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The Extracellular Matrix Receptor Discoidin Domain Receptor 1 Regulates Collagen Transcription by Translocating to the Nucleus

Manuel Chiusa,1 Wen Hu,1 Hong-Jun Liao,1 Yan Su,1 Corina M. Borza,1 Mark P. de Caestecker,1 Nataliya I. Skrypnyk,1 Agnes B. Fogo,3 Vadim Pedchenko,1 Xiyue Li,1 Ming-Zhi Zhang,1 Billy G. Hudson,1 Trayambak Basak,1 Roberto M. Vanacore,1 Roy Zent,1,2 and Ambra Pozzi1,2

1Division of Nephrology and Hypertension, Department of Medicine, and 3Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee; and 2Department of Veterans Affairs, Nashville, Tennessee

ABSTRACT Background The discoidin domain receptor 1 (DDR1) is activated by collagens, upregulated in injured and fibrotic kidneys, and contributes to fibrosis by regulating extracellular matrix production, but how DDR1 controls fibrosis is poorly understood. DDR1 is a receptor tyrosine kinase (RTK). RTKs can translocate to the nucleus via a nuclear localization sequence (NLS) present on the receptor itself or a ligand it is bound to. In the nucleus, RTKs regulate gene expression by binding chromatin directly or by interacting with tran- scription factors. Methods To determine whether DDR1 translocates to the nucleus and whether this event is mediated by collagen-induced DDR1 activation, we generated renal cells expressing wild-type or mutant forms of DDR1 no longer able to bind collagen. Then, we determined the location of the DDR1 upon collagen stimulation. Using both biochemical assays and immunofluorescence, we analyzed the steps involved in DDR1 nuclear translocation. Results We show that although DDR1 and its natural ligand, collagen, lack an NLS, DDR1 is present in the nucleus of injured human and mouse kidney proximal tubules. We show that DDR1 nuclear translocation requires collagen-mediated receptor activation and interaction of DDR1 with SEC61B, a component of the Sec61 translocon, and nonmuscle myosin IIA and b-actin. Once in the nucleus, DDR1 binds to chromatin to increase the transcription of collagen IV, a major collagen upregulated in fibrosis. Conclusions These findings reveal a novel mechanism whereby activated DDR1 translates to the nucleus to regulate synthesis of profibrotic molecules.

JASN 30: 1605–1624, 2019. doi: https://doi.org/10.1681/ASN.2018111160

Discoidin domain receptors (DDRs) are receptor tyrosine kinases (RTKs) that undergo tyrosine au- tophosphorylation upon interaction with colla- Received November 25, 2018. Accepted May 20, 2019. gens.1,2 The DDR family consists of two members: fi Published online ahead of print. Publication date available at DDR1 that binds to and is activated by brillar and www.jasn.org. nonfibrillar collagens and DDR2 that binds to and Correspondence: Dr. Ambra Pozzi, Vanderbilt University Medi- fi 3 is activated by brillar collagens. cal Center, Department of Medicine, Division of Nephrology and DDR1 regulates multiple physiologic cellular Hypertension, Room B3115, Nashville, TN 37232. Email: ambra. functions including cytokine secretion,4 collective [email protected] cell migration,5 and extracellular matrix (ECM) Copyright © 2019 by the American Society of Nephrology

JASN 30: 1605–1624, 2019 ISSN : 1046-6673/3009-1605 1605 BASIC RESEARCH www.jasn.org homeostasis6 as well as pathologic conditions including cancer,7 Significance Statement inflammation,8 and fibrosis.6,9 DDR1 activates canonical intra- cellular signaling pathways including ERK and the RhoGTPase The receptor discoidin domain receptor 1 (DDR1) is activated by Cdc42, thus controlling cell migration.10,11 In diabetes, DDR1 collagen, upregulated in injured kidneys, and contributes to kidney fi fi promotes RUNX2 activity and atherosclerotic vascular calcifica- brosis, but how DDR1 controls brosis is poorly understood. The authors show that upon collagen stimulation, DDR1 translocates to 12 tion via Akt activation. In addition to canonical signaling, the nucleus. To do this, DDR1 must bind with SEC61B, a component DDR1 mediates activation of PKCa and STAT3phosphorylation of the Sec61 translocon, as well as with nonmuscle myosin IIA and via TM4SF1-mediated DDR1 coupling to syntenin 2.7 This b-actin. In the nucleus, DDR1 binds to chromatin to increase the noncanonical DDR1-mediated signaling drives metastatic transcription of collagen IV, a major collagen upregulated in fibro- reactivation of breast cancer stem cells in various organs.7 sis. The study reveals a novel mechanism whereby collagen- fi activated DDR1 moves to the nucleus to increase the production of DDR1 expression is upregulated in brosed organs includ- profibrotic molecules. ing the kidney and it contributes to disease progression by regulating inflammatory and fibrotic responses.13 We showed that DDR1 promotes collagen IV production, a major ECM b-actin, which is crucial for DDR1 nuclear translocation. Fi- upregulated in fibrotic diseases. This effect requires collagen nally, we show that NM IIA, actin, and DDR1 interact with binding to DDR1 and receptor kinase domain activation; chromatin to promote the transcription of collagen IV. Thus, however, the molecular mechanisms whereby DDR1 increases DDR1 contributes to fibrosis by translocating to the nucleus ECM synthesis and contributes to fibrosis initiation and/or where it acts as a cotranscription factor. This previously un- progression is poorly defined. recognized nuclear role of DDR1 opens unanticipated thera- RTKs regulate cell function by activating intracellular sig- peutic options for the treatment of fibrotic diseases. naling and by translocating to the nucleus, where they regulate gene expression by binding chromatin or by interacting with transcription factors.14–16 Nuclear localization of cell surface– METHODS bound RTKs has been reported for several RTK subclasses, including subfamilies of epithelial growth factor (EGF), insu- Cells lin, PDGF, and vascular endothelial growth factor receptors.17 Human embryonic kidney (HEK) and human kidney 2 RTKs can translocate to the nucleus as cleaved or full-length (HK-2) cells were obtained from ATCC and cultured in DMEM receptors. In the case of full-length receptors, ligand-activated with 10% FBS. Cells were tested for mycoplasma upon pur- RTKs are internalized via clathrin-coated vesicles. Upon in- chase and were characterized and authenticated by the vendor. ternalization, they undergo retrograde transport from Golgi to the endoplasmic reticulum and are subsequently transported Plasmids into the nucleus via interaction with SEC61B, a member of the pIRES-DDR1-FLAG, pIRES-DDR1, and pIRES-DDR1-R105A Sec61 translocon, and importins.18 If RTKs do not contain a were generated as described.6 For DDR1-EGFP, DDR1 cDNA classic nuclear localization sequence (NLS), they can still was amplified using the primers 59-GATGGAATTCGGAGC- translocate to the nucleus by association with their NLS- TATGGGACCAGAGG-39 and 59-GCTGGATCCGCACC- containing ligands.19 GTGTTGAGTGCATC-39 and pRK5-DDR1 as a template. DDR1 and its ligands lack an NLS motif, questioning The PCR product was digested with EcoRI and BamHI and the ability of this receptor to translocate to the nucleus. In- cloned into pEGFP-N2. RLC-TS/DD-GFP was a gift from terestingly, DDR1 associates with the actin-binding protein Dr. Donna Webb. nonmuscle myosin II (NM II) and this association mediates DDR1-driven cell motility.20,21 In addition to controlling cell FACS motility, NM II and its binding partner G-actin interact with To generate HEK cells expressing DDR1, DDR1-flag, DDR1- and facilitate the nuclear translocation of plasma membrane– R105A, and DDR1-GFP,cells were transfected with 0.4–1 mgof bound receptors. Moreover, they are found in the nucleus plasmid using Lipofectamine 2000 (Life Technologies) and where they regulate gene transcription by interacting with selected with puromycin (Sigma) or G418 (Corning). Cells transcription factors and/or RNA polymerase.22–27 were incubated with antibody to the extracellular domain of The goal of this study was to investigate whether DDR1 can DDR1 (Millipore) followed by PE-conjugated secondary an- translocate to the nucleus, the mechanisms regulating its nu- tibody and sorted using a FACS Aria II sorter (BD Biosciences) clear translocation, and the function of nuclear DDR1. We available through the Research Flow Cytometry Core Labora- show that full-length DDR1 is present in the nuclei of proximal tory at the Nashville VA Medical Center. tubules of injured human and murine kidneys. Using mass spectrometry (MS) and biochemical and cellular assays, we Cell Treatment show that upon collagen stimulation full-length DDR1 inter- Serum-starved cells were incubated with 20 mM blebbistatin acts with SEC61B in endoplasmic reticulum–enriched frac- (cat. B0560; Sigma), 10 mM Leptomycin B (cat. L2913; Sigma), tions. Moreover, DDR1 forms a complex with NM IIA and or 1 mM Latrunculin-B (cat. L5288; Sigma). After 30 minutes,

1606 JASN JASN 30: 1605–1624, 2019 www.jasn.org BASIC RESEARCH cells were treated with vehicle (20 mM acetic acid) or with 150 mM NaCl, and 1% Triton X-100; eluted in sample buffer; collagen I (Corning; 50 mg/ml in 20 mM acetic acid) for 0.5–3 and analyzed by western blot and as indicated above. hours. Cells were then processed as indicated below. Immunohistochemistry and Immunofluorescence Non-Nuclear and Nuclear Cell Fractions Human paraffin kidney sections were stained with rabbit anti- Cells were harvested in 10 mM HEPES pH 7.9, 1.5 mM MgCl2, DDR1 antibody (cat. 5583; Cell Signaling) followed by horse- 10 mM KCl, protease inhibitors (Roche Applied Science), radish peroxidase–conjugated anti-rabbit secondary antibody 5 mM NaVO3, and supernatant (non-nuclear), and pellet (nu- (cat. 711035152; Jackson Immunoresearch) and Sigma Fast clear) fractions were separated by centrifugation (400 3 g for DAB chromogenic tablets (Sigma). For double immunostaining, 4 minutes at 4°C). The nuclear fraction was lysed in the buffer paraffin sections were stained with anti-DDR1 antibody, anti- above containing 25% glycerol. collagen IV antibody (600–401–106–0.5; Rockland), collagen I Kidney cortex (10 mg) was homogenized in 250 mM su- antibody (ab34710; Abcam), and anti–NMHC-IIA antibody crose, 10 mM HEPES pH7.4, 5 mMKCl, 1.5 mMEDTApH8.0, (ab89837; Abcam), together with biotinylated Lotus tetrago- 5mMNa3VO4, and proteases inhibitors. After 15 minutes on nolobus agglutinin (cat. B-1325; Vector Laboratories), fol- ice, tissue lysates were centrifuged as described above. The lowed by secondary antibodies conjugated to AlexaFluor 555 nuclear fraction was resuspended in 20 mM HEPES pH 7.4, and Fluorescein-Streptavidin (cat. SA-5001; Vector Laborato- 0.4 M NaCl, 2.5% glycerol, 1 mM EDTA pH 8.0, 0.5 mM NaF, ries), and mounted using ProLong Gold Antifade Mountant and protease inhibitors. with DAPI (cat. P36931; Thermo Scientific). Serum-starved HEK cells expressing DDR1-GFP or Nuclear Soluble and Chromatin Fractions GFP, plated in multiwell chamber slides (cat. PEZG S0416; Cells were collected in 10 mM HEPES pH 7.9, 10 mM KCl, Millipore), were treated with vehicle or 50 mg/ml collagen I 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, with or without 10 mM Leptomyocin B or 20 mM blebbistatin. 28 proteases inhibitors, and 5 mM NaVO3, as described. After After 1–3 hours, cells were fixed in 4% PFA and slides were addition of Triton X-100 (0.1%), the nuclear pellet (P1) mounted using ProLong Gold Antifade Mountant with DAPI. and non-nuclear fractions were separated by centrifugation For double staining, cells were stained with anti-histone H3 or (700 3 g for 5 minutes at 4°C). P1 was incubated in 3 mM anti–NMHC-IIA antibodies followed by secondary anti- EDTA, 0.2 mM EGTA, 1 mM DTT, protease inhibitors, and bodies, AlexaFluor 555 conjugated, and mounted as indicated 5mMNaVO3. The nuclear soluble fraction and total chromatin above. Serum-starved HEK cells expressing GFP or the con- were collected by centrifuging P1 at 1200 3 g for 5 minutes at 4°C. stitutively active RLC-GFP,plated in multiwell chamber slides, were fixed in 4% PFA, permeabilized with 0.5% Triton-X in Endoplasmic Reticulum Fractionation PBS, and stained with rhodamine phalloidin (cat. Ab235138; Cell pellets were homogenized in 3 ml of 0.25 M sucrose and Abcam) to visualize the cytoskeleton. Slides were mounted as 0.1 M Tris (pH 7.4, 4°C). After sonication, cells were centri- indicated above. Confocal images were collected using an fuged at 10,000 3 g for 20 minutes at 4°C. The supernatant LSM710 META inverted confocal microscope. Confocal underwent further ultracentrifugation at 100,000 3 g for z-stacks consisting of up to 35 images were reconstructed 90 minutes at 4°C and the pellets were saved as microsomal into 3D animations using the ZEN lite microscope image fractions. software.

Western Blotting Image Quantification Cell or tissue lysates were resolved in 8% or 4%–20% SDS- To quantify the degree of nuclear colocalization of DDR1 and PAGE and transferred to nitrocellulose membranes. Mem- histone H3 or NMHC-IIA, we used Manders’ coefficient branes were incubated with the primary antibodies described through the software ImageJ (30–40 images analyzed). To in Supplemental Table 1 followed by horseradish peroxidase– quantify the area of nuclear DDR1-GFP–positive structures, conjugated secondary antibodies. Immunoreactive bands we used the software ImageJ (90–129 cells analyzed). were quantified by densitometry analysis using ImageJ and normalized against the appropriate loading controls. Ischemia-Reperfusion AKI Surgery was performed as described.29 Anesthetized 10– Immunoprecipitation 12-week-old male 129Sv mice were kept on a water bath– Cell fractions (200–500 mg proteins) were precleared using heated platform at 38°C. To induce ischemia-reperfusion 40 ml Protein G Sepharose (cat. SE-75184; GE healthcare). AKI, mice underwent left renal pedicle clamping for 31 min- The lysates were then incubated with 20 ml Protein G Sephar- utes. Three days later, mice were euthanized and the left kidney ose together with 1 mg anti–NMHC-IIA, 1 mganti–b-actin, or removed and used for analysis of nuclear and non-nuclear 20 ml anti-FLAG M2 affinity gel agarose beads (Sigma); or 1 mg DDR1 levels by western blot. Uninjured mice served as con- anti-SEC61B or 1 mg of IgG control antibody. After 18 hours, trol. Protocols were approved by the Vanderbilt Institutional the immunoprecipitates were washed with 50 mM Tris pH 7.2, Animal Care and Use Committee.

JASN 30: 1605–1624, 2019 Discoidin Domain Receptor 1 Translocates to Nucleus 1607 BASIC RESEARCH www.jasn.org

A control Tx-AKI1

Tx-AKI2 Tx-AKI3

B LTA DDR1 DAPI Merge C Tx-AKI2 control

0.8 1.0 p<0.05 D uninjured 3d-I/R uninjured 3d-I/R E p<0.05 F 0.8 125 kDa- - DDR1 0.6 0.6 116 kDa- - PARP1 0.4 0.4

37 kDa- - GAPDH DDR1/PARP1 DDR1/GAPDH 0.2 0.2 non nuclear nuclear 0.0 0.0 uninjured 3d-I/R

G Collagen I non nuclear I B 0 60 min HK-2 B B B B B Collagen I 125 kDa- - DDR1 Biotin B B B B nuclear 116 kDa- - PARP1 B B

50 kDa- - α-tubulin J Collagen I K 50 kDa- - α-tubulin 0 600 60 min ++++++++--biotin input nuclear p<0.05 HK-2 125 kDa- - DDR1 1.00 0.75 H 116 kDa- - PARP1 0.50 1.2 p<0.05 0.25 250 kDa- HRP-avidin

DDR1/PARP1 0.00 0 60 min 0.8 100 kDa- nuclear Collagen I 0.4

DDR1/PARP1 L Collagen I M 0.0 0 60 0 60 0 60 min 0 60 min p<0.05 ++++ +++ + + + biotin Collagen I --M 2.0 IP: avidin 125 kDa- 1.5 IB: DDR1 1.0 Coomassie 0.5 biot-DDR1/ Coomassie 0.0 nuclear Lysates 0 60 min 37C 4C Collagen I

Figure 1. DDR1 is upregulated in injured kidney proximal tubules. (A) Paraffin kidney sections from control and biopsy specimens from three different patients with transplant AKI (Tx-AKI) were stained with anti-DDR1 antibody. Upregulated DDR1 expression is evident in the tubules of injured kidneys. (B) Paraffin kidney sections from control or one patient with Tx-AKI were stained with anti-DDR1

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LC-MS/MS Analyses (Bio-Rad) and rqPCR was performed with the SYBR green Lysates of HEK cells expressing DDR1-GFP treated with collagen method using iQ Real-Time Sybr Green PCR Supermix Kit I(50mg/ml) or acetic acid for 1 hour were immunoprecipitated (Bio-Rad). Fluorescence was acquired at each cycle on a with GFP-binding protein coupled magnetic beads (Chromo- CFX96 system (Bio-Rad) using the following cycling con- Tek). Proteins were eluted by heating magnetic beads in gel ditions: 94°C/2 min, 94°C/30 s and 60°C/30 s for 40 cycles, loading buffer and subjected to short-stack SDS-PAGE and in- and 72°C/10 min. The Cq values were analyzed using gel digestion with trypsin. Tryptic peptides were analyzed by LC- the CFX96 system and normalized to GAPDH levels. Pri- MS/MS and the resulting Thermo .raw files were searched with mers used for human collagen IV a1chain(COL4A1, MyriMatch algorithm against a human database (20,232 entries; NM_001845.5) were: 59-TGTGCAACTTTGCATCACGAA-39 Uniprot). Identified peptide-spectrum matches were imported (forward) and 59-TCACACACAGCACACCTACTAA-39 (re- into IDPicker (version 3.1) for the parsimonious grouping of verse); and for human GAPDH 59-AAGGTGAAGGTCG- identified proteins at a 0% protein global FDR.30 Proteins iden- GAGTCAA-39 (forward) and 59-AATGAAGGGGTCA- tified in vehicle- and collagen I–treated samples were manually TTGATGG-39 (reverse).34 curated to identify proteins only present in cells stimulated with collagen I. To discriminate between potential bona fide interac- Chromatin Immunoprecipitation tors or background contaminants (e.g., proteins that interact Serum-starved HEK-DDR1b-GFP or HK-2 cells were treated with magnetic beads or GFR protein), the resulting protein sub- with acetic acid (20 mM) or collagen I (50 mg/ml in 20 mM set was further curated by querying against the Contaminant acetic acid) for 30 or 60 minutes, respectively. Cells were Repository for Affinity Purification (CRAPome).31 Proteins harvested and the nuclear DDR1-DNA complex was isolated showing a high frequency of occurrence in control experiments using ChIP-IT Express Enzymatic Magnetic Chromatin Im- were eliminated as they were likely to be background contami- munoprecipitation Kit (cat. 53009; Active Motif) according to nants. Spectral counting was used to assess relative abundance of manufacturer instructions. Briefly, proteins and DNA were proteins in both vehicle- and collagen I–treated samples. To crosslinked with 1% paraformaldehyde. After 15 minutes, identify phosphorylation of tyrosine residues on DDR1, Thermo the crosslink reaction was stopped by incubation with Glycine .raw files were subjected to a second search using the subset of Stop-Solution and, after chromatin shearing, DDR1 was im- identified proteins and setting phosphotyrosine [Tyr+79.9663] munoprecipitated using GFP-TRAP beads (Cromteck, for as variable modification as described.32,33 The raw data can be HEK-DDR1-GFP) or rabbit anti-DDR1 (Santa-Cruz) or rab- accessed at https://massive.ucsd.edu/ProteoSAFe/static/massi- bit IgG (Cell Signaling) antibodies with Protein G agarose ve.jsp?redirect=auth bearing a massive ID “MSV000082595” beads (for HK-2 cells). DNA was eluted from the beads with (username- “basakt,” password–“vanderbilt2015”). Elution Buffer AM2 and, after incubation with Proteinase K for 1 hour, the DNA was further purified using phenol- RT-rqPCR chloroform extraction. DNA amplification was performed RNAwas isolated from cells with AgilentTotal RNA Isolation using the following human collagen IV promoter primers: Kit (Agilent Technologies). cDNA synthesis was performed 59-AAATACGCCCAAAGCTGCTC-39 (forward) and 59-GG- using 0.5 mg RNA with iScript cDNA Synthesis Kit ACCGAGCCTCCTTTGTAT-39 (reverse).

antibody and Lotus tetragonolobus agglutinin (LTA, a marker of proximal tubule) and analyzed by confocal microscopy. Expression of DDR1 is evident both in the cytoplasm and in the nuclei of injured proximal tubules (arrow). (C) Orthogonal projection of confocal images of kidney sections from the patient shownin (B)was performed using theimaging program Zen (black edition).Red, DDR1; blue,DAPI. (D)Non- nuclear and nuclear fractions (20 mg/lane) from kidney cortices of wild-type mice uninjured or 3 days after ischemia-reperfusion (3d-I/R) were analyzed by western blot for levels of DDR1. (E and F) Non-nuclear DDR1 and GAPDH (E) or nuclear DDR1 and PARP1 (F) bands were quantified by densitometry. Values represent DDR1/GAPDH or DDR1/PARP1 ratio and are the mean6SD of four animals. (G) Serum- starved HK-2 cells were treated with collagen I (50 mg/ml) for the time indicated. Time 0 represents cells incubated with vehicle (20 mM acetic acid) for 60 minutes. Nuclear fractions (20 mg/lane) were analyzed by western blot for levels of DDR1. (H) Nuclear DDR1 and PARP1 bands were quantified by densitometry. Values represent DDR1/PARP1 ratio and are the mean6SD of two experiments performed in triplicate. PARP1 (nuclear marker), GAPDH, or a-tubulin (non-nuclear markers) was used to evaluate fraction purity. (I) Schematic repre- sentation of the biotinylation assay performed on HK-2 cells. See text for details. (J) Nuclear fractions of HK-2 cells biotinylated (+ biotin) and treated at 37°C with collagen I for the time indicated were analyzed for levels of DDR1 or total biotinylated proteins using HRP-avidin. Nonbiotinylated (- biotin) cells treated with collagen I for the times indicated served as control. (K) Nuclear DDR1 and PARP1 of bio- tinylated cells were quantified and expressed as indicated above. (L) Nuclear fractions (200 mg) of biotinylated HK-2 cells treated at 37°C with collagen I for the times indicated were immunoprecipitated using streptavidin beads. Immunoprecipitated biotinylated proteins were analyzed for levels of DDR1. Cells treated at 37°C with collagen I for the time indicated in the absence of biotinylation (- biotin) or biotinylated by kept at 4°C served as negative (background for streptavidin beads) and positive (total biotinylated DDR1) controls, respectively. (M) Nuclear biotinylated DDR1 was quantified to the Coomassie protein band shown. IP, immunoprecipitation; IB, immunoblot.

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A B Collagen I Collagen I 01530 60 180 360 0 15 30 60 180 360 min 125 kDa- - DDR1 HEK GFP DDR1-GFP 125 kDa- - pY513 150 kDa- - DDR1-GFP 150 kDa- - DDR1-GFP 125 kDa- - pY792

116 kDa- - PARP1 25 kDa- - GFP α 50kDa- - -tubulin 50kDa- -α-tubulin

non nuclear nuclear

C HEK-DDR1-GFP D --++ - - + + Collagen I DDR1-GFP DAPI Merge 150 kDa- - DDR1-GFP

150 kDa- - DDR1-GFP 0h

116 kDa- - PARP1 50kDa- α

- -tubulin 3h E non nuclear nuclear 0h GFP DAPI Merge 200

100 0h 0 3h 200

100 3h Intensity Intensity 0 024610 8 μm F G DDR1-GFP DDR1-GFP 0.010 p<0.05

0.008 H HEK-DDR1 II Abs 0.006 300 HEK-DDR1 HEK-DDR1 0.004 200 -R105A

0.002 100 Arbitrary units

DDR1-GFP area/nuclear area 0.000 0 0h 3h 0h 3h 100 101 102 103 104 PE I J p<0.05p<0.05 1.0 0.8 WT R105A WT R105A 0.6 --++- + - + Coll I 0.4 125 kDa- - DDR1 0.2 nDDR1/nnDDR1 116 kDa- - PARP1 0.0 - + - + Coll l non nuclear nuclear WT R105A

Figure 2. Activated DDR1 translocates to the nucleus. (A) Serum-starved HEK-DDR1 cells were treated with collagen I (50 mg/ml) for the times indicated. Time 0 represents cells incubated with 20 mM acetic acid for 360 minutes. Non-nuclear and nuclear fractions were analyzed by western blot for total (DDR1) and phosphorylated (pY513 and pY792) DDR1. PARP1 and a-tubulin were used to verify purity of nuclear versus non-nuclear fractions, respectively. (B) Cell lysates of triplicate samples of HEK cells expressing GFP (HEK-GFP)

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Biotinylation Assay agglutinin (a marker of proximal tubules) showed that DDR1 Serum-starved HK-2 cells were rinsed with cold PBS and in- localized mainly in proximal tubules (Figure 1B). Surprisingly, cubated with freshly prepared EZ-Link Sulpho-NHS-SS- we detected cytoplasmic and nuclear DDR1 staining in injured Biotin (cat. 21331; ThermoFisher Scientific) at 0.5 mg/ml in proximal tubule cells (Figure 1, B and C). To confirm DDR1 PBS pH 8.0. After 20 minutes, unreacted biotinylating reagent nuclear localization, we evaluated DDR1 expression in non- was quenched with 100 mM glycine in PBS and rinsed with PBS nuclear and nuclear fractions of kidney cortices of mice un- to remove glycine. After biotinylation, cells were treated with injured or 3 days after proximal tubule injury induced by vehicle or collagen I (50 mg/ml) at 37°C (to allow internaliza- ischemia-reperfusion. Full-length DDR1 expression was sig- tion) or at 4°C (to prevent internalization and detect total nificantly increased in non-nuclear and nuclear fractions of levels of cell surface biotinylated proteins). Cells treated with kidney cortices from injured mice (Figure 1, D–F). Thus, vehicle or collagen I at 37°C in the absence of biotinylation DDR1 expression is upregulated in the injured kidney and served as negative control. After 1 hour, nuclear fractions the full-length receptor can translocate to the nucleus. (200 mg for cells incubated at 37°C) were incubated with strep- tavidin agarose resin (cat. 20357; ThermoFisher Scientific). After DDR1 Translocates to the Nucleus upon Collagen I 2 hours, the resin was washed with Tris 50 mM, NaCl 150 mM, Treatment and Triton 1% and Igepal 1%, followed by a wash with 2% SDS RTKs can translocate to the nucleus upon ligand binding.39 in Tris 50 mM. Biotinylated proteins were eluted with reducing Thus, we analyzed nuclear fractions of human proximal tu- SDS-PAGE sample buffer and analyzed by western blot analysis bule HK-2 cells (which express endogenous DDR1 and are the for levels of DDR1 or total biotinylated proteins. main cell types affected in AKI) treated with vehicle or soluble collagen I (which activates DDR1, but not other collagen- Statistical Analyses binding receptors such as integrins40–42). Nuclear levels of Data are shown as mean6SD. Unpaired two-tailed t test was DDR1 were significantly increased in collagen I–treated com- used to evaluate statistically significance differences (P,0.05) pared with vehicle-treated cells (Figure 1, G and H). To further between two groups. GraphPad Prism software (GraphPad) confirm that endogenous DDR1 translocates to the nucleus ANOVA followed by Bonferroni’s multiple comparison test upon collagen I stimulation, we performed cell-surface pro- when appropriate was used to evaluate statistically significant tein biotinylation in HK-2 cells followed by treatment with differences (P,0.05) among multiple groups. collagen I, nuclei isolation, streptavidin pulldown, and west- ern blot analysis (Figure 1, I–M). Nuclear fractionation of biotinylated NK-2 cells treated with vehicle or collagen I RESULTS showed that nuclear levels of DDR1 were significantly in- creased in collagen I–treated compared with vehicle-treated Nuclear DDR1 Expression in Proximal Tubules of cells, indicating that biotinylation did not compromise DDR1 Injured Human Kidneys trafficking (Figure 1, J and K). Streptavidin pulldown of nu- DDR1 levels are upregulated in humans with kidney dis- clear fractions, followed by western blot analysis of DDR1 ease35,36 and in several mouse models of kidney in- levels, revealed the presence of biotinylated DDR1 primarily jury.4,35,37,38 To determine where DDR1 is expressed, we in the nuclei of cells treated with collagen I, but not vehicle stained transplant kidney biopsy specimens with AKI with (Figure 1, L and M). anti-DDR1 antibody. Tubular DDR1 was increased in AKI To define how activated DDR1 translocates to the nucleus, we compared with control healthy subjects (Figure 1A). Double generated HEK cells (which express low levels of endogenous staining with anti-DDR1 antibody and Lotus tetragonolobus DDR1) that express the human DDR1b isoform (HEK-DDR1).

or the fusion DDR1-GFP (HEK-DDR1-GFP) cDNA were analyzed by western blot using anti-DDR1 (upper panel) or anti-GFP (middle panel) antibody. a-tubulin was used to verify loading. (C) Serum-starved HEK-DDR1-GFP cells were treated with vehicle or collagen I (50 mg/ml) for 3 hours. Nuclear and non-nuclear fractions were analyzed by western blot using anti-DDR1 (upper panel) or anti-GFP (second panel) antibody. PARP1 and a-tubulin were used to verify the fraction purity. (D) Confocal images of HEK-DDR1-GFP cells treated with collagen I (50 mg/ml; 3h) or acetic acid (20 mM; 0h) for 3 hours. (E) Line scanning profile showing intensity of DDR1-GFP (green) and DAPI (blue) pixels, measured along the white line in (D). (F) Orthogonal projection of confocal images of HEK-DDR1-GFP cells was performed using the imaging program Zen (black edition). (G) The area occupied by DDR1-GFP–positive pixels/nuclear area was evaluated in HEK-DDR1-GFP cells. Circles represent single cells (n=90 for 0 hours and n=92 for 3 hours), whereas the bars represent the mean6SD. (H) HEK cells were transfected with DDR1 or DDR1-R105A cDNA and cell populations expressing comparable levels of DDR1 were sorted by FACS. PE, phycoerythrin. (I) Serum-starved HEK cells expressing DDR1 (WT) or DDR1-R105A (R105A) were treated with collagen I (50 mg/ml) for 0 and 3 hours. Time 0 is as indicated above. Non-nuclear and nuclear fractions were analyzed by western blot for total DDR1. PARP1 was used to verify the purity of nuclear fractions. (J) Nuclear DDR1 (nDDR1) and non-nuclear DDR1 (nnDDR1) bands were quantified by densitometry. Values represent nDDR1/nnDDR1 ratio and are the mean6SD of three experiments.

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A B C

p<0.05

input anti-FLAG lgG 8 input anti-SEC61B 0 15 30 0 15 30 min 0 15 15 0 15 15 min 6 p<0.05 -125 kDa -125 - DDR1 4 kDa -125 - DDR1 2

14 kDa - - SEC61B SEC61B/DDR1 0 kDa -14 - SEC61B 0 15 30 mins HEK-DDR1b-FLAG - + + Coll l HK-2

-++LMB- D 3 p<0.05 E F --+ + Coll l 2.5 p<0.05 2 kDa -125 - DDR1 2.0 1.5 p<0.05 1 kDa -116 - PARP1 1.0

DDR1/SEC61B 0.5 DDR1/PARP1 0 0.0 0 15 mins -++LMB- - + Coll l --+ + Coll l

G DDR1-GFP DAPl Merge H 200

100 0h Intensity 0

200

100 Intensity

0h+LMB 0

200

100 Intensity 0

200

100 Intensity

3h+LMB 3h 0 0246 810 m

I J p<0.05 0.035 0.030 0.025 0.020 0.015 p<0.05 0.010 0.005 0.000 DDR1-GFP area/nuclear area -+-+LMB Collagen l Collagen l + LMB +-- + Coll l

Figure 3. Inhibition of exportins increases collagen I–induced DDR1 nuclear retention and levels. (A) Serum-starved HEK cells transfected with human DDR1-FLAG cDNA were treated with collagen I (50 mg/ml) for 15 or 30 minutes. Time 0 represents cells in- cubated with 20 mM acetic acid for 30 minutes. Equal amounts of microsomes (200 mg) were immunoprecipitated with anti-FLAG antibody and the immunoprecipitates were analyzed by western blot for levels of DDR1 and SEC61B. (B) SEC61B and DDR1 bands

1612 JASN JASN 30: 1605–1624, 2019 www.jasn.org BASIC RESEARCH

We focused on DDR1b because, among the five DDR1 isoforms, carrying this signal. To identify these interactors, we immu- it is well characterized in terms of collagen-mediated activation noprecipitated DDR1-GFP in HEK-DDR1-GFP cells treated and downstream signaling.6,43 DDR1 was detected in non- with vehicle or collagen I and then analyzed the immunopre- nuclear, but not nuclear, fractions of unstimulated cells (Figure cipitated proteins by MS. 2A). Upon collagen I treatment, full-length nuclear DDR1 be- MS analysis resulted in the identification of 629 and 563 came evident 15 minutes after treatment and it remained in proteins from vehicle- and collagen I–treated DDR1-GFP im- nuclear fractions for at least 360 minutes (Figure 2A). Phosphor- mune complexes, respectively. DDR1 was the most abundant ylation of the receptor, measured with antibodies to pY513 (spe- protein identified in immunoprecipitates of both vehicle- and cific to DDR1b) and Y792 (in the activation loop of the receptor), collagen I–treated samples. DDR1 peptides containing phos- was evident only after collagen I treatment and was present in both photyrosine resides (Y484, Y513, Y520, Y792) were present the nuclear and non-nuclear fractions (Figure 2A). only in collagen I–treated samples, confirming that DDR1- To confirm DDR1 nuclear translocation upon collagen I GFPundergoesphosphorylationatsitesknowntoactivate stimulation, we generated HEK cells expressing human DDR1 upon collagen stimulation43 (Supplemental Figure 1). DDR1-GFP cDNA (HEK-DDR1-GFP) (Figure 2B) and ana- We identified 73 proteins in collagen I–treated cells, two of lyzed DDR1-GFP localization by western blot and confocal which were protein transport protein Sec61 subunit b microscopy upon collagen I treatment. Full-length DDR1- (SEC61B) and histone-binding protein RBBP4 (Supplemental GFP was evident in non-nuclear fractions of vehicle- and Table 2). We observed similar spectral counts for b-actin pep- collagen I–treated cells (Figure 2C, non-nuclear); however, tides in both vehicle- and collagen I–treated samples. More- nuclear DDR1-GFP was detected only in collagen I–stimulated over, twice as many spectral counts for nonmuscle myosin cells (Figure 2C, nuclear). In unstimulated cells, DDR1-GFP heavy chain II A (NMHCII-A, encoded by the MYH9 gene) was present at the cell membrane and in perinuclear regions peptides were present in collagen I–treated samples (Supple- only (Figure 2, D–F). After collagen I treatment, DDR1-GFP mental Table 2). This result suggests selective interaction with was visualized in aggregates in both cytoplasm and nuclei (Fig- SEC61B and increased interaction of NMHCII-A with DDR1 ure 2, D–F) and there was significantly more nuclear DDR1- upon collagen I treatment. SEC61B regulates the nuclear GFP in collagen I– than vehicle-treated HEK-DDR1-GFP cells translocation of full-length growth factor receptors.45 NM (Figure 2G). IIA associates with DDR1 at the plasma membrane,21 is coex- To determine whether collagen I binding is required for pressed with DDR1 in the inner ear in vivo,20 interacts with DDR1 nuclear translocation, we generated HEK cells express- DDR1 via its C-terminal kinase domain,46 drives internaliza- ing the collagen I–binding–deficient DDR1-R105A mutant tion of growth factor receptors,47 and interacts with b-actin (HEK-DDR1-R105A)44 (Figure 2H). Collagen I treatment and regulates gene expression upon nuclear translocation.22 failed to promote nuclear translocation of DDR1-R105A (Fig- ure 2, I and J), suggesting that the collagen I/DDR1 interaction DDR1 Forms a Complex with SEC61B is a key step in promoting receptor nuclear translocation. To validate the MS finding, we generated HEK-DDR1-FLAG cells and isolated microsomes from cells treated with vehicle or Identification of Proteins Involved in DDR1 Nuclear collagen I. Immunoprecipitation assays using anti-FLAG an- Translocation tibody revealed that collagen Itreatment significantly increased DDR1 does not contain an NLS, suggesting that its nuclear the DDR1/SEC61B interaction compared with that detected in translocation is mediated by its interaction with chaperones vehicle-treated cells (Figure 3, A and B). Tovalidate the finding

were quantified by densitometry. Values represent SEC61B/DDR1 ratio and are the mean6SD of three experiments. (C) Serum-starved HK- 2 cells were treated with 20 mM acetic acid or collagen I (50 mg/ml) for 15 minutes. Equal amounts of microsomes (200 mg) were im- munoprecipitated with anti-SEC61B antibody or IgG control and the immunoprecipitates were analyzed by western blot for levels of DDR1 and SEC61B. (D) SEC61B and DDR1 bands were quantified and expressed as indicated above. Values represent DDR1/SEC61B ratio and are the mean6SD of six experiments. (E) Serum-starved HEK-DDR1 cells were treated with 20 mM acetic acid or collagen I (50 mg/ml) for 3 hours in the presence or absence of Leptomycin B (LMB, 10 ng/ml). Nuclear fractions (20 mg/lane) were analyzed by western blot for levels of DDR1 or PARP1 (to verify equal loading of nuclear fractions). (F) Nuclear DDR1 and PARP1 bands were quantified by densitometry. Values represent DDR1/PARP1 ratio and are the mean6SD of three experiments normalized to samples treated with collagen I only. (G) Confocal images of serum-starved HEK-DDR1-GFP cells treated with collagen I (50 mg/ml) for 3 hours in the presence or absence of LMB (10 ng/ml). Time 0 represents cells incubated with 20 mM acetic acid for 3 hours. (H) Line scanning profile showing intensity of DDR1-GFP (green) and DAPI (blue) pixels (measured along the white line in [G]) in HEK-DDR1-GFP cells treated as described in (E). (I) Orthogonal projection of confocal images of serum-starved HEK-DDR1-GFP cells treated as indicated above was performed using the imaging program Zen (black edition). Green, DDR1-GFP; blue, DAPI. (J) The area occupied by DDR1-GFP–positive pixels/nuclear area was evaluated in HEK-DDR1-GFP cells treated as indicated in (E) using ImageJ. Circles represent single cells (n=90 for 0 hour, n=27 for 0 hour+LMB, n=92 for 3 hour, and n=87 for 3 hour+LMB), whereas the bars represent the mean6SD. Coll I, collagen I.

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A B 1.5 --++ BLB - + - + Coll l p<0.05 p<0.05 1.0 125 kDa- - DDR1

0.5 DDR1/PARP1 166 kDa- - PARP1 0.0 -- ++ BLB --++Coll l

C D 0h DDR1-GFP DAPI Merge 200

100

0h 0 3h 200

100 3h

0 0h+BLB 200

100 0h

0

BLB 3h+BLB 200 3h 100 Intensity Intensity Intensity Intensity

0 1086420 μm E F

p<0.05 p<0.05 0.010

0.008

0.006

0.004

0.002

DDR1-GFP area/nuclear area 0.000 - + - + BLB Collagen I Collagen I + BLB -- ++ Coll l

Figure 4. Inhibition of nonmuscle myosin II ATPase activity decreases collagen I–induced DDR1 nuclear translocation. (A) Serum- starved HEK-DDR1 cells were treated with vehicle or collagen I (50 mg/ml) for 3 hours in the presence or absence of blebbistatin (BLB, 20 mM). Nuclear fractions (20 mg/lane) were analyzed by western blot for levels of total DDR1 and PARP1 (to verify equal loading of nuclear fractions). (B) Nuclear DDR1 and PARP1 bands were quantified by densitometry. Values represent DDR1/PARP1 ratio and are the mean6SD of three experiments normalized to samples treated with collagen I only. (C) Confocal images of serum-starved

1614 JASN JASN 30: 1605–1624, 2019 www.jasn.org BASIC RESEARCH to endogenously expressed DDR1, we isolated micro- the cytoskeleton) and observed presence of cortical actin only somes from HK-2 cells treated with vehicle or collagen I. Im- in cells expressing constitutively active RLC (Figure 5B). We munoprecipitation assays using anti-SEC61B antibody or IgG treated these cells with vehicle or collagen I and analyzed the control antibody followed by western blot for DDR1 showed nuclear level of DDR1. We detected nuclear localization of pronounced DDR1/SEC61B interaction in cells treated with GFP and RLC-TS/DD-GFP at baseline and after collagen I collagen compared with that detected in vehicle-treated cells stimulation (Figure 5C); however, the levels of RLC-TS/ (Figure 3, C and D). No DDR1/SEC61B interaction was ob- DD-GFP were significantly higher in collagen I–treated cells served in lysates of collagen-treated cells immunoprecipitated compared with vehicle-treated cells (Figure 5E). We detected with IgG control antibody (Figure 3, C and D). nuclear DDR1 in HEK-GFP cells treated with collagen I, whereas no visible signal was evident in vehicle-treated cells Inhibition of Nuclear Exports Increases Collagen (Figure 5, C and E). By contrast, we detected baseline nuclear I–Mediated DDR1 Nuclear Levels DDR1 in vehicle-treated HEK-RLC-TS/DD-GFP cells (consis- To confirm that activated DDR1 is translocated to the nucleus, tent with the increased baseline of nuclear RLC-TS/DD-GFP we stimulated HEK-DDR1 cells with collagen Iwith or without levels) that significantly increased upon collagen I treatment the exportin inhibitor Leptomycin B. Western blot of nuclear (consistent with the further increased levels of RLC-TS/DD- fractions showed that Leptomycin B significantly increased the GFP after collagen I treatment) (Figure 5, C and E). nuclear levels of DDR1 in collagen I–stimulated, but not ve- To confirm that RLC-TS/DD–mediated DDR1 nuclear hicle-treated, cells (Figure 3, E and F). Confocal microscopy translocation is potentiated by collagen I stimulation, we an- confirmed a significant increase in DDR1-GFP–positive nu- alyzed the nuclear localization of DDR1-R105A in vehicle- or clear clusters in cells treated with collagen I together with collagen I–treated HEK-DDR1-R105A cells expressing GFP or Leptomycin B (Figure 3, G–J). RLC-TS/DD-GFP. Although we detected both GFP and RLC- TS/DD-GFP in the nuclei, we failed to detect nuclear DDR1- Collagen I–Mediated DDR1 Nuclear Translocation Is R105A in either vehicle- or collagen I–treated cells (Figure 5, D Acto-Myosin Dependent and E). We identified NMHCII-A as a potential DDR1 interactor (Sup- plemental Table 2). To determine whether NMHCII-A medi- G-Actin Regulates DDR1 Nuclear Translocation upon ates DDR1 nuclear translocation, we stimulated HEK-DDR1 Collagen I Stimulation cells with collagen I with or without blebbistatin, which in- NM IIA is an actin-binding protein. Increased actin depoly- hibits myosin ATPase activity. Western blot of nuclear frac- merization leads to the translocation of actin monomers from tions and confocal microscopy showed that treatment with cytoplasm to the nucleus 50 and actin can regulate gene ex- blebbistatin significantly decreased collagen I–mediated pression upon nuclear translocation.22 On the basis of the MS DDR1 nuclear translocation, but had no effect in vehicle- data showing that DDR1 interacts with b-actin (Supplemental treated cells (Figure 4, A–F). Table 2), we investigated the contribution of actin in DDR1 nuclear translocation. HEK-DDR1 cells were treated with col- Activation of NM II Activity Enhances Collagen I– lagen I with or without Latrunculin-B, and nuclear levels of Mediated DDR1 Nuclear Translocation DDR1 were significantly increased in cells treated with colla- NM II activity is regulated by the regulatory light chains gen I in the presence of Latrunculin-B compared with cells (RLCs).48 RLCs contain two amino acids, threonine 18 treated with collagen I only (Figure 5, F and G). A plausible (T18) and serine 19 (S19) that, when phosphorylated, relieve reason for this result is that Latrunculin-B disassembles the inhibition imposed on the myosin molecule.48 We there- F-actin and promotes G-actin formation. fore generated HEK-DDR1 cells expressing GFP or the con- stitutively active RLC-GFP (T18 and S19 mutated to aspartic DDR1 Forms a Nuclear Complex with NMHC-IIA and acid, RLC-TS/DD-GFP) (Figure 5A). Because myosin motors Actin upon Collagen I Stimulation generate contractile stresses promoting the formation of cor- To determine whether DDR1 forms a nuclear complex with tical actin filaments,49 we stained HEK-DDR1-GFP or HEK- NM IIA and actin, we stimulated HEK-DDR1-FLAG cells DDR1-RLC-GFP cells with rhodamine phalloidin (to visualize with collagen I for different time points and then performed

HEK-DDR1-GFP cells treated with collagen I (50 mg/ml) for 0 or 3 hours in the presence or absence of BLB (20 mM). Time 0 represents cells incubated with 20 mM acetic acid for 3 hours. (D) Line scanning profile showing intensity of DDR1-GFP (green) and DAPI (blue) pixels (measured along the white line in [C]) in HEK-DDR1-GFP cells treated as described in (C). (E) Orthogonal projection of confocal images of serum-starved HEK-DDR1-GFP cells treated as indicated above was performed using the imaging program Zen (black edition). Green, DDR1-GFP; blue, DAPI. (F) The area occupied by DDR1-GFP–positive pixels/nuclear area was evaluated in HEK-DDR1-GFP cells treated as indicated above using ImageJ. Circles represent single cells (n=90 for 0 hour, n=107 for 0 hour+BLB, n=92 for 3 hour, and n=129 for 3 hour+BLB), whereas the bars represent the mean6SD. Coll I, collagen I.

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A HEK-DDR1 B phalloidin GFP Merge

50 kDa- - RLC-TS/DD-GFP GFP

25 kDa- - GFP RLC 50 kDa- - -tubulin -TS/DD-GFP

C HEK-DDR1 D HEK-DDR1-R105A GFP HEK-DDR1 HEK-DDR1-R105A GFP GFP RLC-TS/DD-GFP RLC-TS/DD-GFP GFP GFP RLC-TS/DD-GFP RLC-TS/DD-GFP GFP GFP RLC-TS/DD-GFP RLC-TS/DD-GFP GFP RLC-TS/DD-GFP RLC-TS/DD-GFP ----++++ ----++ -- Coll I 125 kDa- - DDR1

50 kDa- - RLC-DD-GFP

25 kDa- - GFP

116 kDa- - PARP1

50 kDa- --tubulin

nuclear input nuclear

E p<0.05 10 p<0.05 8 GFP 8 p<0.05 RLC-TS/DD-GFP 6 p<0.05 6 GFP/PARP1 p<0.05 4 4 2

DDR1/PARP1 0.6 2 0.3 0.0 0 --++ --++ Coll I

F - - --+ + + + LATR G p<0.05 2.5 - - ++ --++ Coll I 2.0 1.5 125 kDa- - DDR1 0.5 p<0.05 0.4 0.3 116 kDa- - PARP1

DDR1/PARP1 0.2 0.1 0.0 HEK-DDR1 - - ++Latr. - - + Coll I

Figure 5. Nonmuscle myosin II A and b-actin promote DDR1 nuclear translocation. (A) HEK-DDR1 cells were transiently transfected with empty vector (GFP) or constitutively active nonmuscle myosin II RLC (RLC-TS/DD-GFP) cDNA. Cell lysates (20 mg/lane) of cells transfected in duplicate were analyzed by western blot using anti-GFP antibody. a-tubulin was used to verify equal loading. (B)

1616 JASN JASN 30: 1605–1624, 2019 www.jasn.org BASIC RESEARCH immunoprecipitation assays using anti-FLAG antibody (Fig- but not HEK-DDR1-R105A, cells (Figure 7, D–G). Collagen ure 6A). No immunoprecipitated nuclear DDR1 was evident I–stimulated HEK-DDR1 cells also showed significantly more in cells treated with vehicle; however, immunoprecipitated chromatin-bound NMHC-IIA and b-actin compared with ve- nuclear DDR1 was evident 15 minutes after collagen I stimu- hicle-treated cells (Figure 7, D and E). Similar results were ob- lation, which increased at 60 minutes and reached a plateau tained when analysis was performed on HK-2 cells treated with at 360 minutes (Figure 6A). Anti-FLAG antibody also immu- vehicle or collagen I, suggesting that endogenous DDR1 can noprecipitated NMHCII-A, which was particularly evident form a complex with nuclear chromatin upon activation (Figure starting 60 minutes after collagen I stimulation (Figure 6A). 7, D and E). Confocal microscopy of vehicle- and collagen I– Immunoprecipitation of HEK-DDR1 nuclear extracts with treated HEK-DDR1-GFP cells stained with anti-histone H3 or anti–b-actin antibody revealed b-actin, NMHCII-A, and anti–NMHC-IIA antibody further confirmed that upon collagen DDR1inacomplexstartingat15minutesaftercollagenI I treatment nuclear DDR1 is associated with chromatin and stimulation, which persisted up to 360 minutes (Figure 6B). colocalizes with NMHC-IIA (Figure 7, H–J). Thus, nuclear Finally, immunoprecipitation of nuclear extracts with anti– DDR1 exists as a complex with these two proteins primarily in NMHCII-A antibody confirmed that the three proteins chromatin-rich fractions. form a complex only in collagen I–stimulated cells (Figure 6C). No proteins were visible in samples incubated with IgG Nuclear DDR1 Associates with Chromatin and controls (Figure 6C). Regulates Gene Transcription Activated DDR1 induces the synthesis of collagens at both the Nuclear DDR1 Associates with Chromatin RNA and protein level.6,43 The finding that DDR1, NMHCII- RTKs can regulate gene transcription by forming a nuclear A, and b-actin exist as a complex in nuclear chromatin frac- complex with chromatin, chaperone proteins, and transcrip- tions led to the hypothesis that nuclear DDR1 may function tion factors.17 MS analysis of proteins associated with DDR1 as a cotranscription factor by binding NMHCII-A, which can in collagen I–treated cells revealed the presence of histone- act as an actin-based nuclear motor to regulate gene transcrip- binding protein RBBP4 (Supplemental Table 2). This nuclear tion. Analysis of the human bidirectional promoter of collagen protein is present in protein complexes involved in both his- IV a1anda2 chains revealed the presence of four AGCTCC tone acetylation and chromatin assembly.51 Tovalidate the MS sequences, which have been identified as putative NM IIA analysis, we stimulated HEK-DDR1-GFP with vehicle or col- binding motifs.52,53 To determine whether DDR1 forms a lagen I and then analyzed the association between nuclear complex with the collagen IV promoter and whether this is DDR1-GFP and RBBP4 by performing immunoprecipitation dependent on collagen I stimulation, we performed the chro- assays using GFP-TRAP beads. Collagen I treatment signifi- matin immunoprecipitation (ChIP) assay on HEK-DDR1- cantly increased the RBBP4/DDR1 nuclear interaction com- GFP and HK-2 cells treated with vehicle or collagen I. To do pared with that detected in vehicle-treated cells (Supplemental this, nuclei of cells untreated or treated with collagen I were Figure 2). Interestingly, histone H3 (a marker of chromatin) immunoprecipitated with GFP-TRAP beads or anti-DDR1 also formed a complex together with DDR1-GFP and RBBP4 antibody and the immunoprecipitated DNA was amplified in collagen I–treated cells (Supplemental Figure 2), suggesting using primers spanning an NM IIA binding motif at positions that nuclear DDR1 forms a complex with chromatin which is 1351–1357 (Figure 7, A–D). ChIP analysis showed a signifi- mediated by collagen I. To validate these findings, we stimu- cant increase in binding of DDR1 to this collagen IV promoter lated HEK-DDR1 or HEK-DDR1-R105A cells with collagen I region in cells stimulated with collagen I compared with cells and then analyzed DDR1 levels in nuclear soluble and/or chro- treated with vehicle only (Figure 7, A–D). matin fractions. Undetectable and/or low levels of DDR1 were The ChIP analysis suggests that collagen IV might be transcrip- present in nuclear soluble or chromatin fractions of vehicle- tionally controlled by DDR1. To determine the functionality of our treated HEK-DDR1 or HEK-DDR1-R105A cells (Figure 7, finding, we analyzed the contribution of DDR1/NM IIAinteraction D–G). Upon collagen I treatment, DDR1 levels were significantly in driving collagen IV mRNA expression. To do this, we measured upregulated primarily in chromatin fractions in HEK-DDR1, collagen IV mRNA in HEK-DDR1 and HEK-DDR1-R105A cells

Representative image of HEK-DDR1 cells transfected as indicated above stained with rhodamine phalloidin showing the presence of cortical actin in cells transfected with RLC-TS/DD-GFP cDNA. (C and D) Serum-starved HEK-DDR1 or HEK-DDR1-R105A cells, transfected with GFP or RLC-TS/DD-GFP cDNA, were treated with vehicle or collagen I (50 mg/ml) for 3 hours. Nuclear fractions (20 mg/lane) were analyzed by western blot for levels of DDR1 (with anti-DDR1 antibody) or RLC-TS/DD-GFP and GFP (with anti-GFP antibody). PARP1 and a-tubulin were used to verify equal loading and purity of nuclear fractions. (E) DDR1, RLC-TS/DD-GFP, and PARP1 bands of cells in (B) were quantified by densitometry. Values represent DDR1/or RLC-TS/DD-GFP/PARP1 ratio and are the mean6SD of three experiments. (F) Serum-starved HEK-DDR1 cells were treated with vehicle or collagen I (50 mg/ml) for 3 hours in the presence or absence of Latrunculin B (LATR, 10 mM). Nuclear fractions (20 mg/lane) were analyzed by western blot for levels of DDR1 and PARP1 (used to verify equal loading of nuclear fractions). (G) Nuclear DDR1 and PARP1 bands were quantified by densitometry. Values represent DDR1/PARP1 ratio and are the mean6SD of one experiment performed in triplicate. Coll I, collagen I.

JASN 30: 1605–1624, 2019 Discoidin Domain Receptor 1 Translocates to Nucleus 1617 BASIC RESEARCH www.jasn.org IIA

A Collagen I B Collagen I C IP: NMHC- IP: IgG 0 15 30 60 180 360 min 01560180 360 min -++Collagen I

226 kDa- - NMHC-IIA 226 kDa- - NMHC-IIA 226 kDa- - NMHC-IIA 125 kDa- - DDR1 125 kDa- - DDR1 IP: FLAG 125 kDa- IP:  -actin 44 kDa- - -actin - DDR1 125 kDa- - DDR1 125 kDa- - DDR1 44 kDa- - -actin 60 kDa- - HDAC2 116 kDa- - PARP1 Input Input 50 kDa- - -tubulin 50 kDa- - -tubulin 116 kDa- - PARP1 Input

D Collagen I F Collagen I 0 180 0 180 0 30 60 0 30 60 min 0 180 min 125 kDa- - DDR1 WTR105A WT R105A - DDR1 226 kDa- - NMHC-IIA 125 kDa- 44 kDa- --actin 50 kDa- - Histone H3 15 kDa- - Histone H3 50 kDa- - -tubulin Chromatin 15 kDa- Ponceau Nuclear Soluble ChromatinNuclear Soluble Chromatin HEK-DDR1 HK-2 G WT R105A 15 p<0.05 p<0.05 E DDR1 2.5 NMHC-IIA * 10 2.0 -actin * 5

1.5 * Histone H3

* * Chromatin bound/ 0 1.0 * * * - + - + Collagen I Histone H3 * 0.5 Chromatin bound/

0.0 -+-+-+ - + + - + + - + + Collagen I I 0 0 0 0 0 0

30 60 30 60 30 60 150 180 180 180 HEK-DDR1 HK-2 100 50 H DDR1-GFP Histone H3Merge Merge Intensity 0 DDR1-GFP Histone H3 200 0h 100 Intensity 0 DDR1-GFP NMHC-IIA 0246810 m 1h J 1.2 p<0.05 DDR1-GFP NMHC-IIA Merge Merge p<0.05 0.9

0.6 0h 0.3

0.0

nuclear DDR1 colocalized 0h 1h 0h 1h

1h H3 NMHC- IIA

Figure 6. DDR1, NM IIA, and b-actin exist as a nuclear complex in chromatin fractions. (A) Serum-starved HEK cells transfected with DDR1-FLAG cDNA were treated with collagen I (50 mg/ml) for the times indicated. Time 0 represents cells incubated with 20 mM acetic acid for 360 minutes. Nuclear lysates (200 mg) were immunoprecipitated with anti-FLAG antibody and the immunoprecipitates were

1618 JASN JASN 30: 1605–1624, 2019 www.jasn.org BASIC RESEARCH treated with vehicle or collagen I. Treatment with collagen I signif- complex with Sec61B, NM-IIA, and b-actin. This complex icantly enhanced collagen IV mRNA synthesis only in HEK-DDR1 mediates DDR1 translocation to the nucleus where it inter- cells (Figure 7E), consistent with the findingthatcollagenIpro- acts with chromatin and regulates collagen IV transcription motes nuclear translocation and chromatin association of DDR1, (Figure 8). Our study reveals a novel mechanism whereby ac- but not the DDR1-R105A receptor (Figures 2, I and J and 5, C–E). tivated DDR1 regulates gene transcription by directly trans- To analyze the contribution of NM IIA in driving DDR1- locating to the nucleus where it acts as a cotranscription factor. mediated collagen IV mRNA synthesis, we measured collagen Activated DDR1 undergoes aggregation followed by cyto- IV mRNA levels in HEK-DDR1 cells expressing RLC-TS/DD- plasmic internalization and incorporation into early endo- GFP treated with vehicle or treated with collagen I. Collagen I somes.42 DDR1 is internalized alone or together with other treatment significantly increased collagen IV mRNA levels in RTKs, because IGF-I induces DDR1 tyrosine phosphoryla- HEK-DDR1 cells expressing RLC-TS/DD-GFP compared with tion, cointernalization with the IGF-I receptor, and incorpo- collagen I–stimulated HEK-DDR1 cells (Figure 7E). This re- ration into early endosomes.54 Internalized RTKs can recycle sult is consistent with our finding that collagen I treatment back to the plasma membranes, be degraded, or undergo an significantly increased DDR1 nuclear levels in HEK-DDR1 endosome/Golgi/endoplasmic reticulum retrograde pathway. cells expressing RLC-TS/DD-GFP compared with collagen Once in the endoplasmic reticulum, RTKs can be transported I–stimulated HEK-DDR1 cells (Figure 5, C and E). to the nucleus via an interaction with Sec61B. We provide MS Finally, to determine the relevance of our finding in vivo,we and biochemical evidence that activated DDR1 interacts with stained control kidneys and transplant kidney biopsy speci- Sec61B in endoplasmic reticulum–enriched fractions. Sec61B mens with AKI with anti-NM IIA, -collagen IV, or -collagen I regulates the retrograde pathway and nuclear translocation of antibody. NM IIA and collagen expression was evident in both full-length growth factor receptors via two distinct retrograde uninjured and injured proximal tubules (Supplemental Figure pathways.45 Both pathways require Sec61B and a cytosolic or 3). However, in the tubules of transplant patients with AKI, an endoplasmic reticulum–associated importin. Our MS anal- NM IIA was evident in both cytoplasm and nuclei, reassem- ysis revealed the presence of importin-b in vehicle- and col- bling the pattern observed for DDR1 (Figure 1B). In addition, lagen I–treated DDR1-expressing cells, and importin-5 only in increased expression of the two fibrotic collagens I and IV was collagen I–stimulated cells (Supplemental Table 2). Importin- observed in the basement membranes and surrounding in- 5 is a member of the importin b family,55 whichactsasa jured proximal tubules (Supplemental Figure 3). nuclear transport receptor for nuclear myosin 1,56 transcrip- tion factors such as Jun and SMADS,57,58 and G-protein– coupled receptors.59 Thus, it is conceivable that importin-b DISCUSSION or importin-5, together with Sec61B, regulates collagen I–induced DDR1 nuclear translocation. DDR1 is a nonclassic RTK that activates intracellular signaling DDR1, NMHC-IIA, and b-actin exist as a nuclear complex pathways upon collagen-induced receptor activation. In this associated with chromatin. DDR1, NMHC-IIA, and b-actin study, we show that activated full-length DDR1 is able to form a do not contain a putative NLS domain, yet NMHC-IIA and

analyzed by western blot for levels of NMHC-IIA and DDR1. Input represents non-nuclear lysates (20 mg/lane) analyzed for total levels of DDR1, or nuclear lysates analyzed for total levels of HDAC2 (nuclear marker) and b-tubulin (cytoplasmic marker). (B) Serum-starved HEK- DDR1 cells were treated as in (A). Nuclear lysates (200 mg) were immunoprecipitated with anti–b-actin antibody and the immunopre- cipitates were analyzed by western blot for levels of NMHC-IIA, DDR1, and b-actin. Input represents non-nuclear lysates (20 mg/lane) analyzed for total levels of DDR1, or nuclear lysates analyzed for total levels of PARP1 and b-tubulin. (C) To verify specificity of the im- munoprecipitation, serum-starved HEK-DDR1 cells were treated with vehicle and collagen I for 3 hours. Nuclear lysates (200 mg) were immunoprecipitated with anti–NMHC-IIA or IgG control and the immunoprecipitates were analyzed by western blot for levels of NMHC- IIA, DDR1, and b-actin. Input represents nuclear lysates (20 mg/lane) analyzed for levels of PARP1. (D) Serum-starved HEK-DDR1 cells or HK-2 cells were treated with collagen I (50 mg/ml) for the times indicated. Time 0 represents cells incubated with 20 mM acetic acid for 60 or 180 minutes. Nuclei were separated into nuclear soluble and chromatin fractions which were analyzed by western blot (20 mg/lane) for levels of DDR1, NMHC-IIA, b-actin, and histone H3. (E) Chromatin-bound DDR1, NMHC-IIA, b-actin, and histone H3 bands in HEK-DDR1 or HK-2 cells were quantified by densitometry. Values represent DDR1/, NMHC-IIA/, or b-actin/histone H3 ratio and are the mean6SD of three experiments. (F) Serum-starved HEK-DDR1 and HEK-DDR1-R105A cells were treated with collagen I (50 mg/ml) for the times in- dicated. Chromatin-bound fractions (20 mg/lane) were analyzed by western blot for level of DDR1 and histone H3. (G) Chromatin bound DDR1 and histone H3 bands were quantified by densitometry. Values represent DDR1/histone H3 ratio and are the mean6SD of two experiments performed in triplicate. (H) Confocal images of serum-starved HEK-DDR1-GFP cells treated with collagen I (50 mg/ml) for 0 or 1 hour and stained with anti-histone H3 (left panel) or anti–NMHC-IIA (right panel) antibodies. Time 0 represents cells incubated with 20 mM acetic acid for 3 hours. (I) Line scanning profile showing intensity of DDR1-GFP and histone-3 or NMHC-IIA pixels, measured along the white line in (E). (J) The degree of nuclear colocalization between DDR1 and histone H3 or NMHC-IIA was performed using Manders’ coefficient. Circles represent single cells, whereas the bars represent the mean6SD.

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A HEK-DDR1-GFP DDR1

NM-IIA Actin GFP CIV promoter AGCTCC C 282 bp p<0.05 anti-GFP 1.2 -- --++ ++ CI (50 g/ml) 0.8 282bp - - CIV ChIP

0.4 282bp - - CIV Input CHIP/input 0.0 -+Coll I

B HK-2 DDR1

NM-IIA Actin

AGCTCC CIV promoter D 282 bp p<0.05 1.5 anti-DDR1 IgG ---++++CI (50 g/ml) 1.0 282bp - - CIV ChIP 0.5 CHIP/input 282bp - - CIV Input 0.0 - + Coll I

E 6 p<0.05

4 p<0.05 p<0.05

p<0.05

2 CIV mRNA/GAPDH mRNA

0 -+-+-+ Coll l

WT R105A WT + RLC-TS/DD-GFP

Figure 7. DDR1 forms a complex with the human collagen IV promoter and promotes transcription. (A–D) Overview of the ChIP assay performed in (A) HEK-DDR1-GFP or (B) HK-2 cells. Crosslinked DNA-protein complexes from cells treated with acetic acid or collagen I for 30 or 60 minutes, respectively, were immunoprecipitated with (A) GFP-TRAP beads or (B) anti-DDR1 or IgG control antibody. After DNA elution and purification, DNA fragments were amplified using collagen IV (CIV) promoter primers spanning the putative NM-IIA binding site. Input represents amplified CIV promoter in total DNA isolated from untreated or collagen I–treated cells before

1620 JASN JASN 30: 1605–1624, 2019 www.jasn.org BASIC RESEARCH

A BC

pY PM Sec61B Actin DDR1 ER CIV pY NM-IIA Nucleus + collagen

Figure 8. Schematic representation of the steps and molecules involved in nuclear translocation of activated DDR1. (A) In unstimulated cells, DDR1 interacts with NM-IIA and b-actin at the plasma membrane (PM). (B) Upon collagen stimulation and DDR1 auto- phosphorylation (pY), DDR1/NM-IIA/b-actin complex interacts with Sec61B in the endoplasmic reticulum (ER). (C) This interaction promotes DDR1 nuclear translocation and its association to chromatin where, together with NM-IIA and b-actin, it promotes the transcription of collagen IV (CIV). b-actin are known to translocate to the nucleus. b-actin inter- that the mutant DDR1-R105A fails to translocate to the nu- acts with cofilin which has an NLS and promotes the nuclear cleus after collagen I treatment, suggests that both a collagen I/ translocation of G-actin.60 We identified cofilin 1 peptides by MS DDR1 interaction and DDR1 activation are required for in vehicle- and collagen I–treated cells (Supplemental Table 2), DDR1 nuclear translocation. Our finding that DDR1 interacts suggesting that an interaction between b-actin and cofilin occurs with NM-IIA agrees with the finding that these two proteins and promotes translocation of a DDR1/NMHC-IIA/b-actin colocalize in the inner ear of mice.20 Furthermore, DDR1 and complex into the nucleus. NMHC-IIA interaction, which requires the kinase domain of Nuclear NMHC-IIA and b-actin act as nuclear chaperones DDR1, regulates the assembly of NMHC-IIA into filaments and cotranscription factors. NMHC-IIA regulates annexin I and promotes cell migration.21 Our study reveals a novel func- nuclear translocation 61 and, together with a and b actin, tion for NMHC-IIA, which is to localize DDR1 to the nucleus. regulates ICAM transcription via interaction with its pro- The finding that blocking NM-IIA function by blebbistatin moter.52 Nuclear b-actin promotes eNOS transcription by reduces nuclear DDR1 localization further supports a role of binding repeats that have a cis-acting role in eNOS promoter NMHC-IIA in regulating DDR1 nuclear translocation. Inter- function.62 On the basis of our findingthatuponcollagen estingly, in cells expressing artificially activated NM IIA, stimulation DDR1 interacts with the collagen IV promoter DDR1 but not DDR1-R105A translocates the nucleus also in and with RBBP4, we propose that DDR1 association with the absence of collagen stimulation. A plausible reason is that chromatin enhances NMHC-IIA and b-actin transcription low basal (yet undetectable at western blot level) activation of activity. Consistent with this statement, the transcription of DDR1 by endogenously produced collagen can promote nu- collagen IV, whose promoter contains putative NM II respon- clear translocation of DDR1 in the absence of exogenously sive elements, is enhanced in cells expressing DDR1 able to administered collagen and in the presence of activated NM translocate to the nucleus after collagen I stimulation, and is IIA. On the contrary, DDR1-R105A cannot be activated by further stimulated in cells expressing both DDR1 and a con- endogenous collagen and it cannot be translocated to the nu- stitutively active myosin RLC. cleus also in the presence of activated NM IIA. RTKs translocate to the nucleus in a ligand-dependent and Upon collagen Istimulation, DDR1 regulates the expression ligand-independent manner. Our MS analysis suggests that of several genes by promoting the activation of transcrip- b-actin and NMHC-IIA associate with DDR1 in the absence tion factors. In breast cancer cells, DDR1 increases NFkB-DNA of collagen; however, nuclear DDR1 is only evident after col- binding thus promoting the expression of cyclooxygenase 2.63 lagen I stimulation. This observation, together with the fact In colon cancer cells, DDR1 promotes Notch1 cleavage by

immunoprecipitation. (C and D) CIV ChIP and input CIV bands were quantified by densitometry. Values represent ChIP/input ratio and are the mean6SD of four samples. (E) Serum-starved HEK-DDR1 (WT), HEK-DDR1-R105A, or HEK-DDR1 cells expressing RLC-TS/DD-GFP cDNA were treated with vehicle or collagen I (50 mg/ml). After 24 hours, the levels of collagen IV a1 mRNA were analyzed by RT-rqPCR. Bars and errors are the mean6SD of two to three experiments performed at least in duplicate.

JASN 30: 1605–1624, 2019 Discoidin Domain Receptor 1 Translocates to Nucleus 1621 BASIC RESEARCH www.jasn.org g-secretase, thus enhancing nuclear translocation of cleaved DISCLOSURES Notch1 and transcription of prosurvival genes such as Hes1 and Hes2.64 In this study, we provide evidence that nuclear None. DDR1 can regulate gene transcription by interacting with chromatin and acting as cotranscription factors together with NMHCII-A and b-actin. FUNDING We show that DDR1 is upregulated by injured tubular cells and that nuclear translocation of DDR1 regulates the synthesis This work was in part supported by Veterans Affairs Merit Reviews of collagen IV, a DDR1 ligand found in basement mem- 1I01BX002025 (Dr. Pozzi) and 1I01BX002196 (Dr. Zent); National Institutes 65 of Health grants R01-DK119212 (Dr. Pozzi, Dr. Borza), R01-DK069921 branes. Collagen IV, together with collagen I, is a major (Dr. Zent), R01-DK112688 (Dr. de Caestecker), R01-DK099467 (Dr. Vana- ECM component upregulated in fibrosis.66 Collagen IV is pro- core), R01-DK56942 (Dr. Fogo), and P30-DK114809 (Dr. Pozzi, Dr. Zhang, duced primarily by epithelial cells and, in models of kidney Dr. Zent, Dr. de Caestecker, Dr. Fogo); and the Department of Defense grant tubule–mediated injury, is deposited primarily along injured DOD-PR161028 (Dr. de Caestecker). tubules (see also Supplemental Figure 3). Collagen I is also upregulated in kidney fibrosis and is considered the classic hallmark of tubule-interstitial fibrosis. A key question is why SUPPLEMENTAL MATERIAL DDR1 is also upregulated in injured tubules. A plausible hy- pothesis is that increased DDR1 could strengthen the adhesion This article contains the following supplemental material online of injured proximal tubule cells to collagen IV, thus protecting at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018111160/-/ them from injury or promoting repair. This event might par- DCSupplemental. adoxically contribute to the development of fibrosis by stimu- Supplemental Table 1. List of primary antibodies used for western blot. lating excessive production of collagen upon DDR1 activation Supplemental Table 2. Mass spectrometry identification of some of by its two major ligands. the immunoprecipitated DDR1-interacting proteins from HEK- In conclusion, we provide evidence that activated DDR1 DDR1-GFP expressing cells treated with either vehicle (acetic acid) or translocates to the nucleus, localizes to chromatin, and regulates collagen I. the transcription of profibrotic genes, including collagen IV. Supplemental Figure 1. Mass spectrometry identification of DDR1 These findings, together with the observation that increased nu- phosphotyrosine peptides in collagen I–treated HEK-DDR1-GFP cells. clear levels of DDR1 are evident in injured human and injured Supplemental Figure 2. Nuclear DDR1 forms a complex with murine kidneys, reveal a previously unrecognized mechanism RBBP4. whereby DDR1 promotes the synthesis of profibrotic molecules. Supplemental Figure 3. Analysis of collagens and NMHC-IIA lo- calization in injured human kidneys.

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1624 JASN JASN 30: 1605–1624, 2019 Chiusa et al. Supplementary Information

Supplementary Table of Content

Supplementary Table 1

Supplementary Table 2

Supplementary Figure 1

Supplementary Figure 2

Supplementary Figure 3 Supplementary Table 1. List of primary antibodies used for Western blot

Antibody Vendor Catalog # β-actin Cell Signaling 4970 DDR1 Santa Cruz Biotechnology sc-532 pY792-DDR1 Cell Signaling 5174 pY513-DDR1 Cell Signaling 14531 GAPDH Cell Signaling 5174 GFP Roche 11814460001 HDAC2 Santa Cruz Biotechnology sc-6296 histone H3 Cell Signaling 3638 NMHC-IIA Santa Cruz Biotechnology sc-98978 or sc-47199 PARP1 Cell Signaling 9532 RBBP4 Abcam ab1765 SEC61β Cell Signaling 14648 α-tubulin Santa Cruz Biotechnology sc-8035

Supplementary Table 2. Mass spectrometry identification of some of the immunoprecipitated

DDR1-interacting proteins from HEK-DDR1-GFP expressing cells treated with either vehicle

(acetic acid) or collagen I.

Spectral Counts1 DDR1-interacting candidate Vehicle Collagen I Group assignment ACTBL (actin β like) 0 8 Collagen I2 IPO5 (importin-5) 0 2 Collagen I RBBP4 0 2 Collagen I SC61B 0 2 Collagen I

DDR1 296 264 Common3 ACTB (β-actin) 26 27 Common KPNB1 (importin-β) 25 16 Common CFL1 (cofilin 1) 10 13 Common MYH9 (NMHCII-A) 2 4 Common

1 Total number of filtered spectra for all identified peptides in each protein group

2 Proteins uniquely present in DDR1 immunoprecipitates of collagen I-treated cells

3 Proteins present in DDR1 immunoprecipitates from both vehicle- and collagen I-treated cells

Chiusa M. et al., Supplmentary Figure 1

Unmodified (Y484) Phosphopeptide (pY484)

1.6E5 7.8E5 +2 +2 100 723.3343 100 683.3508 y*10 y*9 y*8 y*7 y6 y5 y4 y2 y1 y10 y9 y8 y7 y6 y5 y4 y2 y1 480 490 480E P P P Y Q E P R P R490 E P P P Y Q E P R P R b b b b b b b b1 b2 b3 b10 1 2 3 4 6 7 10 *

50 50 * * * * Relative Intensity (%) Relative Intensity (%) Intensity Relative * * *

0 0 100 500m/z 800 1200 100 500m/z 800 1200 Unmodified (Y513) Phosphopeptide (pY513)

5.5E5 2.4E6 +3 100 +3 100 830.4193 * 857.0669 * * y*15 y*14 y*13 y*12 y*11 y*10 y*9 y*8 y*7 y*6 y*5 y*4 y*3 y*2 y1 491 514 y15 y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 G N P P H S A P C V P N G S A L L L S N P A Y R 491 514 G N P P H S A P C V P N G S A L L L S N P A Y R b2 b3 b5 b6 b7 b8 b10 b11 b12 b13 b15 b16 b23 * * 50 b2 b3 b4 b5 b6 b7 b11 b14 b15 b16 b22 50 * * Relative Intensity (%) Intensity Relative Relative Intensity (%) * * * * * * 0 0 100 500800 1200 1500 100 500800 1200 1500 m/z m/z

Supplementary Figure 1 Mass spectrometry identification of DDR1 phosphotyrosine peptides in collagen I-treated HEK-DDR1-GFP cells. Peptide spectrum matches (PSMs) of unmodified and phosphorylated peptides (Y484 and Y513) from human DDR1. The b-ion (blue) and y-ion (red) series in each PSM were annotated using pLABEL v2.4.0.5 (Wang L.H. et al., Rapid Commun Mass Spectrom 21, 2985-2991, 2007). The asterisk (*) indicates fragments containing the phosphorylated tyrosine. Phosphorylated tyrosine residues are highlighted in red within the peptide sequence shown above each MS/MS spectrum. Chiusa M. et al., Supplmentary Figure 1

Unmodified (Y520) Phosphopeptide (pY520)

5.8E6 3.2E6 100 100 450.9169+3 y10 y9 y8 y7 y6 y5 y3 y1 * 424.2610+3 515 525 * * * * * L L L A T Y A R P P R y10 y9 y8 y7 y6 y5 y4 y3 y2 y1

b b b9 515 525 2 3 * L L L A T Y A R P P R b2 b3

50 50

Relative Intensity (%) *

Relative Intensity (%) Intensity Relative * * * *

0 0 100 500 900 100 500m/z 1000 m/z

Unmodified (Y792) Phosphopeptide (pY792) 2.3E8 1.8E6 100 100 y* y* y y y y y y y y y y y y y 8 7 6 5 4 3 2 1 607.7498+2 7 6 5 4 3 2 1 567.7661+2 790 798 790N L Y A G D Y Y R798 N L Y A G D Y Y R b b1 b2 b3 b2 3 * 50 50 * Relative Intensity (%) Intensity Relative Relative Intensity (%) Intensity Relative * * 0 0 100 500 1000 100 500m/z 1000 m/z Supplementary Figure 1 Mass spectrometry identification of DDR1 phosphotyrosine peptides in collagen I-treated HEK-DDR1-GFP cells. Peptide spectrum matches (PSMs) of unmodified and phosphorylated peptides (Y520 and Y792) from human DDR1. The b-ion (blue) and y-ion (red) series in each PSM were annotated using pLABEL v2.4.0.5 (Wang L.H. et al., Rapid Commun Mass Spectrom 21, 2985-2991, 2007). The asterisk (*) indicates fragments containing the phosphorylated tyrosine. Phosphorylated tyrosine residues are highlighted in red within the peptide sequence shown above each MS/MS spectrum. Chiusa M. et al., Supplmentary Figure 2

A HEK-DDR1-GFP Lysates Nuclear IP:GFP - - + + - - + + - - + + Collagen I 150 kDa- - DDR1-GFP 51 kDa- - RBBP4

50kDa- --tubulin

15 kDa- - Histone H3

p<0.05 B 0.6 0.4

0.2

0.0 RBBP4/DDR1-GFP - + Collagen I

Supplementary Figure 2 Nuclear DDR1 forms a complex with RBBP4. (A) Serum-starved HEK-DDR1-GFP cells were treated with acetic acid (20 mM) or collagen I (50 μg/ml) for 1 hour. Nuclear lysates (200 μg) were immunoprecipitated with GFP-TRAP beads and the immunoprecipitates were analyzed by Western blot for levels of DDR1-GFP and RBBP4. α-tubulin and histone H3 were used to evaluate purity of the fractions. Histone H3 co-immunoprecipitated with DDR1-GFP and RBBP4 only in cells stimulated with collagen I. Lysates and nuclear represent total and nuclear (both 20 μg) proteins analyzed for total levels of DDR1-GFP, RBBP4, α-tubulin or histone H3. (B) DDR1-GFP and RBBP4 bands were quantified by densitometry. Values represent RBBP4/DDR1-GFP ratio and are the mean ± SD of 2 experiments performed in duplicate. Chiusa M. et al., Supplmentary Figure 3

control Tx-AKI

A NM-IIA LTA DAPI

50 m

5 m

Collagen I B LTA DAPI

C Collagen IV LTA DAPI

Supplementary Figure 3 Analysis of collagens and NMHC-IIA localization in injured human kidneys. Paraffin kidney sections from control or 2 patients with Tx-AKI were stained with NM-IIA antibody and LTA (A), collagen I antibody and LTA (B), or collagen IV antibody and LTA (C) and analyzed by confocal microscopy. Expression of NM IIA becomes evident in the nuclei of injured proximal tubules (arrow). Note increaed expression of both collagen IV and collagen I around injured proximal tubules.