased results are in support of our concept of FKBP10 as a po- FBLN1 or decreased type VI collagen as the underlying tential drug target for IPF. mechanism of decreased adhesion and migration, we compared effects of FKBP10 knockdown on adhesion Abbreviations FKBP10: FK506-binding Protein 10; IPF: Idiopathic Pulmonary Fibrosis; and migration in absence and presence of 2- ECM: Extracellular matrix; phLF: Primary Human Lung Fibroblast; TGF- phosphoascorbate, a stable analogue of ascorbic acid. β1: Transforming Growth Factor-β1; IF: Immunofluorescence; FAK: Focal Notably, neither migration nor adhesion were changed Adhesion Kinase; FA: Focal Adhesions; Src: SRC Proto-Oncogene, Non- Receptor Tyrosine Kinase; ERK: Extracellular Signaling Related Kinase; upon loss of FKBP10 when the cell culture medium was MAP: Mitogen-activated Protein; FN: Fibronectin; ER: Endoplasmic Reticulum; ascorbate-deficient (Fig. 5a, b). These results strongly in- α-SMA: α-smooth muscle actin; ACTB: β-actin; CAPNS1: Calpain-4; dicated that the effect of FKBP10 deficiency on adhesion CAV1: Caveolin-1; COL6A1: Collagen VI α1; COL6A3: Collagen VI α3; CORO1C: Coronin 1C; ERp57: ER protein 57 (Protein disulfide-isomerase A3); and migration was collagen-dependent. While coating FBLN1: Fibulin-1; ITGB1: Integrin-β1; TLN1: Talin 1; COL6A1: Collagen VI alpha with collagen VI abolished the effect of FKBP10 knock- 1; COL6A2: Collagen VI alpha 2; COL6A3: Collagen VI alpha 3; PTM: Post- down on migration completely, coating with collagen I translational Modification; sc: scrambled; kd: knockdown only did so marginally (Fig. 6). Overall, this indicated Acknowledgements that FKBP10 knockdown inhibits lung fibroblast migra- We thank Elisabeth Hennen for excellent technical assistance, and Gerald tion by reduced collagen VI biosynthesis rather than Burgstaller for his support with single cell migration assays. Human samples reduced collagen I biosynthesis, an effect of FKBP10 for derivation of phLF were kindly provided by the CPC-M BioArchive. deficiency which we have reported previously [22]. Funding This work was supported by the Friedrich-Baur-Stiftung (grant to CASW, 51/16), Conclusion the Helmholtz Association, and the German Center for Lung Research (DZL). Collagen VI is an important regulator of the ECM or- Availability of data and materials ganizing the three-dimensional meshwork of collagen The datasets used or analyzed during the current study are available from and FN fibers [71, 72] and the topography of the fibrillar the corresponding author on reasonable request. ECM network guides directional cell migration [73]. Therefore, our observations suggest that loss of FKBP10 Authors’ contributions Conception and design: LK, KH, ML, RH, JB, OE, CSW. Experimental work, results in reduced biosynthesis of collagen VI and, pos- analysis, and interpretation: LK, CSW. Drafting the manuscript and intellectual sibly in combination with decreased extracellular colla- content: LK, OE, CSW. All authors read and approved the final manuscript. gen I levels, leads to a disorganized ECM with changed adhesion ligand density, stiffness and composition, Ethics approval and consent to participate The study was approved by the local ethics committee of the Ludwig- which may not provide sites for integrin clustering and Maximilians University of Munich, Germany (333-10), and all participants gave does not favor directional cell migration. Upregulation written informed consent. Knüppel et al. Respiratory Research (2018) 19:67 Page 13 of 14 Competing interests pathogenic mechanism for Hirschsprung's disease. J Clin Invest. 2015;125: The authors declare that they have no competing interests. 4483–96. 20. Theocharidis G, Drymoussi Z, Kao AP, Barber AH, Lee DA, Braun KM, Connelly JT. Type VI Collagen Regulates Dermal Matrix Assembly and Publisher’sNote Fibroblast Motility. J Invest Dermatol. 2016;136:74–83. Springer Nature remains neutral with regard to jurisdictional claims in 21. Ishikawa Y, Vranka J, Wirz J, Nagata K, Bachinger HP. The rough endoplasmic published maps and institutional affiliations. reticulum-resident FK506-binding protein FKBP65 is a molecular chaperone that interacts with collagens. J Biol Chem. 2008;283:31584–90. Author details 22. Staab-Weijnitz CA, Fernandez IE, Knuppel L, Maul J, Heinzelmann K, Juan- 1Comprehensive Pneumology Center, Ludwig-Maximilians-Universität and Guardela BM, Hennen E, Preissler G, Winter H, Neurohr C, et al. FK506- Helmholtz Zentrum Munich, Max-Lebsche-Platz 31, 81377 Munich, Germany. Binding Protein 10, a Potential Novel Drug Target for Idiopathic Pulmonary 2Member of the German Center of Lung Research (DZL), Munich, Germany. Fibrosis. Am J Respir Crit Care Med. 2015;192:455–67. 3Asklepios Fachkliniken Munich-Gauting, Munich, Germany. 23. Knüppel L, Ishikawa Y, Aichler M, Heinzelmann K, Hatz R, Behr J, Walch A, 4Thoraxchirurgisches Zentrum, Klinik für Allgemeine-, Viszeral-, Bachinger HP, Eickelberg O, Staab-Weijnitz CA. A Novel Antifibrotic Transplantations-, Gefäß- und Thoraxchirurgie, Klinikum Großhadern, Mechanism of Nintedanib and Pirfenidone. Inhibition of Collagen Fibril Ludwig-Maximilians-Universität, Munich, Germany. 5Medizinische Klinik und Assembly. Am J Respir Cell Mol Biol. 2017;57:77–90. Poliklinik V, Klinikum der Ludwig-Maximilians-Universität, Munich, Germany. 24. Undyala VV, Dembo M, Cembrola K, Perrin BJ, Huttenlocher A, Elce JS, Greer 6Colorado Anschutz Medical Campus, Pulmonary and Critical Care Medicine PA, Wang YL, Beningo KA. The calpain small subunit regulates cell-substrate University, Denver, Colorado, USA. mechanical interactions during fibroblast migration. 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Nat Rev Mol Cell Biol. 2014;15:813–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ÝSSHO/,VKLNDZD<$LFKOHU0+HLQ]HOPDQQ.+DW]5%HKU-:DOFK$%ÇFKLQJHU+3 (LFNHOEHUJ 2 6WDDE:HLMQLW] &$ 1RYHO $QWLILEURWLF 0HFKDQLVP RI 1LQWHGDQLE DQG 3LUIHQLGRQH ,QKLELWLRQRI&ROODJHQ)LEULO$VVHPEO\$PHULFDQ-RXUQDORI5HVSLUDWRU\&HOO DQG0ROHFXODU%LRORJ\ S 7KH$PHULFDQ-RXUQDORI5HVSLUDWRU\&HOODQG0ROHFXODU%LRORJ\LVDQRIILFLDOMRXUQDORI WKH$PHULFDQ7KRUDFLF6RFLHW\ ORIGINAL RESEARCH A Novel Antifibrotic Mechanism of Nintedanib and Pirfenidone Inhibition of Collagen Fibril Assembly Larissa Knuppel ¨ 1, Yoshihiro Ishikawa2,3, Michaela Aichler4, Katharina Heinzelmann1, Rudolf Hatz5,6,J¨urgen Behr6,7, Axel Walch4, Hans Peter Bachinger ¨ 2,3, Oliver Eickelberg1,8, and Claudia A. Staab-Weijnitz1 1Comprehensive Pneumology Center, and 4Research Unit Analytical Pathology, Helmholtz-Zentrum Munchen, ¨ Munich, Germany; 2Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon; 3Research Department, Shriners Hospital for Children, Portland, Oregon; 5Thoraxchirurgisches Zentrum, Klinik fur ¨ Allgemeine-, Viszeral-, Transplantations-, Gefaß- ¨ und Thoraxchirurgie, Klinikum Großhadern, and 7Medizinische Klinik und Poliklinik V, Klinikum der Ludwig-Maximilians-Universitat, ¨ Ludwig-Maximilians-Universitat, ¨ Munich, Germany; 6Asklepios Fachkliniken Munchen-Gauting, ¨ Munich, Germany; and 8Pulmonary and Critical Care Medicine University, Colorado Anschutz Medical Campus, Denver, Colorado ORCID ID: 0000-0002-1211-7834 (C.A.S.-W.). Abstract drugs act on important regulatory levels in collagen synthesis and processing. Nintedanib was more effective in down-regulating Idiopathic pulmonary fibrosis (IPF) is characterized by excessive profibrotic gene expression and collagen secretion. Importantly, deposition of extracellular matrix, in particular, collagens. Two both drugs inhibited collagen I fibril formation and caused a IPF therapeutics, nintedanib and pirfenidone, decelerate lung reduction in and an altered appearance of collagen fibril function decline, but their underlying mechanisms of action are bundles, representing a completely novel mechanism of action poorly understood. In this study, we sought to analyze their effects for both drugs. on collagen synthesis and maturation at important regulatory levels. fi Primary human broblasts from patients with IPF and healthy Keywords: fi – m idiopathic pulmonary brosis; extracellular matrix; donors were treated with nintedanib (0.01 1.0 M) or pirfenidone nintedanib; pirfenidone (100-1,000 mM) in the absence or presence of transforming growth factor-b1. Effects on collagen, fibronectin, FKBP10, and HSP47 expression, and collagen I and III secretion, were analyzed by Clinical Relevance quantitative polymerase chain reaction and Western blot. The appearance of collagen fibrils was monitored by scanning electron Accumulation of extracellular matrix, mainly collagen, is microscopy, and the kinetics of collagen fibril assembly was assessed a main feature of idiopathic pulmonary fibrosis (IPF). using a light-scattering approach. In IPF fibroblasts, nintedanib Nintedanib and pirfenidone, two recently approved reduced the expression of collagen I and V, fibronectin, and FKBP10 therapeutics for IPF, decelerate disease progression, but and attenuated the secretion of collagen I and III. Pirfenidone also their antifibrotic mechanisms of action are incompletely down-regulated collagen V but otherwise showed fewer and less understood. To the best of our knowledge, this study provides pronounced effects. By and large, the effects were similar in donor the first evidence for the inhibition of collagen fibril formation fibroblasts. For both drugs, electron microscopy of IPF fibroblast as a major mechanism of action for nintedanib and pirfenidone cultures revealed fewer and thinner collagen fibrils compared and puts forward extracellular collagen self-assembly as a with untreated controls. Finally, both drugs dose-dependently druggable target in IPF. delayed fibril formation of purified collagen I. In summary, both (Received in original form July 12, 2016; accepted in final form March 3, 2017 ) This work was supported by the Helmholtz Association, the German Center for Lung Research, and the Shriners Hospital for Children (Grant No. 85100). Author Contributions: Conception and design: L.K., Y.I., K.H., R.H., J.B., A.W., H.P.B., O.E., and C.A.S.-W.; experimental work, analysis, and interpretation: L.K., Y.I., M.A., H.P.B., and C.A.S.-W.; and drafting of the manuscript and revision for intellectual content: L.K., Y.I., M.A., H.P.B., O.E., and C.A.S.-W. Correspondence and requests for reprints should be addressed to: Claudia A. Staab-Weijnitz, Ph.D., Comprehensive Pneumology Center, Ludwig-Maximilians- Universitat ¨ and Helmholtz Zentrum Munchen, ¨ Max-Lebsche-Platz 31, 81377 Munchen, ¨ Germany. E-mail: [email protected] This article has an online data supplement, which is accessible from this issue’s table of content online at www.atsjournals.org Am J Respir Cell Mol Biol Vol 57, Iss 1, pp 77–90, Jul 2017 Copyright © 2017 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2016-0217OC on March 3, 2017 Internet address: www.atsjournals.org Knuppel, ¨ Ishikawa, Aichler, et al.: IPF Drugs Inhibit Collagen Fibril Formation 77 ORIGINAL RESEARCH Idiopathic pulmonary fibrosis (IPF) is a knockdown of FKBP10 in IPF fibroblasts Materials and Methods progressive and fatal interstitial lung disease decreases collagen type I synthesis and with a median survival of 3–5 years after secretion (10). In addition, PTMs such as For more details on materials and methods, diagnosis (1). The underlying pathogenic hydroxylation of lysyl or prolyl residues, or see the online supplement. Statistical processes are poorly understood, but the glycosylation of hydroxylysines, are analysis was performed in GraphPadPrism aberrant fibrotic response is likely initiated essential for proper stability, assembly, and 7.02 (GraphPad Software, San Diego, CA). by repeated microinjuries to the airway and secretion of procollagen, as well as for the the alveolar epithelium (2). This leads to final supramolecular structure of these the secretion of fibrotic mediators, molecules (13). For example, MTT Cytotoxicity Assay including transforming growth factor b hydroxylation of proline residues on See the online supplement. (TGF-b), which results in the accumulation position three (3-Hyp) might play a role fi of myo broblasts in alveolar regions. in inter-triple-helical interactions and Human Lung Material and Culture Multiple progenitor cells may contribute to probably assists in the assembly of of phLF fi fi the myo broblast population, but the most supramolecular collagen and lateral bril Primary human lung fibroblasts, isolated well-established source is the interstitial growth (14, 15). from human lung explant material of fi fi broblast (3). Myo broblasts synthesize Nintedanib and pirfenidone were patients with IPF or healthy donors, were and deposit excessive amounts of approved recently for IPF therapy, because obtained from the BioArchive CPC-M extracellular matrix (ECM) proteins, such both drugs have been shown to slow down for lung diseases at the Comprehensive as collagen type I, III, and V, and disease progression as measured by Pneumology Center (CPC, Munich, fi bronectin (4). The resulting accumulation reduced lung function decline. Despite their Germany). All participants gave written of ECM in the alveolar region is the widespread application in IPF in recent informed consent, and the study was ultimate pathological feature of lung times, their mechanisms of action are approved by the local ethics committee fi brosis, leading to progressive lung poorly understood and remain to be fully of the Ludwig-Maximilians University function decline (5). elucidated (16, 17). Nintedanib, originally of Munich, Germany. Isolation of A recent study highlights that collagens developed as an anticancer drug, is a phLF was performed as described previously are the main components of newly receptor tyrosine kinase inhibitor of (10). For more details, see the online fi synthesized ECM in lung brosis (6), but platelet-derived growth factor receptor, supplement. large-scale quantitative proteome fibroblasts growth factor receptor, and approaches have also demonstrated that the vascular endothelial growth factor receptor, ECM composition (the matrisome) is far all of which play important roles in the Cotreatment of IPF and Donor phLF more complex than previously assumed (7). pathogenesis of IPF (5). Pirfenidone is an with TGF-b1 and Nintedanib or In addition, the complexity of collagen antifibrotic, antiinflammatory, and Pirfenidone biosynthesis and maturation is rarely antioxidant compound with beneficial effects Cells were seeded at a density of 2 taken into account in mechanistic in lung, hepatic, kidney, and cardiac 20,000–25,000 cells/cm , followed by studies of the evaluation of antifibrotic fibrosis, but its direct targets are unknown starvation for 24 hours in starvation strategies. Collagen modification and (18, 19, 20). Several studies have investigated medium (Dulbecco’s modified Eagle folding in the rough endoplasmic the effects of either nintedanib or medium/F12, 0.5% fetal bovine serum, reticulum (rER) requires several enzymes pirfenidone on collagen type I expression or penicillin/streptomycin, 0.1 mM 2- and molecular chaperones essential for secretion in several cell types (10, 21–26). No phospho-L-ascorbic acid). Subsequently, post-translational modifications (PTMs) study to date, however, has directly cells were treated with or without TGF-b1 and the processing of procollagen into compared both drugs on the multiple stages (2 ng/ml) (R&D Systems, Minneapolis, triple helices, where one of the rate-limiting of intracellular collagen synthesis and MN) and with nintedanib (0.01 mM, steps is the cis-trans isomerization of extracellular maturation in the relevant cell 0.1 mM, 1.0 mM) or pirfenidone (100 mM, proline residues catalyzed by rER resident type (i.e., the primary human lung 500 mM, 1,000 mM) (both Selleck, Houston, peptidyl prolyl isomerases (8). Collagen fibroblast). TX) for 48 hours in starvation medium. triple helix formation is followed by its Therefore, the aim of our study was Nintedanib and pirfenidone were dissolved secretion, extracellular fibril formation, to comprehensively assess and directly in dimethyl sulfoxide. The final dimethyl and fiber assembly (9). Two endoplasmic compare the effects of nintedanib and sulfoxide concentration in the medium was reticulum proteins participating in this pirfenidone on the different steps of collagen always 1%. multistep process are the collagen synthesis and maturation in primary human fi chaperones FK506-binding protein 10 lung broblasts (phLF) from patients with RNA Isolation and Real-Time (FKBP10, also called FKBP65) and heat- IPF and healthy donors. We analyzed the Quantitative Reverse-Transcriptase shock protein 47 (HSP47, also called expression of various collagens and the Polymerase Chain Reaction Analysis SerpinH1) (9). Notably, both FKBP10 and collagen chaperones FKBP10 and HSP47,as See the online supplement. HSP47 are up-regulated in bleomycin- well as collagen secretion in IPF and induced lung fibrosis and in IPF (10, 11). healthy phLF. In addition, we examined the Secretion of procollagen from HSP47- effects of both drugs on the levels of Protein Isolation and Western Blot deficient fibroblasts is reduced compared selected PTMs of collagen in IPF fibroblasts Analysis with control cells (12) and similarly, and on collagen fibril formation. See the online supplement. 78 American Journal of Respiratory Cell and Molecular Biology Volume 57 Number 1 | July 2017 ORIGINAL RESEARCH Quantification of Secreted Collagen Initially, we analyzed the effect of protein levels, in both IPF and donor Collagen I and III were precipitated from increasing doses of nintedanib (0.01–1 mM) fibroblasts (Figure E2). In contrast, cell culture supernatant of cultured IPF and and pirfenidone (100-1,000 mM) on the pirfenidone reduced FN1 mRNA, but not donor phLF as described previously (10). viability of IPF fibroblasts in an MTT assay. protein levels, in IPF fibroblasts (Figures For more details, see the online supplement. The used concentrations of nintedanib and E2A, E2C, and E2E) and had no significant pirfenidone were well tolerated for the effect in donor phLF (Figures E2B, E2D, Collagen Precipitation and Analysis treatment period of 48 hours (see Figure E1 and E2F). Interestingly, pirfenidone of PTM in the online supplement). actually showed a trend to increase FN1 See the online supplement. protein levels in both IPF and donor phLF Nintedanib Reduced the Expression (Figures E2E and E2F). Expression of Scanning Electron Microscopy for and Secretion of ECM Components PAI-1, a classic TGF-b–induced gene, was Assessment of Fibrils in the ECM More Effectively Than Did Pirfenidone reduced significantly by nintedanib in of phLF in phLF IPF and donor fibroblasts, with a more IPF phLF were grown on glass slides, treated Next, we assessed the effect of different pronounced effect in the absence of with nintedanib (1 mM) or pirfenidone concentrations of both drugs on the TGF-b1 (Figure E3). Pirfenidone did not (1,000 mM) in combination with TGF-b1 expression and secretion of collagen I affect PAI-1 expression. (2 ng/ml) for 48 hours and fixed with (COL1A1), III (COL3A1), V (COL5A1), In summary, in comparison with paraformaldehyde and glutaraldehyde, 3% fibronectin 1 (FN1), and plasminogen nintedanib, pirfenidone showed fewer each, in 0.1% sodium cacodylate buffer pH activator inhibitor 1 (PAI-1) in lysates and effects on collagen expression and secretion 7.4 (Electron Microscopy Sciences, Munich, cell culture supernatants from primary and FN1 and PAI-1 expression. Notably, Germany). The specimens were dehydrated human IPF and donor fibroblasts. exceeding the effective concentration of in gradual ethanol and dried by the critical- Nintedanib consistently down-regulated nintedanib at by least 500 times, a m point method, using CO2 as the transitional transcript and protein levels of basal and concentration of 500 to 1,000 M fluid (Polaron Critical Point Dryer CPC TGF-b1–induced collagen I in IPF phLF pirfenidone was necessary to achieve E3000; Quorum Technologies, Ringmer, (Figures 1A, 1C, 1E, and 1G). Pirfenidone significant effects (e.g., on collagen I, UK). Specimens were sputter coated with a reduced TGF-b1–induced COL1A1 III, and V and FN1 expression) in thin layer of platinum by a sputtering transcripts only marginally (Figures 1A and TGF-b1–treated IPF fibroblasts (Figures device (Emitech K575; Quorum 1C), and collagen I protein remained largely 1A, 2A, 3E, and E2A). Technologies) and observed by scanning unchanged (Figures 1E and 1G). Similar electron microscopy (JSM 6300F; JEOL, tendencies were observed in phLF isolated Nintedanib, but Not Pirfenidone, Eching, Germany). Fibril thickness was from healthy donor lungs (Figures 1B, 1D, Reduces the Expression of the assessed by measuring the diameter of the and 1F), with the exception of COL1A1 Collagen Chaperone FKBP10 in smallest unit of fiber forming fibrils, using transcript (Figure 1B) and levels of basal IPF Fibroblasts the length measurement tool of the open secreted collagen I (Figure 1H), which both, Next, we investigated the effects of source software ImageJ 1.50i (W.S. in contrast to IPF, appeared unaffected by nintedanib and pirfenidone on the Rasband, National Institutes of Health, nintedanib in donor fibroblasts. expression of FKBP10 and HSP47, two Bethesda, MD). Similarly, the expression of collagen III collagen I chaperones. Nintedanib was consistently down-regulated by moderately, but significantly, down- fi Collagen I Fibril Formation Assay nintedanib in IPF broblasts on transcript regulated protein levels of FKBP10 in b – This assay was performed essentially as and protein levels, whereas pirfenidone mock- as well as in TGF- 1 treated IPF fi described previously (27). For more details, merely regulated COL3A1 transcripts, an broblasts (Figure 4E). This effect was see the online supplement. effect which again did not translate to already visible on the transcript level for protein level (Figures 2A, 2C, and 2E). TGF-b–treated samples (Figures 4A and Interestingly, in donor fibroblasts, 4C). In contrast, pirfenidone did not Results nintedanib increased and pirfenidone influence FKBP10 expression in IPF decreased COL3A1 transcription (Figures fibroblasts (Figures 4A, 4C, and 4E). Applied Concentrations of Nintedanib 2B and 2D), whereas the amount of Interestingly, clearly different results and Pirfenidone Were Well Tolerated secreted collagen III remained largely were obtained with donor fibroblasts by IPF phLF unchanged or even tended to anticorrelate here, where nintedanib failed to For in vitro experiments, we selected a with transcript levels (Figure 2F). regulate FKBP10 expression, and range of nintedanib and pirfenidone As for collagen V, TGF-b1–induced pirfenidone down-regulated FKBP10 concentrations similar to those used in collagen V was significantly reduced in IPF transcript but up-regulated FKBP10 published studies (22, 23, 25). Notably, for fibroblasts by both drugs, an effect that was protein levels (Figures 4B, 4D, and 4F). pirfenidone, in an effort to adhere to captured only partly at the transcript level Regarding the expression of the major physiologically relevant concentrations, we (Figures 3A, 3C, and 3E); similar trends collagen I chaperone HSP47, neither drug used 1,000 mM as the highest were observed in donor phLF (Figures 3B, reduced HSP47 protein levels in phLF concentration, although others have used 3D, and 3F). (Figures 5E and 5F), even if both pirfenidone in concentrations of up to Expression of FN1 was consistently therapeutics decreased basal HSP47 mRNA 10 mM in similar experiments (21, 26, 28). decreased by nintedanib on transcript and (Figures 5A–5D). Knuppel, ¨ Ishikawa, Aichler, et al.: IPF Drugs Inhibit Collagen Fibril Formation 79 ORIGINAL RESEARCH A IPF B Donor nintedanib pirfenidone nintedanib pirfenidone 14 **** ** * 14 12 12 10 10 8 8 Ct (COL1A1-DHX8) Ct (COL1A1-DHX8) Δ Δ - - 6 6 ctrl ctrl ctrl ctrl ctrl ctrl ctrl ctrl TGF-β ––––++++ –––– ++++ TGF-β ––––++++ –––– ++++ C nintedanib pirfenidone D nintedanib pirfenidone 2.0 *** * 2.0 1.5 1.5 1.0 1.0 COL1A1 COL1A1 0.5 0.5 transcript fold change 0 transcript fold change 0 TGF-β ––– +++ ––– +++ TGF-β ––– +++ ––– +++ E nintedanib pirfenidone F nintedanib pirfenidone * 1.5 ** 1.5 1.0 1.0 -actin) -actin) β β 0.5 0.5 (Col I/ (Col I/ realtive band intensity 0 realtive band intensity 0 ctrl ctrl ctrl ctrl ctrl ctrl ctrl ctrl β TGF- ––––++++ ––––++++ TGF-β ––––++++ –––– ++++ Col I ~ 250 kDa ~ 250 kDa Col I ~ 130 kDa ~ 130 kDa β -actin β-actin nintedanib pirfenidone G H nintedanib pirfenidone 4 ** * 4 3 3 2 2 Col I Col I 1 1 realtive band intensity realtive band intensity 0 0 ctrl ctrl ctrl ctrl ctrl ctrl ctrl ctrl TGF-β ––––++++ –––– ++++ TGF-β ––––++++ ––––++++ Col I ~ 130 kDa Col I ~ 130 kDa Figure 1. Nintedanib decreases collagen I expression and secretion more potently than does pirfenidone in idiopathic pulmonary fibrosis (IPF) fibroblasts. (A–D) Quantitative RT-PCR analysis of primary human lung fibroblasts (phLF) isolated from (A and C) patients with IPF or (B and D) healthy donors treated for 48 hours with increasing concentrations of nintedanib (0.01, 0.1, and 1 mM) or pirfenidone (100, 500, and 1,000 mM) in the absence or presence of transforming growth factor b (TGF-b) 1 (2 ng/ml). Transcript levels of COL1A1 are shown as 2DCt values (A and B) as well as transcript fold changes (C and D) to show the effect normalized to control (ctrl). DEAH (Asp-Glu-Ala-His) box polypeptide 8 (DHX8) was used as endogenous control. Data are based on 80 American Journal of Respiratory Cell and Molecular Biology Volume 57 Number 1 | July 2017 ORIGINAL RESEARCH Selected Post-Translational appearance than in control samples, and synthesis of ECM components and collagen Modifications of Collagen I Are Not the fibril thickness was significantly secretion, nintedanib was clearly more Affected by Nintedanib or Pirfenidone reduced in nintedanib- and pirfenidone- effective, because it (1) exerted its effects at PTMs have a major impact on essential treated samples compared with control substantially lower concentrations (up to collagen properties such as the three- fibrils (Figure 6B). 1,000-fold) than did pirfenidone, (2) dimensional structure, thermodynamic affected the expression and secretion of stability, and biological functions (13, 14, Nintedanib and Pirfenidone Inhibit more ECM and ECM-related genes 15), but, to date, whether antifibrotic drugs Spontaneous Collagen I Fibril (i.e., fibronectin, FKBP10, and collagen I), affect the PTMs of collagen secreted by IPF Formation and (3) showed more consistent effects on fibroblasts has not been assessed. Here, we It seemed unlikely that the effects on transcript and protein levels. With few fi tested whether nintedanib and pirfenidone collagen bril formation observed by exceptions, these effects were mostly similar affected the levels of selected PTMs of scanning electron microscopy could be in IPF and donor fibroblasts. Importantly, fi collagen I. More speci cally, we compared accounted for by the effects of both drugs on both drugs strongly inhibited extracellular the levels of prolyl-3-hydroxylation of the collagen synthesis and secretion only, in fibril formation, and assessment of A1 site (Pro-986) and the A3 site (Pro-707) particular in the case of pirfenidone. spontaneous fibril assembly using purified a of the collagen 1 chain (15), prolyl-3- Therefore, we investigated the direct effect collagen I indicated that both drugs directly hydroxylation of the A3 site (Pro-707) of of both drugs on spontaneous collagen I inhibited this process with comparable a fi fibril formation in a light-scattering the collagen 2 chain and, nally, the efficiency. approach. This assay is a well- established glycosylation site of hydroxylysine (Lys- Both nintedanib and pirfenidone method of studying the formation of 174) of collagen type I. Neither drug inhibited TGF-b–induced transcription of collagen fibrils in a solution of purified appeared to affect the levels of the assessed COL1A1, COL3A1, and FN1. In agreement, soluble collagen I. It relies on the principle PTMs (Figure E4). previous reports by others have shown that collagen I, dissolved initially in dilute that nintedanib and pirfenidone counteract acid, spontaneously forms fibrils on TGF-b signaling and down-regulate these Nintedanib and Pirfenidone Affect neutralization in a self-driven process. The TGF-b target genes (23, 24, 32). Although Collagen Fibril Formation in IPF resulting fibrils are similar to those formed Fibroblasts pirfenidone showed these effects only in the in vivo, and the process can be visualized by b Scanning electron microscopy was used to dynamic light scattering at 313 nm (30, 31). presence of exogenously added TGF- 1, assess the number, morphology, and We found that both therapeutics were able nintedanib also affected basal levels of fi thickness of extracellular fibrils formed in to considerably delay fibril formation of COL1A1 in IPF broblasts and basal levels b cultures of TGF-b1–treated IPF fibroblasts purified collagen I already at micromolar of another TGF- target gene, PAI-1,in fi in the absence and presence of nintedanib concentrations in a dose-dependent both IPF and donor broblasts (Figures 1A or pirfenidone. Extracellular collagen fibers manner (Figure 7 and Table 1). and E3). The platelet-derived growth factor were identified as unbranched and dense receptor (PDGFR) and the fibroblast bundles of thread-like–looking twisted growth factor receptor are known targets of fibrils of variable length, which were Discussion the receptor tyrosine kinase inhibitor oriented randomly, and with a maximal nintedanib, and this might reflect the diameter of 1 mm (Figure 6A) (29, 31). Cell In this study, we demonstrated that inhibition of autocrine platelet-derived cultures that had been treated with 1.0 mM nintedanib and pirfenidone affect collagen growth factor or fibroblast growth factor nintedanib or 1.0 mM pirfenidone synthesis and maturation on several signaling, which has been shown to regulate displayed a markedly reduced number of regulatory levels, including the inhibition of collagen gene expression (5, 33, 34) via both fibers and changes in overall fiber structure. collagen gene expression, collagen secretion, the ERK and the PI3K/Akt signaling In the presence of both drugs, fibers were and, most importantly, fibril formation. pathways (35). In agreement, shorter overall and showed a more frayed In terms of intracellular regulation of the phosphorylation of Akt was decreased in Figure 1. (Continued). seven (IPF) or three (donor) completely independent experiments and are presented as mean 6 SEM. Statistical significance between control and different concentrations of nintedanib or pirfenidone is indicated by horizontal brackets and asterisks for 2DCt values and by asterisks only for fold changes relative to 1. (E and F) Western blot analysis of phLF isolated from (E) patients with IPF or (F) donors treated for 48 hours with increasing concentrations of nintedanib (0.01, 0.1, and 1 mM) or pirfenidone (100, 500, and 1,000 mM) in the absence or presence of TGF-b1(2ng/ml). Densitometric analysis and representative blots show the effect of nintedanib and pirfenidone on collagen I (Col I) protein expression relative to b-actin as loading control. Data are based on eight (IPF) or three (donor) completely independent experiments and are presented as mean 6 SEM. Statistical significance between control and different concentrations of nintedanib or pirfenidone is indicated by horizontal brackets and asterisks.(G and H) Western blot analysis of secreted Col I precipitated from cell culture supernatant of (G) IPF or (H) donor fibroblasts treated for 48 hours with increasing concentrations of nintedanib (0.01, 0.1, and 1 mM) or pirfenidone (100, 500, and 1,000 mM) in the absence or presence of TGF-b1 (2 ng/ml). Densitometric analysis and representative blots show the effects of nintedanib and pirfenidone on secreted Col I after 48 hours. Data are based on seven (IPF) or three (donor) completely independent experiments and are given as mean 6 SEM. Statistical significance between control and different concentrations of nintedanib or pirfenidone is indicated by horizontal brackets and asterisks. Statistical analysis was performed by one-way analysis of variance (post-test, Bonferroni‘s multiple comparison test: comparison against control). *P , 0.1, **P , 0.01, ***P , 0.001, ****P , 0.0001. The well-known effect of TGF-b1 on these transcripts and proteins was significant, but it is not specified in the interest of clarity. Knuppel, ¨ Ishikawa, Aichler, et al.: IPF Drugs Inhibit Collagen Fibril Formation 81 ORIGINAL RESEARCH A B IPF Donor nintedanib pirfenidone nintedanib pirfenidone p= 0.19 10 p= 0.15 * 10 * * 8 8 6 6 Ct (COL3A1-DHX8) 4 Ct (COL3A1-DHX8) 4 Δ Δ - - 2 2 ctrl ctrl ctrl ctrl ctrl ctrl ctrl ctrl TGF-β – –––++++ –––– ++++ TGF-β ––––++++ – ––– ++++ C D nintedanib pirfenidone nintedanib pirfenidone 2.0 * 2.0 p= 0.19 1.5 1.5 1.0 1.0 COL3A1 COL3A1 0.5 0.5 transcript fold change transcript fold change 0 0 TGF-β ––– +++ ––– +++ TGF-β ––– +++ ––– +++ E F nintedanib pirfenidone nintedanib pirfenidone 3 * 3 2 2 Col III Col III 1 1 relative band intensity relative band intensity 0 0 ctrl ctrl ctrl ctrl ctrl ctrl ctrl ctrl TGF-β ––––++++ –––– ++++ TGF-β ––––++++ –––– ++++ Col III ~ 130 kDa Col III ~ 130 kDa Figure 2. COL3A1 transcription is decreased by nintedanib in IPF fibroblasts and increased in donor fibroblasts, whereas collagen III (Col III) secretion is decreased in both. (A–D) Quantitative RT-PCR analysis of phLF isolated from (A and C) patients with IPF or (B and D) healthy donors treated for 48 hours with increasing concentrations of nintedanib (0.01, 0.1, and 1 mM) or pirfenidone (100, 500, and 1,000 mM) in the absence or presence of TGF-b1 (2 ng/ml). Transcript levels of COL3A1 are shown as 2DCt values (A and B) as well as transcript fold changes (C and D) to show the effect normalized to control. DHX8 was used as endogenous control. Data are based on seven (IPF) or three (donor) completely independent experiments and are given as mean 6 SEM. Statistical significance between control and different concentrations of nintedanib or pirfenidone is indicated by horizontal brackets and asterisks for 2DCt values and by asterisks only for fold changes relative to 1. The well-known effect of TGF-b1 on these transcripts was significant, but it is not specified in the interest of clarity. (E and F) Western blot analysis of secreted Col III precipitated from cell culture supernatant of (E) IPF or (F) donor fibroblasts after treatment of phLF isolated from patients with IPF treated for 48 hours with increasing concentrations of nintedanib (0.01, 0.1, and 1 mM) or pirfenidone (100, 500, and 1,000 mM) in the absence or presence of TGF-b1 (2 ng/ml). Densitometric analysis and representative blots show the effects of nintedanib and pirfenidone on secreted Col III after 48 hours. Data are based on seven (IPF) or three (donor) completely independent experiments and are given as mean 6 SEM. Statistical significance between control and different concentrations of nintedanib or pirfenidone is indicated by horizontal brackets and asterisks. Statistical analysis was performed by one-way analysis of variance (posttest, Bonferroni‘s multiple comparison test: comparison against control). *P , 0.1. 82 American Journal of Respiratory Cell and Molecular Biology Volume 57 Number 1 | July 2017 ORIGINAL RESEARCH A B IPF Donor nintedanib pirfenidone nintedanib pirfenidone 8 p= 0.16 p= 0.15 8 * 6 6 4 4 2 2 0 0 Ct (COL5A1-DHX8) Ct (COL5A1-DHX8) Δ Δ - –2 - –2 ctrl ctrl ctrl ctrl ctrl ctrl ctrl ctrl TGF-β ––––++++ –––– ++++ TGF-β ––––++++ –––– ++++ C D nintedanib pirfenidone nintedanib pirfenidone 2.0 * 2.0 1.5 1.5 1.0 1.0 COL5A1 COL5A1 0.5 0.5 transcript fold change transcript fold change 0 0 TGF-β ––– +++ ––– +++ TGF-β ––– +++ ––– +++ E F nintedanib pirfenidone nintedanib pirfenidone 2.0 * * 2.0 1.5 1.5 -actin) -actin) β 1.0 β 1.0 0.5 (Col V/ 0.5 (Col V/ relative band intensity 0 relative band intensity 0 ctrl ctrl ctrl ctrl ctrl ctrl ctrl ctrl TGF-β ––––++++ –––– ++++ TGF-β ––––++++ –––– ++++ Col V ~ 250 kDa Col V ~ 250 kDa β-actin β-actin Figure 3. Nintedanib and pirfenidone down-regulate collagen V (Col V). (A–D) Quantitative RT-PCR analysis of phLF isolated from (A and C) patients with IPF or (B and D) healthy donors treated for 48 hours with increasing concentrations of nintedanib (0.01, 0.1, and 1 mM) or pirfenidone (100, 500, and 1,000 mM) in the absence or presence of TGF-b1 (2 ng/ml). Transcript levels of (A and C) IPF and (B and D) donor phLF of COL5A1 are shown as 2DCt values (A and B) as well as transcript fold changes (C and D) to show the effect normalized to control (ctrl). DHX8 was used as endogenous control. Data are based on seven (IPF) or three (donor) completely independent experiments and are given as mean 6 SEM. Statistical significance between control and different concentrations of nintedanib or pirfenidone is indicated by horizontal brackets and asterisks for 2DCt values and by asterisks only for fold changes relative to 1. (E and F) Western blot analysis of phLF isolated from (E) patients with IPF or (F) healthy donors treated for 48 hours with increasing concentrations of nintedanib (0.01, 0.1, and 1 mM) or pirfenidone (100, 500, and 1,000 mM) in the absence or presence of TGF-b1 (2 ng/ml). Densitometric analysis and representative blots show the effect of nintedanib and pirfenidone on Col V protein expression relative to b-actin. Data are based on eight (IPF) or three (donor) completely independent experiments and are given as mean 6 SEM. Statistical significance between control and different concentrations of nintedanib or pirfenidone is indicated by horizontal brackets and asterisks. Statistical analysis was performed by one-way analysis of variance (posttest, Bonferroni‘s multiple comparison test: comparison against control). *P , 0.1. The well-known effect of TGF-b1onthese transcripts and proteins was significant, but it is not specified in the interest of clarity. Knuppel, ¨ Ishikawa, Aichler, et al.: IPF Drugs Inhibit Collagen Fibril Formation 83 ORIGINAL RESEARCH AB IPF Donor nintedanib pirfenidone nintedanib pirfenidone ** p= 0.15 6 6 4 4 Ct (FKBP10-DHX8) Ct (FKBP10-DHX8) Δ Δ - - 2 2 ctrl ctrl ctrl ctrl ctrl ctrl ctrl ctrl TGF-β ––––++++ –––– ++++ TGF-β ––––++++ –––– ++++ C nintedanib pirfenidone D nintedanib pirfenidone 2.0 * *** 2.0 1.5 1.5 1.0 1.0 FKBP10 FKBP10 0.5 0.5 transcript fold change transcript fold change 0 0 β TGF-β –––+++ ––– +++ TGF- –––+++ ––– +++ E F nintedanib pirfenidone nintedanib pirfenidone p= 0.19 p= 0.19 * 2.0 2.0 * 1.5 1.5 -actin) -actin) β β 1.0 1.0 0.5 0.5 FKBP10/ (FKBP10/ relative band intensity relative band intensity 0 0 ctrl ctrl ctrl ctrl ctrl ctrl ctrl ctrl TGF-β ––––++++ –––– ++++ TGF-β ––––++++ –––– ++++ FKBP10 ∼ 70 FKBP10 ∼ 70 kDa kDa β-actin β-actin Figure 4. Expression of the collagen chaperone FKBP10 is consistently down-regulated by nintedanib in IPF but regulated more dynamically by pirfenidone in donor fibroblasts. (A–D) Quantitative RT-PCR analysis of phLF isolated from (A and C) patients with IPF or (B and D) healthy donors treated for 48 hours with increasing concentrations of nintedanib (0.01, 0.1, and 1 mM) or pirfenidone (100, 500, and 1,000 mM) in the absence or presence of TGF-b1 (2 ng/ml). Transcript levels of (A and C)IPFand(B and D) donor phLF of FKBP10 are shown as 2DCt values (A and B)as well as transcript fold changes (C and D) to show the effect normalized to control. DHX8 was used as endogenous control. Data are based on seven (IPF) or three (donor) completely independent experiments and are given as mean 6 SEM. Statistical significance between control and different concentrations of nintedanib or pirfenidone is indicated by horizontal brackets and asterisks for 2DCt values and by asterisks only for fold changes relative to 1. (E and F) Western blot analysis of phLF isolated from (E )patientswithIPFor(F ) healthy donors treated for 48 hours with increasing concentrations of nintedanib (0.01, 0.1, and 1 mM) or pirfenidone (100, 500, and 1,000 mM) in the absence or presence of TGF-b1 84 American Journal of Respiratory Cell and Molecular Biology Volume 57 Number 1 | July 2017 ORIGINAL RESEARCH response to nintedanib in all our and 3F). To our knowledge, down- TGF-b–induced collagen III secretion. experiments (Figures E5A and E5B). In regulation of collagen V levels in response Pirfenidone showed only weak inhibitory contrast, phosphorylation of ERK was not to nintedanib or pirfenidone has not effects on basal collagen I secretion consistently changed, either in IPF or in been reported before. Type V collagen is (Figure 1G), and, notably, no significant donor fibroblasts (Figures E5C and E5D). a minor component of collagen type I effects on collagen III secretion (Figure 2E). In light of the time point studied (48 h after fibrils that plays an important role in Given the observed inhibitory effect of treatment start), this argues for a stronger fibrogenesis and regulation of fiber size nintedanib on total collagen secretion in and more sustained inhibition of the (37–39). In IPF lungs, collagen V is heavily our previous study (10), this suggests that PI3K/Akt signaling pathway by nintedanib overexpressed compared with that in nintedanib-induced down-regulation of in our studies. Inhibition of PDGFR normal lungs (38). Importantly, in the collagen III secretion contributes more signaling, however, cannot explain all our context of the observed effects on fibril strongly to the decrease of total secreted in vitro results, because a previous study thickness (Figure 6B), collagen V has been collagen than does collagen I in IPF from our laboratory (34) found that small shown to be crucial for the initiation of fibroblasts. interfering RNA–mediated down- collagen fibril assembly (39). Therefore, FKBP10 and HSP47 are rER-resident regulation of PDGFR-a actually increased down-regulation of collagen V by both chaperones, critical for the proper folding of levels of collagen V drastically in primary drugs likely contributes to the phenomenon triple-helical procollagen (9, 12). Deficiency human lung fibroblasts, which is in contrast of fewer and thinner fibrils in the of both proteins leads to changes in the to what we observed in the presence of extracellular space of primary human IPF extracellular appearance of collagen fibrils nintedanib. fibroblasts described here (Figure 6). as, for example, reduced collagen It is striking that many of the observed Interestingly, Hostettler and colleagues (25) crosslinking (41, 42) or aberrant fibril effects translated to the protein level only found that matrix metalloprotease 2, an formation (12). Both collagen chaperones in the presence of nintedanib, but not ECM metalloprotease that cleaves collagen are increased in animal models of pirfenidone. For instance, both drugs inhibit V, is up-regulated, and its inhibitor, bleomycin-induced lung fibrosis and in TGF-b–induced COL1A1, COL3A1, and metalloproteinase inhibitor 2 (TIMP2), is patients with IPF (7, 10, 11), and we FN1 transcription, whereas the levels of down-regulated in response to nintedanib. showed recently that small interfering collagen I protein, secreted collagen III, and This provides indirect evidence for RNA–mediated down-regulation of fibronectin protein were reduced only by increased extracellular degradation of FKBP10 attenuates the expression and nintedanib in IPF fibroblasts (Figures 1, 2, collagen V, and, collectively, this suggests secretion of collagen in pHLF (10). and E2). These results suggest that post- that nintedanib may decrease collagen V Interestingly, it had been reported in two transcriptional regulation mechanisms are via both an intracellular and an independent studies that pirfenidone affected differently by the drugs and extracellular mechanism. down-regulated the expression of HSP47 in highlight the importance of analysis at the To date, few studies have assessed the A549 cells and human lung fibroblasts, protein level in this context. Nevertheless, effects of nintedanib and pirfenidone on which suggested that pirfenidone exerted its other studies have reported effects of collagen secretion. Previously, using Sirius antifibrotic effects in part via the inhibition pirfenidone on collagen I and/or Red–based quantification of total collagen of intracellular collagen folding (21, 26). fibronectin protein levels in normal phLF in cell culture supernatant, we showed Therefore, we also assessed the regulation (26), alveolar epithelial cells (21), and nasal that nintedanib, but not pirfenidone, dose of HSP47 and FKBP10 by nintedanib and polyp fibroblasts (28). These discrepancies, dependently inhibited collagen secretion in pirfenidone in IPF and donor fibroblasts. however, may be the result of the use of IPF fibroblasts (10). Similarly, Hostettler Notably, nintedanib marginally, but substantially higher concentrations of and colleagues reported a reduction of total significantly, down-regulated TGF- pirfenidone in those studies (1.6–10 mM). secreted collagens in IPF and control b–induced FKBP10 expression on Notably, during standard treatment with fibroblasts on nintedanib treatment (25). transcript and protein levels in IPF pirfenidone of patients with IPF (three daily Because collagen I and III are the most fibroblasts, whereas pirfenidone had no doses of 801 mg pirfenidone), serum levels abundant fibrillar collagens in the lung effect (Figures 4A, 4C, and 4E). Strikingly, a of pirfenidone do not exceed 100 mM (36), interstitium and both are known to different pattern was observed here for a concentration at which we did not be increased in IPF (40), we further donor fibroblasts: FKBP10 expression observe any effect on phLF gene expression. characterized the inhibition of collagen remained unaffected by nintedanib, but Both nintedanib and pirfenidone secretion by both drugs, looking at these decreased at the transcript and increased significantly down-regulated collagen V in two specific collagen subtypes in this study. at the protein level by pirfenidone IPF fibroblasts, and a similar trend was Clearly, nintedanib was more effective (Figures 4B, 4D, and 4F). Regarding HSP47 observed in donor fibroblasts (Figures 3E in inhibiting basal collagen I and expression, neither drug had an effect on Figure 4. (Continued). (2 ng/ml). Densitometric analysis and representative blots show the effect of nintedanib and pirfenidone on FKBP10 protein expression relative to b-actin. Data are based on eight (IPF) or three (donor) completely independent experiments and are given as mean 6 SEM. Statistical significance between control and different concentrations of nintedanib or pirfenidone is indicated by horizontal brackets and asterisks. Statistical analysis was performed by one-way analysis of variance (posttest, Bonferroni‘s multiple comparison test: comparison against control). *P , 0.1, **P , 0.01, ***P , 0.001. The well-known effect of TGF-b1 on these transcripts and proteins was significant, but it is not specified in the interest of clarity. Knuppel, ¨ Ishikawa, Aichler, et al.: IPF Drugs Inhibit Collagen Fibril Formation 85 ORIGINAL RESEARCH ABIPF Donor nintedanib pirfenidone nintedanib pirfenidone **** * 10 * 10 8 8 6 6 Ct (HSP47-DHX8) Ct (HSP47-DHX8) Δ Δ - - 4 4 ctrl ctrl ctrl ctrl ctrl ctrl ctrl ctrl TGF-β ––––++++ –––– ++++ TGF-β ––––++++ –––– ++++ C nintedanib pirfenidone D nintedanib pirfenidone 1.5 * *** 1.5 1.0 1.0 HSP47 HSP47 0.5 0.5 transcript fold change transcript fold change 0 0 TGF-β ––– +++ ––– +++ TGF-β ––– +++ ––– +++ E F nintedanib pirfenidone nintedanib pirfenidone 1.5 1.5 1.0 1.0 -actin) -actin) β β 0.5 0.5 (HSP47/ (HSP47/ relative band intensity relative band intensity 0 0 ctrl ctrl ctrl ctrl ctrl ctrl ctrl ctrl TGF-β ––––++++ –––– ++++ TGF-β ––––++++ –––– ++++ ∼ ∼ HSP47 55 HSP47 55 kDa kDa β-actin β-actin Figure 5. Expression of the major collagen I chaperone HSP47 is reduced only on the transcript level by both drugs in IPF fibroblasts. (A–D) Quantitative RT-PCR analysis of phLF isolated from (A and C) patients with IPF or (B and D) healthy donors treated for 48 hours with increasing concentrations of nintedanib (0.01, 0.1, and 1 mM) or pirfenidone (100, 500, and 1,000 mM) in the absence or presence of TGF-b1 (2 ng/ml). Transcript levels of (A and C) IPF and (B and D) donor phLF of HSP47 are shown as 2DCt values (A and B) as well as transcript fold changes (C and D) to show the effect normalized to control. DHX8 was used as endogenous control. Data are based on seven (IPF) or three (donor) completely independent experiments and are given as mean 6 SEM. Statistical significance between control and different concentrations of nintedanib or pirfenidone is indicated by horizontal brackets and asterisks for 2DCt values and by asterisks only for fold changes relative to 1. (E and F) Western blot analysis of phLF isolated from (E) patients with IPF or (F) healthy donors treated for 48 hours with increasing concentrations of nintedanib (0.01, 0.1, and 1 mM) or pirfenidone (100, 500, and 1,000 mM) cotreated with or without TGF-b1 (2 ng/ml). Densitometric analysis and representative blots show the effect of nintedanib and pirfenidone on FKBP10 protein expression relative to b-actin. Data are based on eight (IPF) or three (donor) completely independent experiments and are given as mean 6 SEM. Statistical significance between control and different concentrations of nintedanib or pirfenidone is indicated by horizontal brackets and asterisks. Statistical analysis was performed by one-way analysis of variance (posttest, Bonferroni‘s multiple comparison test: comparison against control). *P , 0.1, ***P , 0.001, ****P , 0.0001. The well-known effect of TGF-b1 on these transcripts and proteins was significant, but it is not specified in the interest of clarity. 86 American Journal of Respiratory Cell and Molecular Biology Volume 57 Number 1 | July 2017 ORIGINAL RESEARCH A control nintedanib pirfenidone After cleavage of the propeptides, the collagen molecules become competent for A B C fibril formation. We took advantage of the fact that this process can be studied in a straightforward manner using purified 250x pepsin-digested collagen (27) and found that low micromolar concentrations of both 50 μm 50 μm 50 μm drugs inhibited collagen I fibril formation with comparable efficiencies in a dose- dependent manner (Figure 7). Even if the exact molecular mechanisms remain obscure, it can be speculated that 1000x pirfenidone and nintedanib directly bind to collagen triple helices and mask or μ μ μ 10 m 10 m 10 m alter interaction sites caused by changes in hydrophobicity or charges on the surface B ** of the triple helix. Moreover, considering ** that higher concentrations of pirfenidone 300 than of nintedanib were necessary to achieve the same amplitude of effect, 200 nintedanib likely displays a stronger affinity to collagen than does pirfenidone. 100 Finally, because pepsin-digested solubilized collagen is used in our Fibril thickness [nm] cell-free system, it can be concluded that 0 – control Nintedanib Pirfenidone the direct drug collagen interaction takes place in the collagenous region of Figure 6. Nintedanib and pirfenidone decrease the number and thickness of collagen fibrils and alter collagen and not in the telopeptides their appearance. (A) Scanning electron microscopy of extracellular matrix fibrils deposited by phLF treated for 48 hours with nintedanib (1 mM, middle panel) or pirfenidone (1,000 mM, right panel)and and propeptides, which are typically TGF-b (2 ng/ml) showed fewer, thinner, and more frayed fiber bundles when compared with control removed by pepsin digestion. (left panel). Magnification is indicated on the left side (upper panel: 250 3 ; lower panel: 1000 3 ). Interestingly, inhibition of collagen I Results shown are representative images of three independent experiments with similar results. (B) self-assembly has been proposed as a Thickness of single fibrils was measured in scanning electron microscopy pictures in three strategy for antifibrotic therapy, but this experiments using independently derived IPF fibroblasts. Statistical analysis was performed by a paired t concept has received little attention in the test. Scale bars,50mm in top panels of A–C,and10mminbottompanelsofA–C.**P , 0.01. field of lung fibrosis thus far (44). Instead, efforts have been undertaken to evaluate the inhibition of collagen crosslinking by protein levels (Figures 5E and 5F), even if 3-Hyp has been shown to have major the enzyme lysyl oxidase-like 2, notably a both drugs showed effects on the transcript effects on lateral fibril growth (15), we step subsequent to spontaneous fibril level (Figures 5A and 5C). Again, this is in also assessed hydroxylation of three formation that stabilizes existing fibrils contrast to previously reported results, 3-Hyp sites in collagen I, a comparatively (31, 45). Recently, however, a phase II study but may be due to higher pirfenidone rare collagen PTM, next to a lysyl with a monoclonal anti–lysyl oxidase-like concentrations used in those studies glycosylation site in collagen I. We did 2 antibody was terminated because of a (21, 26). not observe any effect of nintedanib or lack of efficacy (46). Here we show, to Importantly, we found much fewer, pirfenidone on these PTMs of collagen I our knowledge for the first time, that thinner, and aberrantly structured collagen (Figure E4). However, there are clearly nintedanib and pirfenidone down-regulate fibrils in the extracellular space of IPF many more collagen PTMs to consider collagen V, a minor collagen important fibroblasts treated with nintedanib or and, in light of the recently reported for the initiation of extracellular pirfenidone (Figure 6). This was profibrotic properties of ECM of patients fibrillogenesis, and directly inhibit collagen particularly unexpected for pirfenidone, with IPF (43), a broader PTM fibril formation. This suggests that both because we had observed only minor fingerprinting of collagens in the context of therapeutics exert their antifibrotic effects on collagen synthesis and secretion. fibrotic disease would undoubtedly be actions at least in part via the inhibition As pointed out earlier in the text, warranted. of collagen fibril formation, which provides a partial explanation for this result may Extracellular collagen fibril formation additional support for the concept of be the observed down-regulation of type is mainly an entropy-driven self-assembly inhibition of collagen self-assembly as a V collagen, which, even if it represents a process (31). The so-called collagen promising antifibrotic strategy. This is a minor constituent of collagen fibrils, D-stagger is formed by specific interactions particularly interesting finding for appears to be crucial for collagen of the residues along the triple-helical pirfenidone, in which the well-known fibrogenesis (39). Because deficiency of molecules with regularly staggered ends. antifibrotic effects in vivo to date stay in Knuppel, ¨ Ishikawa, Aichler, et al.: IPF Drugs Inhibit Collagen Fibril Formation 87 ORIGINAL RESEARCH A B 0.14 Type I collagen 0.14 Type I collagen + DMSO + DMSO + 0.5 μM Nintedanib + 1.25 μM Pirfenidone + 1.0 μM Nintedanib + 2.50 μM Pirfenidone 0.07 0.07 + 10.0 μM Pirfenidone A (313 nm) A (313 nm) 0.00 0.00 0 2040 60 80 100 0 2040 60 80 100 Time (min) Time (min) Figure 7. Spontaneous collagen fibril formation is inhibited by both drugs in a dose-dependent manner. A collagen type I stock solution in 50 mM acetic acid was diluted to a final concentration of 0.1 mM into a 0.1 M NaHCO3 buffer (pH 7.8) containing 0.15 M NaCl and 1 mM CaCl2 and heated up to 348C, followed by monitoring of absorbance (light scattering) at 313 nm. (A) Nintedanib (red, 0.5 mM[n = 2]; blue, 1.0 mM[n = 5]) and (B) pirfenidone (red, 1.25 mM[n = 3]; blue, 2.50 mM[n = 2]; green, 10.0 mM[n = 3]) on collagen type I fibril formation in comparison to dimethyl sulfoxide control (black [n = 4]) are shown. The resulting half-time values for fibril formation are given in Table 1. sharp contrast to concentrations in the the expression of the studied targets. The down-regulated basal collagen I secretion millimolar range required to observe most striking difference was observed in the only in IPF fibroblasts and not in donor effects on fibrotic marker expression regulation of the collagen chaperone fibroblasts. Apart from these differences, by (19–21, 26, 36). Notably, we believe our FKBP10, the expression of which was and large, the tendencies of the effects of study is the first to offer evidence for consistently down-regulated by nintedanib nintedanib and pirfenidone between IPF an antifibrotic effect of pirfenidone in IPF fibroblasts without evidence for and donor fibroblasts on fibrotic markers, in vitro, which occurs at a micromolar down-regulation in donor fibroblasts. Even collagen chaperones, and collagen concentration. For nintedanib, which affects more surprisingly, pirfenidone, which did secretion were similar. Notably, in a the intracellular collagen pathway more not affect FKBP10 expression in IPF previous study, we also observed that the strongly and in physiologically relevant fibroblasts, decreased FKBP10 transcript effects on collagen synthesis and secretion concentrations, the reduced fibril formation is and increased FKBP10 protein levels in were similar in IPF and normal control probably a combined result of intracellular donor fibroblasts. Another difference was fibroblasts, and we ultimately pooled those and extracellular events. Our findings that nintedanib had a stronger negative results for data presentation (10). This is furthermore emphasize that collagen V plays effect on COL1A1, COL3A1, and HSP47 also in agreement with the findings of two a hitherto underestimated role in fibrogenesis. transcription in IPF fibroblasts than in previously published studies (25, 47). We observed only few differences donor fibroblasts; at the same time, Lehtonen and colleagues (47) examined the between IPF and donor fibroblasts regarding however, protein levels were unchanged or effect of nintedanib and pirfenidone on the effects of nintedanib and pirfenidone on regulated similarly. Finally, both drugs fibroblast and myofibroblast properties and also observed only marginal differences between cells from control and IPF lungs. Table 1. Half-Time Values for Fibril Formation (Fibril Formation50) Hostettler and colleagues (25) studied collagen secretion by nintedanib in phLF Concentration of and IPF fibroblasts and found that Indicated Drug (mM) Fibril Formation50 (min) P Value* collagen secretion was down-regulated to the same extent in both cell types. 0 (DMSO control) 49.6 6 2.1 In conclusion, our findings provide Nintedanib an overview and a direct comparison 0.5 55.6 6 2.1 n.d. of the effects of the Food and Drug 1.0 59.1 6 2.0 0.000218† Pirfenidone Administration/European Medicines 1.25 54.2 6 4.7 0.133 Agency-approved IPF drugs nintedanib 2.5 57.3 6 4.6 n.d. and pirfenidone on different stages of ‡ 10 61.6 6 3.1 0.00165 expression and maturation of collagen in primary human lung fibroblast derived Definition of abbreviation: n.d., not determined (n = 2). from patients with IPF as well as from Nintedanib and pirfenidone increase half-time values for fibril formation (fibril formation50) dose dependently. Half-time values for fibril formation (fibril formation50) are defined as the time at which the healthy donors. Nintedanib clearly was absorbance reaches half the value of the total absorbance change. Data are derived from graphs more efficient than pirfenidone in shown in Figure 7 and are presented as mean 6 SD. inhibiting profibrotic gene expression and *Statistical analysis was performed on half-time values relative to DMSO control using one-way analysis of variance. collagen secretion, both in terms of the †P , 0.001. required effective concentration as well ‡P , 0.01. as in the number, consistency, and 88 American Journal of Respiratory Cell and Molecular Biology Volume 57 Number 1 | July 2017 ORIGINAL RESEARCH magnitude of its effects in independently of action for both drugs. We suggest direct interaction of the drugs with derived IPF fibroblast lines. 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Effects of FKBP10 deficiency. FKBP10 deficiency leads to several antifibrotic effects based on altered collagen or fibrotic marker expression. FKBP10 loss reduces collagen secretion and collagen I, III, V, FN expression. Collagen V and FN in turn are needed for extracellular collagen I fibril formation in vivo. FKBP10 downregulates collagen VI expression which in turn leads to reduced cell migration. FKBP10 might be needed to prevent HSP47 aggregation, which is needed for collagen triple helix formation, like FKBP10. P3H4 forms a complex with P3H3 and Cyclophilin B (CYPB) influencing LH1 activity. LH1 is needed for the generation of PTM in the helical regions of collagen. FKBP10 is a known modulator of LH2 activity. LH2 is responsible for PTMs in the telopeptide regions of collagen I, which are important in collagen fibril formation. Missing PTMs result in aberrant collagen fibril formation, followed by impaired collagen crosslinking and ultimately in an altered ECM topography. 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