Small proline-rich protein 2B drives stress-dependent degradation and fibroblast proliferation in heart failure

Ryan M. Burkea, Janet K. Lighthousea, Pearl Quijadaa, Ronald A. Dirkx Jr.a, Alexander Rosenbergb,c, Christine S. Moravecd, Jeffrey D. Alexise, and Eric M. Smalla,e,f,g,1

aAab Cardiovascular Research Institute, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642; bDivision of Allergy, Immunology, and Rheumatology Research, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642; cDepartment of Microbiology, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294; dDepartment of Molecular Cardiology, Cleveland Clinic, Cleveland, OH 44195; eDepartment of Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642; fDepartment of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642; and gDepartment of Biomedical Engineering, University of Rochester, Rochester, NY 14642

Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved March 1, 2018 (received for review October 5, 2017) Heart disease is associated with the accumulation of resident leading to self-destruction. The mechanisms controlling the bal- cardiac fibroblasts (CFs) that secrete extracellular matrix (ECM), ance between -autoubiquitination and MDM2-dependent leading to the development of pathological fibrosis and heart p53 degradation are complex, context dependent, and incompletely failure. However, the mechanisms underlying resident CF pro- understood (9). liferation remain poorly defined. Here, we report that small proline- We conducted an RNA sequencing (RNA-seq) screen to identify rich protein 2b (Sprr2b) is among the most up-regulated in genes that are differentially expressed in mouse CFs during physi- CFs during heart disease. We demonstrate that SPRR2B is a regula- ological (swim training) or pathological [transverse aortic constric- tory subunit of the USP7/MDM2-containing ubiquitination complex. tion (TAC)] remodeling. Small proline-rich protein 2b (Sprr2b)was SPRR2B stimulates the accumulation of MDM2 and the degradation among the genes that were most profoundly up-regulated in path- of p53, thus facilitating the proliferation of pathological CFs. Fur- ological remodeling and lost in physiological remodeling. Here, we thermore, SPRR2B phosphorylation by nonreceptor tyrosine kinases describe an interaction between Sprr2b and a nuclear complex of in response to TGF-β1 signaling and free-radical production poten- USP7, MDM2, and p53. Sprr2b promotes MDM2 accumulation tiates SPRR2B activity and cell cycle progression. Knockdown of the and p53 degradation, and accelerates cell cycle progression spe- Sprr2b or inhibition of SPRR2B phosphorylation attenuates cifically in CFs. We demonstrate that TGF-β1 and reactive oxygen USP7/MDM2 binding and p53 degradation, leading to CF cell cycle species (ROS) signaling potentiates both Sprr2b SPRR2B arrest. Importantly, expression is elevated in cardiac tissue and nonreceptor tyrosine kinase (NRTK)-dependent phosphory- from human heart failure patients and correlates with the prolifer- lation of the Sprr2b protein, facilitating USP7/MDM2 interaction ative state of patient-derived CFs in a process that is reversed by and the degradation of p53. Importantly, we show that SPRR2B is insulin growth factor-1 signaling. These data establish SPRR2B as a significantly enriched within the cardiac interstitium in human HF. unique component of the USP7/MDM2 ubiquitination complex that Indeed, SPRR2B expression positively correlates with proliferation drives p53 degradation, CF accumulation, and the development of of CFs isolated from human HF patients. Taken together, our study pathological cardiac fibrosis.

fibroblast | heart | p53 | proliferation | SPRR2B Significance

Heart disease is associated with the development of fibrosis, a eart disease is accompanied by hypertrophic growth and type of scarring that impedes cardiac function. The primary cel- pathological remodeling of the myocardium and is the leading H lular source of cardiac fibrosis is the resident cardiac fibroblast. We cause of death in the United States (1, 2). Activated cardiac fi- found that cardiac fibroblasts from human heart failure patients broblasts (CFs), often called myofibroblasts, accumulate in heart or a mouse model of heart disease express excessive amounts of disease and secrete extracellular matrix (ECM), driving the devel- the SPRR2B protein. We provide evidence that SPRR2B is a signal- opment of cardiac fibrosis (3, 4). Cardiac fibrosis is a form of responsive regulatory subunit of the p53 ubiquitination complex scarring that replaces necrotic tissue and provides structural support that stimulates the destruction of p53 and the accumulation of to the expanding myocardium, but does so at the expense of cardiac pathological fibroblasts. This study defines a unique mechanism function and ultimately leads to heart failure (HF) (1). In contrast, of cell cycle control that is dysfunctional in heart disease and may physiological hypertrophic growth in response to exercise improves drive the development pathological fibrosis. cardiac function and attenuates the development of fibrosis. The mechanisms that control fibroblast accumulation and activation Author contributions: R.M.B. and E.M.S. designed research; R.M.B., J.K.L., P.Q., and R.A.D. specifically in pathological remodeling are poorly defined. performed research; C.S.M. and J.D.A. contributed new reagents/analytic tools; R.M.B., Similarities between pathological fibroblast activation and J.K.L., P.Q., R.A.D., A.R., and E.M.S. analyzed data; and R.M.B. and E.M.S. wrote the paper. unrestrained cell proliferation in cancerous tumors are evident Conflict of interest statement: E.M.S. is the recipient of a research grant from Novartis Pharmaceuticals. (5); the p53 tumor suppressor has recently been linked to lung This article is a PNAS Direct Submission. and cardiac fibrosis, and is also thought to impact CF plasticity Published under the PNAS license. during pressure overload and ischemic injury (6, 7). p53 activates Data deposition: The data reported in this paper have been deposited in the Gene Ex- cell cycle exit genes such as cyclin-dependent kinase inhibitor 1a pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. (Cdkn1a) as well as proapoptotic genes such as BAX (8). p53 is a GSE89885). substrate for the E3 ligase MDM2, which targets 1To whom correspondence should be addressed. Email: [email protected]. p53 for -mediated degradation, relieving constraints This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. on cell cycle. USP7 is an MDM2 deubiquitinase that stabilizes 1073/pnas.1717423115/-/DCSupplemental. MDM2, whereas MDM2 might otherwise ubiquitinate itself, Published online March 26, 2018.

E3436–E3445 | PNAS | vol. 115 | no. 15 www.pnas.org/cgi/doi/10.1073/pnas.1717423115 Downloaded by guest on September 26, 2021 describes a unique stress-dependent mechanism regulating strated robust expression of SPRR2B following TAC, localizing to PNAS PLUS MDM2-dependent p53 degradation that controls pathological both the cytoplasm and nucleus of cardiac interstitial cells, but CF accumulation. not CMs (Fig. 1E). In contrast, SPRR2B-expressing cells were rarely found in the heart of control animals and were undetect- Results able in swim-trained animals. Sprr2b Expression Is Induced in CFs During Pathologic Remodeling. To identify genes that might underlie the divergent fibrotic re- TGF-β1 and H2O2 Synergistically Induce Sprr2b Expression, Which Is sponse of the heart to pathological and physiological hypertro- Required for CF Proliferation. Since HF is associated with TGF-β phic growth, we performed RNA-seq on CFs isolated from mice signaling and increased ROS burden, we evaluated the potential subjected to 10 days of left ventricle pressure overload or swim contribution of SPRR2B to TGF-β and ROS-induced changes in training (Fig. S1A and B) (10–14). This study identified 2,454 fibroblast plasticity. Treatment of CFs with TGF-β1 (10 ng/mL) genes that changed in endurance exercise and 4,051 genes that and H2O2 (50 μM) was found to maximize fibroblast activation changed in response to TAC (Fig. 1A). One of the most affected and proliferation while minimizing cell death (Fig. S2). TGF-β1and genes was Sprr2b (Fig. 1 A and B), which was 4.3-fold up- H2O2 synergistically stimulated the expression of Sprr2b (Fig. 2A) regulated in TAC relative to controls [fragments per kilobase and also led to a synergistic increase in CF proliferation (Fig. 2B). per million reads (FPKM) 8.099 versus 1.890] and 9.1-fold down- In contrast, myofibroblast genes were not synergistically induced regulated in swim relative to controls (FPKM 0.207 versus 1.890). by TGF-β1andH2O2 (Fig. 2C). To determine whether SPRR2B We also identified five others members of the Sprr2 family (Sprr2a, impacts fibroblast activation and/or proliferation, we transfected -d, -e, -h, -i) that were only moderately up-regulated in TAC vehicle or TGF-β1/H2O2–treated CFs with a plasmid driving the compared with swim (Fig. 1B and Fig. S1C). qRT-PCR confirmed expression of human SPRR2B. SPRR2B did not have an effect on the changes in Sprr2b expression during physiological and patho- Acta2 or Col1a1 expression (Fig. 2D), but significantly increased logical remodeling and revealed that Sprr2b is specifically enriched proliferation in both control and TGF-β1/H2O2–treated CFs (Fig. in CFs and not detected in cardiomyocytes (CMs) (Fig. 1C). 2E). In contrast, knockdown of endogenous Sprr2b expression using Separation of cardiac interstitial cells by FACS followed by qRT- two distinct siRNA oligos (Fig. S3 A and B) suppressed TGF-β1/ − PCR confirmed that Sprr2b is enriched following TAC in CD31 / H2O2–induced CF proliferation (Fig. 2F and Fig. S3C). These re- − CD45 mesenchymal cells/fibroblasts (Fig. 1D). Western blot sults reveal that induction of Sprr2b expression in CFs by TGF-β1/ − confirmed that SPRR2B protein is also enriched in the CD31 / H2O2 at least partially underlies the ability of this treatment to − CD45 population (Fig. S1D). Immunostaining further demon- induce cell proliferation.

AB CD Sprr2b Sprr2b

Sprr2b

Sprr2b Sprr2a Sprr2d Sprr2e Sprr2i E

Fig. 1. RNA-seq reveals the Sprr2b gene is differentially regulated by exercise and disease specifically in CFs. (A) Comparison of genes expressed in CFs with fragments per kilobase per million reads (FPKM) > 1 for any condition that are significantly altered by exercise (swim, Left column) or pressure overload (TAC, Right column) compared with controls (q < 0.05). Genes that are altered in both treatment groups (n = 2,075) are indicated by connecting lines with Sprr2b highlighted in red showing inverse regulation. (B) FPKM values reveal the expression of Sprr2b in CFs following 4 or 10 days of a swim and 3 or 10 days after TAC relative to controls. (C) qRT-PCR confirms changes in Sprr2b expression in CFs isolated by differential plating. Sprr2b is not detected in CMs. (D) qRT-PCR for Sprr2b expression in cardiac + + − − endothelial cells (CD31 ), circulating cells (CD45 ), and mesenchymal/fibroblasts (CD31 /CD45 ) isolated by FACS. qRT-PCR data normalized to Gapdh. All data rep- resent mean ± SEM. *P < 0.05; ****P < 0.0001, one-way ANOVA compared with control. n = 3(swimandTAC),n = 4 (control). ND, not detected. (E) Immunostaining CELL BIOLOGY of sections from mouse hearts of the indicated treatment for SPRR2B (red), cardiac troponin T (blue), WGA (green), and nuclei (DAPI, blue). (Scale bar:25μm.)

Burke et al. PNAS | vol. 115 | no. 15 | E3437 Downloaded by guest on September 26, 2021 A B C D Sprr2b Acta2 Col1a1 40 Acta2Col1a1 Sprr2b 25 5 8 *#& **** * #& #& 20 4 * 30 6 * * 15 3 * * 4 * 20 # *# 10 2 *** 2 10 5 1 Relative expression

Relative expression ** Relative expression **

0 Fluorescence intensity (AU) 0 0 0 - - ++ - - ++ - - ++ - - ++ - - ++ - - ++ T/H2O2 - - ++ - ++- - ++- - ++- H2O2 - ++- H2O2 - ++- H2O2 - ++- - ++- SPRR2B

E 6 *#& F 2.5 * G H 60 80 Control * Vehicle + NC si 2.0 # *# SPRR2B T/H2O2 + NC si 60 4 T/H2O2 + siSprr2b 1.5 40 *# *** 40 * 1.0 2 20 *** 20 * % of Population

0.5 % of Population # Fluorescence intensity (AU) 0 Fluorescence intensity (AU) 0 0 0 T/H2O2 - - ++ T/H2O2 - + + G1 S G2/M G1 S G2/M SPRR2B - ++- NC si + -- siSprr2b -- +

IJControl FLAG-SPRR2B K 1.0 3 *** *** 0.8 2 0.6 FLAG / pH3 Dapi 0.4 1 Ki67 + cells (%) pH3 + cells (%) 0.2

0.0 0 Control -- Control -- SPRR2B - SPRR2B - +

FLAG / Ki67 Dapi +

Fig. 2. Synergistic activation of Sprr2b by TGF-β1 and H2O2 is required for CF proliferation. (A–C) Primary neonatal CFs were treated with combinations of TGF-β1 (10 ng/mL) and H2O2 (50 μM) for 24 h. (A) qRT-PCR was performed to evaluate Sprr2b expression (n = 6). One-way ANOVA. (B) CyQuant dye DNA incorporation was performed to evaluate proliferation (n = 8). One-way ANOVA. (C) qRT-PCR for Acta2 and Col1a1 was performed to evaluate myofibroblast activation (n = 6). Two-way ANOVA with Bonferroni post hoc. (D) SPRR2B transfection of CFs followed by qRT-PCR for Acta2 and Col1a1 (n = 6). Two-way ANOVA with Bonferroni post hoc. (E) CyQuant assay was used to evaluate the impact of SPRR2B overexpression on CF proliferation (n = 8). One-way ANOVA.

(F) CyQuant assay was used to evaluate the effect of siRNA-mediated Sprr2b knockdown on TGF-β1/H2O2–induced CF proliferation (n = 8). One-way ANOVA. NC, no-target control siRNA. (G) Flow cytometry shows G2/M and S populations increase in response to SPRR2B overexpression in CFs relative to control (n = 3). One-way ANOVA. (H) Flow cytometry reveals siRNA-mediated knockdown of Sprr2b in CFs blocks the increase in G2/M and S populations by TGF-β1/H2O2 treatment, relative to control (n = 3). One-way ANOVA. (I) Primary CFs were transfected with control or Flag-SPRR2B expression vectors (transfection effi- ciency was ∼36%). Immunofluorescent detection of FLAG and phosphohistone H3 or Ki67 reveals increased mitosis and proliferation in SPRR2B-transfected CFs. (Scale bar: 50 μm.) (J) Quantification of pH3+ cells from I.(K) Quantification of Ki67+ cells from I. n = 6 wells with five nonoverlapping fields of view each. Unpaired Student’s t test. qRT-PCR data normalized to Gapdh. All data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with control. #P < 0.05 compared with second condition; &P < 0.05 compared with third condition.

SPRR2B Accelerates Cell Cycle Progression in CFs. To further evalu- (Fig. S3D). Importantly, forced expression of SPRR2B further ate the role of SPRR2B in CF proliferation, we assessed cell cycle stimulated CF proliferation in response to TGF-β1/H2O2 treat- progression by flow cytometry and immunostaining. TGF-β1/ ment (Fig. 2G). In contrast, siRNA-mediated knockdown of en- H2O2 treatment led to a significant increase of primary CFs in dogenous Sprr2b blocked the increase in cell cycle progression the S and G2/M phases, and decreased the proportion of cells in G1 observed upon TGF-β1/H2O2 treatment (Fig. 2H). Immunostaining

E3438 | www.pnas.org/cgi/doi/10.1073/pnas.1717423115 Burke et al. Downloaded by guest on September 26, 2021 Control SPRR2B PNAS PLUS A 8 B 50 Sprr2b Ccna2 5 Ccnb1 *** # CCNA2 **** **** **** # 40 4 6 CCNB1 30 3 ** * 4 ** CDK1 20 2 * * CDK1 2 Relative expression -pY15 10 1 GAPDH 0 0 0 Control H O Control H O TGF- 1/ 2 2 TGF- 1/ 2 2 Control TGF- 1/H2O2

4 10 1.5 C 8 Ccne1 Cdk1 ** Cdkn1a # *** Control Control 8 3 SPRR2B 6 SPRR2B 1.0 6 2 * 4 ** ** 4 0.5 * * 1 2 ** Relative expression 2 **** *** Relative protein levels

*** 0 0 0 0 Control H O Control TGF- 1/H O Control TGF- 1/H2O2 TGF- 1/ 2 2 2 2 CDK1 CDK1 CCNA2 CCNB1 pY15 D Sprr2b Ccna2 Ccnb1 Ccne1 Cdk1 Cdkn1a 20 5 5 2.5 5 **** 4 *** ** * * **** ** # # 4 4 2.0 4 15 * 3 ** ** # ** ** 3 3 # # 1.5 * 3 * ** # # 10 # # 2 2 2 1.0 2 *

5 1

Relative expression 1 1 0.5 1 * * # # 0 0 0 0 0 0 T/H2O2 - + ++ - + ++ - + ++ - + ++ - + ++ - + ++ Sprr2b-siRNA NCNC 1 2 NC NC 12 NC NC 12 NC NC 12 NC NC 12 NC NC 12

Fig. 3. Sprr2b is necessary and sufficient for alterations in gene expression consistent with accelerated cell cycle. (A) The expression of promitotic cyclins and cyclin-dependent kinases (Ccna2, Ccnb1, Ccne1, Cdk1) and Cyclin-dependent kinase 1 inhibitor (Cdkn1a/p21) were examined by qRT-PCR in primary CFs

overexpressing SPRR2B with or without TGF-β1/H2O2 and analyzed by two-way ANOVA with Bonferroni post hoc (n = 6). (B) Western blot evaluates alter- ations in CCNA2, CCNB1, CDK1 protein levels and an inhibitory phosphorylation of CDK1 (pY15). GAPDH is included as a loading control. (C) Quantification of

B. n = 3 experiments each, analyzed by unpaired Student’s t test. (D) TGF-β1/H2O2–dependent alterations in the expression of mitotic genes were evaluated by qRT-PCR upon knockdown of Sprr2b with two independent siRNAs (1 and 2). n = 3 experiments each, analyzed by two-way ANOVA with Bonferroni post hoc. qRT-PCR data normalized to Gapdh. All data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. #P < 0.05 compared with second condition. NC, nontargeting control siRNA.

for FLAG-tagged SPRR2B and phospho-histone H3 (pH3) or associated with mitosis and cell cycle that cluster with Sprr2b Ki67 to identify SPRR2B-expressing CFs in M or S phases of the expression changes. Several promitotic cyclins and cyclin- cell cycle confirmed the positive influence of SPRR2B on cell cycle dependent kinases are up-regulated in TAC relative to swim, progression (Fig. 2 I–K). while important cyclin-dependent kinase inhibitors (CDKNs) are

Since Sprr2b induction by TGF-β1 and ROS drives CF pro- suppressed (Table S1), consistent with the dysregulation of CELL BIOLOGY liferation in vitro, we evaluated our RNA-seq dataset for genes p53-dependent gene programs identified by Ingenuity Pathway

Burke et al. PNAS | vol. 115 | no. 15 | E3439 Downloaded by guest on September 26, 2021 Analysis (Table S2). Interestingly, the expression of the mitotic We therefore evaluated predicted interacting motifs in the cyclins Ccna2 and Ccnb1,andCdk1 were synergistically induced in SPRR2B protein. Eukaryotic linear motif prediction identified CFs by TGF-β1/H2O2 and SPRR2B (Fig. 3A). In contrast, ex- four motifs potentially related to cell cycle control, including a C- pression of Cdkn1a, a CDK1 inhibitor, was silenced by SPRR2B terminal interacting module for USP7, an MDM2 and p53 deu- overexpression (Fig. 3A). Western blot for the protein products of biquitinase (Fig. 4B) (22). Consistent with our observation of relevant mitotic genes confirmed the alterations upon SPRR2B endogenous SPRR2B protein localization in hearts of mice sub- overexpression (Fig. 3 B and C). In addition to alterations in jected to TAC surgery, immunofluorescent detection of Flag CDK1 protein levels, SPRR2B also led to a reduction in the epitope-tagged SPRR2B in NIH 3T3 cells revealed localization phosphorylation of CDK1 at Y15—a cell cycle-inhibitory phos- both in the cytoplasm and nucleus, where relevant interactions phorylation conferred by Wee1/Myt1 kinases (Fig. 3 B and C) with USP7/MDM2 would likely occur (Fig. 4C). To test the pre- (15). To determine whether SPRR2B is essential for the alter- dicted interaction between SPRR2B and USP7, we overexpressed ations in mitotic regulators by TGF-β1/H2O2, we again used an HA-tagged USP7 construct alone or in combination with Flag- siRNA knockdown of endogenous Sprr2b in CFs, which signifi- SPRR2B in NIH 3T3 cells. SPRR2B coimmunoprecipitated with cantly attenuated the alterations in mitotic regulators (Fig. 3D). USP7, and formation of this unique protein complex was stimu- These data indicate SPRR2B is necessary and sufficient for the lated by TGF-β1/H2O2 treatment (Fig. 4 D and E). The presence acceleration of cell cycle in TGF-β1/H2O2–treated CFs. of SPRR2B also facilitated an interaction between USP7 and endogenous MDM2 and the accumulation of MDM2 protein, SPRR2B Binds USP7, Stabilizing USP7–MDM2 Interaction. To gain indicating SPRR2B is a component of the p53–E3 insight into the potential mechanism whereby SPRR2B drives protein complex in CFs. CF cell cycle progression, we examined the expression of genes encoding proteins known to interact with SPRR2B. The best Tyrosine Phosphorylation of SPRR2B Promotes USP7 Binding and CF characterized role of SPRR2B is as part of the keratinocyte Proliferation. SPRR2B was predicted to be a phosphoprotein in a cornified envelope (CEnv), following cross-linking by TGM1 and high-throughput mass spectrometry screen of non-small cell lung TGM3 (16–21). Since the CEnv program is induced by oxidative cancer cells (23). We find that Flag epitope-tagged SPRR2B is a stress in the skin, we began by evaluating the expression of CEnv substrate for tyrosine (Y) phosphorylation in NIH 3T3 cells and program genes (Lor, Ivl, Flg, Tgm1, and Tgm3) in either CFs or primary mouse CFs (Fig. 5A and Fig. S5). Phosphorylation of primary mouse dermal fibroblasts (DFs) subjected to optimal SPRR2B in both the nuclear and cytoplasmic compartments is doses of TGF-β1 and H2O2 (Figs. S2 and S4). Although the strongly stimulated by TGF-β1/H2O2. SPRR2s have been shown entire CEnv program was present in DFs at baseline and was to interact with the Src family of nonreceptor tyrosine kinases highly enriched in response to TGF-β1/H2O2, only the expression (NRTKs) via SH3 domain repeats, but the consequence of these of Sprr2b was stimulated by TGF-β1/H2O2 in CFs (Fig. 4A). This interactions is unknown (24, 25). Inhibition of NRTK activity finding suggests that SPRR2B may play a noncanonical role in with saracatinib (50 nM) attenuated TGF-β1/H2O2–dependent CFs due to the absence of typical binding partners. USP7-SPRR2B binding (Fig. 5 B and C). Since human SPRR2B

P A Cardiac Fibroblasts Dermal Fibroblasts B 1 SPRR2B 27 25 Lor *** QQYSM KPTPCV KPPYKPSPTVP 20 Ivl BIR-II CDK CSK1 USP7 Flg *** C 15 Tgm1 Flag-SPRR2B Tgm3 Phalloidin Dapi 10 Sprr2b

5 Relative expression 0 Control TGF- 1/ Control TGF- 1/ H2O2 H2O2 D E USP7 Flag MDM2 IgG IP: HA TCL *** 25 # HA-USP7 - + ++ - + ++ Flag-SPRR2B - -++ - -++ 20 H O TGF- 1/ 2 2 -- +- -- +- AH 7PSU 15 (IP / TCL) / (IP GALF GALF 10

*

2MDM 2MDM Relative protein levels 5 # * GAPDH * 0 HA-USP7 + ++ + ++ + ++ Flag-SPRR2B - + + -++ -++ H O TGF- 1/ 2 2 - +- - +- - +-

Fig. 4. SPRR2B is a binding partner for the MDM2 deubiquitinase USP7 in CFs. (A) qRT-PCR for Sprr2b and genes encoding keratinocyte proteins in primary

CFs and DFs with or without TGF-β1/H2O2.(B) Schematic of human SPRR2B protein, highlighting predicted protein–protein interactions related to cell cycle control. The predicted USP7 interaction domain contains a canonical tyrosine phosphorylation site at Y67. (C) Fluorescent detection of phalloidin (green) SPRR2B (red) and nuclei (blue) by confocal microscopy reveals both cytoplasmic and nuclear localization of SPRR2B in CFs. (Scale bar: 10 μm.) (D) NIH 3T3 cells were cotransfected with HA-tagged USP7 with or without Flag-tagged SPRR2B. Immunoprecipitation of HA-USP7, followed by immunoblotting for Flag-

SPRR2B or endogenous MDM2 revealed complex formation particularly in the presence of TGF-β1/H2O2.(E) Quantification of D, analyzed by two-way ANOVA with Bonferroni post hoc (n = 3). qRT-PCR data normalized to Gapdh. All data represent mean ± SEM. *P < 0.05, ***P < 0.001 compared with control. #P < 0.05 compared with second condition.

E3440 | www.pnas.org/cgi/doi/10.1073/pnas.1717423115 Burke et al. Downloaded by guest on September 26, 2021 has only two tyrosine residues (Y3 and Y67), and Y67 is in the played a significant decrease in p53 protein relative to control PNAS PLUS USP7-interacting region, we tested whether USP7 binding is de- cultures (Fig. 6 A and B). The reduction in p53 was reflected in pendent upon SPRR2B-pY67. Although a SPRR2B-Y67F phospho- significant attenuation of the p53 target gene and cell cycle in- + mutant does not completely abolish the USP7 interaction, the Y67F hibitor, CDKN1A/p21. We also made use of a Zn2 -inducible mutation attenuates the stimulation of SPRR2B–USP7 interaction CFP-tagged p53 overexpression construct to test the effect of and accumulation of MDM2 protein upon TGF-β1/H2O2 treatment SPRR2B on p53 protein levels, localization, and target gene ex- (Fig. 5 D and E). Importantly, SPRR2B-Y67F overexpression fails to pression in NIH 3T3 cells. SPRR2B led to reduced levels of both stimulate the TGF-β1/H2O2–dependent changes in cell cycle-related p53-CFP and endogenous p53, specifically in the nucleus, where genes and induction of CF proliferation that are observed upon wild- ubiquitin-mediated degradation of p53 occurs (Fig. 6C). Impor- type SPRR2B overexpression (Fig. 5 F and G). These data suggest tantly, p53-CFP overexpression induced the expression of Cdkn1a that the phosphorylation of SPRR2B by Src family NRTKs at and inhibited the promitotic cyclins, an effect that was reversed Y67 facilitates USP7 interaction, MDM2 accumulation, and cell upon SPRR2B overexpression (Fig. 6D). In contrast, SPRR2B- cycle progression. Y67F failed to facilitate p53 degradation in response to TGF-β1/ H2O2 treatment, confirming the importance of SPRR2B phos- SPRR2B Destabilizes the Nuclear p53 Pool. Since SPRR2B promotes phorylation (Fig. 6 E and F). MG132 blocked the SPRR2B- cell cycle progression and the accumulation of MDM2, we hy- dependent degradation of p53, revealing SPRR2B stimulates pothesized that SPRR2B may influence p53 protein levels in ubiquitin-mediated p53 degradation (Fig. 6 G and H). Indeed, CFs. TGF-β1/H2O2–treated CFs overexpressing SPRR2B dis- SPRR2B overexpression stimulated the ubiquitination of p53 as

ABNucleus Cytoplasm HA-USP7 + + ++ C HA- FLAG- TGF- 1/H O - - 2 2 + + Flag-SPRR2B + +++ 8 USP7 SPRR2B H O *** IP: FLAG TGF- 1/ 2 2 -+ +- Blot: Phos-Y Saracatinib -- ++ 6 TCL: FLAG HA 4

(IP / TCL) / (IP 2 FLAG # #

HDAC1 Relative protein 0 FLAG HA-USP7 + + ++ + + ++ SOD1 Src Flag-SPRR2B +++ + +++ + TGF- 1/H O - ++- - ++-

TCLpY416 IP : HA 2 2 Saracatinib -- ++ -- ++ GAPDH

D E SPRR2B MDM2 IP: HA TCL 2.5 H O TGF- 1/ 2 2 - + +- - + +- **** Flag-SPRR2B + +-- + +-- 2.0 FLAG-Y67F -- ++ -- ++ HA-USP7 ++ ++ ++ ++ 1.5 # HA HA * 1.0 (IP / TCL) / (IP # * ** # FLAG FLAG # Relative protein 0.5 2MDM 2MDM 0 H O - - - + - + GAPDH TGF- 1/ 2 2 + + Flag-SPRR2B ++- - ++- - FLAG-Y67F -- ++ -- ++ F Ccna2 Ccnb1 Ccne1 Cdk1 Cdkn1a G 15 3 Control ** SPRR2B * Y67F # 10 2 ** # #

5 1

Relative expression * * # * # Fluorescence Intensity (AU) # * # 0 0 TGF- 1/H O - + - + - + - + - + - + - + - + - + - + 2 2 Control TGF- 1/H O Flag-SPRR2B ++- - ++- - ++- - ++- - ++- - 2 2 FLAG-Y67F --+ + --+ + --+ + --+ + --+ +

Fig. 5. USP7 binding and cell cycle progression is facilitated by NRTK-dependent tyrosine phosphorylation of SPRR2B. (A) Fractionation of Flag-SPRR2B– transfected NIH 3T3 cells and immunoprecipitation of Flag followed by immunoblot for phosphotyrosine. Representative of three independent experi- ments. Loading controls are HDAC1 (nucleus) and SOD1 (cytoplasm). (B) NIH 3T3 cells transfected with HA-USP7 and Flag-SPRR2B were treated with

saracatinib (50 nM) with or without TGF-β1/H2O2. Coimmunoprecipitation of HA reveals stimulation of Flag-SPRR2B interaction by TGF-β1/H2O2 is blocked by saracatinib treatment. TCL, total cell lysate. (C) Quantification of B. n = 3. (D) Coimmunoprecipitation of HA-USP7/Flag-SPRR2B (wild type or Y67F) reveals USP7 association and MDM2 accumulation is dependent upon SPRR2B-Y67 phosphorylation. (E) Quantification of D. n = 3. (F) Expression of genes encoding

cell cycle regulators was evaluated by qRT-PCR in CFs transfected with SPRR2B or SPRR2B-Y67F with or without TGF-β1/H2O2. n = 6. qRT-PCR data normalized to Gapdh and analyzed by one-way ANOVA. (G) CyQuant assay was used to evaluate proliferation of CFs in response to SPRR2B or SPRR2B-Y67F transfection CELL BIOLOGY with or without TGF-β1/H2O2. n = 8. Analyzed by two-way ANOVA with Bonferroni post hoc. All data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; #P < 0.05 compared with second condition.

Burke et al. PNAS | vol. 115 | no. 15 | E3441 Downloaded by guest on September 26, 2021 AB3 C ControlSPRR2B Control *** Nucleus Cytoplasm SPRR2B p53 SPRR2B - ++- - ++- --+ + 2 p53-CFP --+ + CDKN1A p53-CFP

MDM2 p53 (endog) 1 SOD1 USP7 ***

Relative protein levels *** HDAC1 GAPDH 0 p53 MDM2 USP7 CDKN1A DECdkn1a Ccna2 Ccnb1 Ccne1 F 10 1.5 * Control # TGF- 1/H O - ++- 8 2 2 T/H O SPRR2B ++ -- 2 2 1.0 6 Y67F --+ + p53 4 * * # * 0.5 & * * * * GAPDH 2 * # * # * # ** Relative expression * # & # & # &

0 Relative p53 protein 0 SPRR2B ++-- ++-- ++-- ++-- SPRR2B Y67F p53 -- ++ -- ++ -- ++ -- ++

G HI2.5 4 ** & SPRR2B -- ++ 2.0 & 3 MG132 ++-- p=0.077 * IP:p53 Ub 1.5 2 p53 1.0 1 TCL GAPDH 0.5 Relative Ub / p53 Relative p53 protein

0 0 SPRR2B -- ++ SPRR2B -- ++ MG132 ++-- MG132 ++--

Fig. 6. SPRR2B facilitates ubiquitin-mediated proteasome degradation of p53. (A) Primary CFs were transfected with Flag-SPRR2B or control and analyzed by Western blot. (B) Quantification of A. n = 3. Analyzed by Student’s two-tailed unpaired t test. (C) Nuclear and cytoplasmic fractions were isolated from NIH 3T3 cells that were transfected with indicated combination of SPRR2B and p53-CFP. Western blot was performed to detect exogenous or endogenous p53. Loading controls are HDAC1 (nucleus) and SOD1 (cytoplasm). (D) qRT-PCR was used to evaluate the expression of genes encoding cell cycle regulators in NIH 3T3 cells that were transfected with p53-CFP with or without SPRR2B (n = 6). qRT-PCR data normalized to Gapdh and analyzed by one-way ANOVA. (E)NIH

3T3 cells were transfected with SPRR2B or SPRR2B-Y67F with or without TGF-β1/H2O2, followed by Western blot for endogenous p53. (F) Quantification of E analyzed by two-way ANOVA with Bonferroni post hoc (n = 3). (G–I) Primary CFs were treated with ZnSO4 (50 μM) and transfected with Flag-SPRR2B with or without MG132 (10 μM). (G) Coimmunoprecipitation of Flag was followed by immunoblot for monoubiquitin (Ub). Western blot was also performed to detect endogenous p53 in total cell lysate (TCL). (H) Quantification of total p53 levels in G relative to GAPDH loading controls. (I) Quantification of immunopre- cipitated ubiquitin in G relative to total GAPDH-normalized p53 levels. Analyzed by two-way ANOVA with Bonferroni post hoc (n = 3). All data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05 compared with second condition; &P < 0.05 compared with third condition.

revealed by coimmunoprecipitation (Fig. 6 G and I). These data undetectable in 9 of 16 normal heart samples and significantly link SPRR2B stimulation by TGF-β1/H2O2 to MDM2-dependent enriched in human HF (Fig. 7A). Immunostaining for SPRR2B p53 degradation and CF proliferation. in tissue derived from HF patients (n = 3) supported these data, as SPRR2B expression correlated well with vimentin staining— SPRR2B Expression Correlates with Fibroblast Proliferation in Human suggesting a nonmyocyte localization similar to that seen in HF. We next evaluated the expression of SPRR2B in cardiac tissue the TAC mouse model (Fig. 7B). Importantly, healthy hearts isolated from HF patients undergoing ventricular assist device (n = 3) were largely devoid of SPRR2B expression (Fig. 7B). We implantation, compared with normal heart tissue. SPRR2B was therefore examined the relationship between SPRR2B expression

E3442 | www.pnas.org/cgi/doi/10.1073/pnas.1717423115 Burke et al. Downloaded by guest on September 26, 2021 PNAS PLUS A SPRR2B B Healthy Heart failure 25 * 20

15

10 S/VIM/Dapi S/VIM/Dapi

5

Relative expression 0 Healthy Heart Failure SPRR2B WGA SPRR2B WGA (9/16 = ND) (0/15 = ND) C D 25 Human CF (Healthy) Human CF * 15 20 15 * * * 10 * * * 10 5

1.5 5 1.0 Relative expression * * * 0.5 Relative expression * 0 0 Healthy HF #1 HF #2 HF #3 SPRR2B POSTN ACTA2 COL1A1 COL3A1 FN

POSTN ACTA2 COL1A1 COL3A1 SPRR2B Control TGF- 1/H2O2 IGF-1/ H2O2

E Human CF (HF) F Human CF (HF) G CARDIAC FIBROBLAST 2.0 Control * 1.5 TGF- 1/ROS IGF-1/H O 1.5 2 2 Disease P 1.0 * USP7 Cell 1.0 * SPRR2B p53 Fibrosis ** Cycle MDM2 0.5 Exercise 0.5

Relative expression Ub

Fluorescence intensity IGF-1 0 0 SPRR2BCCNA2 CDK1 CDKN1A IGF-1/H2O2 - +

Fig. 7. SPRR2B is significantly enriched in the failing human heart and positively correlates with promitotic factors in human HF fibroblasts. (A) qRT-PCR was used to evaluate SPRR2B expression in left ventricle tissue isolated HF patients or healthy hearts. n = 16 (Healthy), 15 (HF). ND, not detected. (B) Immuno- histochemistry of representative sections from failing or healthy hearts costained for WGA (white), SPRR2B (green), and VIMENTIN (VIM, red). DAPI (blue) stains nuclei. SPRR2B is associated with vimentin-positive cells in the failing heart, indicated by white arrows. (Scale bar: 25 μm.) (C) qRT-PCR was used to evaluate the expression of SPRR2B and genes encoding myofibroblast markers in human CFs isolated from healthy or failing (HF) hearts (n = 3 HF and one healthy isolate). (D) qRT-PCR was used to evaluate the expression of SPRR2B and myofibroblast markers in healthy human CFs treated with indicated

combinations of TGF-β1 (10 ng/mL), IGF1 (100 ng/mL), and H2O2 (25 μM) (n = 3). (E) qRT-PCR was used to evaluate the expression of SPRR2B and mitotic regulators in human HF CFs treated with vehicle or IGF1/H2O2.(F) CyQuant dye incorporation to measure proliferation of human HF CFs treated with vehicle or IGF1/H2O2. All qRT-PCR data normalized to Gapdh and analyzed by one-way ANOVA (n = 3, unless indicated). CyQuant data analyzed by unpaired Stu- dent’s t test (n= 3). All data represent mean ± SEM. *P < 0.05; **P < 0.01. (G) Model highlighting the role of SPRR2B in regulating cell cycle progression in CFs. Heart disease (in vivo) or TGF-β1/ROS (in vitro) induce, while exercise (in vivo) or IGF1 (in vitro) repress SPRR2B gene expression in CFs. Phosphorylation of SPRR2B facilitates interaction with USP7/MDM2, leading to MDM2 accumulation and degradation of the nuclear p53 pool, relieving constraints on cell cycle progression and promoting fibrosis in heart disease.

and markers of myofibroblast activation in cultures of primary conditions, respectively; IGF1 stimulates physiological hyper- human CFs (hCFs) isolated from three independent HF patients, trophy in skeletal myoblasts and CMs (12). We found that IGF1/ compared with one hCF isolate obtained from healthy cardiac H2O2 significantly reduced SPRR2B expression in healthy hCFs, tissue. SPRR2B expression was significantly increased in HF hCFs whereas TGF-β1/H2O2 elevated SPRR2B expression relative to compared with healthy hCFs, positively correlating with the ex- control treatment, again positively correlating with markers of pression of POSTN, ACTA2, COL1A1,andCOL3A1 (Fig. 7C). myofibroblast activation (Fig. 7D). We therefore asked whether To further evaluate the regulation and function of SPRR2B, IGF1/H2O2 treatment of hCFs from HF patients might attenuate we cultured normal healthy hCFs in a combination of H2O2 SPRR2B expression and normalize the expression of cell cycle

(25 μM) and TGF-β1 (10 ng/mL) or insulin-like growth factor 1 regulators. While all three HF hCF isolates proliferated to a CELL BIOLOGY (IGF1) (100 ng/mL) to mimic pathological and physiological similar extent in response to varying concentrations of H2O2

Burke et al. PNAS | vol. 115 | no. 15 | E3443 Downloaded by guest on September 26, 2021 (Fig. S6A), none of the HF hCF isolates displayed a significant identify additional binding partners for SPRR2B and provide increase in myofibroblast markers upon TGF-β1/H2O2 treat- further insight into the regulation of the SPRR2B–USP7– ment, further indicating a high level of basal activation (Fig. MDM2–p53 complex. Taken together, our findings suggest S6B). IGF1/H2O2 treatment of HF hCFs decreased SPRR2B that SPRR2B controls whether USP7/MDM2 is self-destructive expression, and this reduction correlated with an increase in or acts as the p53 E3 ubiquitin ligase, relieving constraints on cell CDKN1A as well as a decrease in CDK1 expression (Fig. 7E). cycle in CFs. Possible explanations for these findings include re- Importantly, IGF1/H2O2 treatment also significantly reduced duced deubiquitination of p53 by USP7, increased deubiquiti- proliferation of primary HF hCFs (Fig. 7F). We conclude that nation of MDM2 by USP7, or increased MDM2 activity, which SPRR2B dysregulation is a conserved aspect of mouse and hu- may arise either via steric hindrance or alterations in the stoichi- man HF that controls cell cycle regulators and CF proliferation ometry of complex formation by SPRR2B. in disease. The development of cardiac fibrosis is associated with pro- + liferation of Tcf21 resident CFs and the transformation of qui- Discussion + escent CFs into Postn activated fibroblasts (31). Activated This study reveals a unique and conserved molecular mechanism fibroblasts, called myofibroblasts, are characterized by the abun- that stimulates CF accumulation during pathological cardiac dant expression of contractile proteins and secretion of ECM (3, remodeling. Expression of the SPRR2B gene is elevated in hu- 4). Although it is currently not clear whether fibroblast pro- man HF and in a mouse model of pressure overload-induced liferation and myofibroblast activation are coincident or are sep- ventricular hypertrophy and lost during exercise. We demon- arable cellular processes, cell proliferation and differentiation are strate that, within the heart, SPRR2B is restricted to CFs, where it mutually exclusive in many contexts, including progenitor cell self- functions as a regulatory subunit of the USP7/MDM2-containing renewal versus differentiation and smooth muscle cell phenotypic p53 E3 ubiquitination ligase complex, facilitating MDM2 accu- modulation (32–34). Even though SPRR2B gene expression pos- mulation and p53 destruction (Fig. 7G). Thus, SPRR2B relieves itively correlates with both myofibroblast differentiation markers a major constraint on CF cell cycle progression in response to and promitotic factors, SPRR2B only functionally impacts CF pathological signals and may contribute to the development of proliferation. It remains possible that the heart harbors a het- pathological cardiac fibrosis. erogeneous population of CFs, some of which are proliferative SPRR2B function has previously been studied almost exclu- + − sively in the context of keratinocyte differentiation, where it is SPRR2B cells and some of which are SPRR2B myofibroblasts. cross-linked into the cell membrane to form the CEnv during the Further studies using single-cell transcriptomics are required to process of epidermal barrier formation and programmed cell discern the differences between individual CFs in health and death (26). Although SPRR2B has not been directly implicated disease that might differentially influence CF proliferation and in cell death mechanistically, its incorporation into the CEnv ECM secretion. is an essential step leading to cornification death. Interestingly, RNA-seq revealed inverse regulation of Sprr2b expression by the USP7 interaction motif within the C terminus of SPRR2B exercise and disease specifically in CFs. We were able to re- identified in our study is cross-linked by TGM1 and TGM3 in capitulate this dichotomy in CFs derived from HF patients using keratinocytes (21). Thus, an interaction between SPRR2B and in vitro models of physiological (IGF1/H2O2) and pathologic β USP7 that may suppress p53 activity and block death signaling is (TGF- 1/H2O2) signaling. These data suggest that regulation of unlikely in keratinocytes or DFs. In contrast, CFs do not express Sprr2b expression represents a conserved transcriptional response SPRR2B cross-linking or any of the CEnv components to extrinsic signals in CFs. The Sprr2b promoter is complex, with other than SPRR2B. The only transglutaminase we found to be several evolutionarily conserved transcription factor-binding sites significantly expressed in CFs is tissue transglutaminase (TGM2), upstream of the transcriptional start site and within the first intron which has a significantly lower affinity for SPRRs (21, 27, 28). Our that are relevant to cardiac stress signaling (e.g., Smad, NRF2, study reveals that, in the absence of CEnv partners, SPRR2B plays AHR, GABP, and SP1). Further study of this promoter region in a noncanonical role in the nucleus, where it contributes to cell CFs may reveal a paradigm of differential transcriptional regula- cycle acceleration via p53 degradation. More broadly, our study tion by pathological and physiological signaling. expands on a paradigm whereby fibroblasts express gene programs In conclusion, this study defines an evolutionarily conserved that reflect their developmental origin and may influence their mechanism controlling p53 stability that regulates resident CF function (29). proliferation, but not their differentiation into myofibroblasts. The unique interaction between SPRR2B and USP7 that leads Scar formation by myofibroblasts is an essential aspect of cardiac to MDM2 accumulation and p53 degradation suggests a more repair that provides structural support in pressure overload and complex regulation of cell cycle control in CFs. USP7 is a prevents ventricle rupture after myocardial infarction (35, 36). context-dependent deubiquitinase that stabilizes either MDM2 However, accumulation of fibroblasts in the injured heart ulti- or p53, accelerating or blocking cell cycle progression, re- mately leads to pathological fibrosis and deterioration of cardiac spectively (9). Thus, regulation of MDM2–USP7 interaction is a function. Our findings indicate a provocative therapeutic strategy primary determinant of p53 stability and cell cycle control. may be blocking excessive CF proliferation and accumulation MDM2 interacts with USP7 via an N-terminal MATH domain while allowing preexisting resident CFs to differentiate into (competitive interaction with p53) and at a C-terminal Ubl domain ECM-producing cells. Chemotherapeutic agents typically target (responsible for p53 deubiquitination) (9, 30). Motifs present in replication checkpoint controls, a general approach that would SPRR2B predict the interaction with USP7 is through the C- not be amenable to cardiac therapeutics. Targeting CF-enriched terminal Ubl domain, and not through the MATH domain that and stress-responsive promitotic factors such as SPRR2B may might preclude MDM2-dependent p53 degradation (22). We also prevent pathological cardiac remodeling while limiting off- found that NRTK-dependent phosphorylation of SPRR2B at Y67 target effects. facilitated complex formation with USP7 and p53 degradation. We therefore anticipate SPRR2B may be the target of signaling Materials and Methods pathways and additional binding partners that coordinate the All animal work was approved by the University Committee on Animal Re- regulation of cell cycle progression. Eukaryotic linear motif anal- search at the University of Rochester. Human tissue samples were collected ysis predicted interaction sites for CDKs and CKS1, which suggest following informed consent, and all experiments involving human subjects that cyclin–CDK–CKS1 complexes may directly interact with were performed under the auspices of protocols approved by University of SPRR2B once in the nucleus (22). A proteomics approach might Rochester or Cleveland Clinic Research Subjects Review Board.

E3444 | www.pnas.org/cgi/doi/10.1073/pnas.1717423115 Burke et al. Downloaded by guest on September 26, 2021 Animal Models. TAC and swim training models were performed as described in Western Blotting and Immunoprecipitation. Western blotting and immuno- PNAS PLUS SI Materials and Methods. precipitation were performed as described in SI Materials and Methods. Antibodies used are listed in Table S5. Primary Cell Isolations. Isolation of adult mouse ventricular fibroblasts, neonatal mouse ventricular and dermal fibroblasts, and human CFs is described in SI Ma- Immunofluorescence. Immunofluorescence was performed as described in SI terials and Methods. CFs were used between passages 1 and 7 for experiments. Materials and Methods. Primary antibody dilutions may be found in Table S5.

Constructs and Mutagenesis. Constructs and their sources may be found in Image Analysis. Masson Trichrome staining was quantified with the use of the Tables S3 and S4. K-means clustering algorithm in NIH ImageJ. Nuclear counting was per- formed by particle analysis with NIH ImageJ. Western blot densitometry was RNA Isolation and Analyses. RNA-seq and qRT-PCR analysis are described in SI performed in NIH ImageJ using the Gels plugin. Materials and Methods. Statistical Analysis. Data are presented as the mean ± SEM. Statistical dif- Proliferation Assays. Proliferation was assessed by CyQuant assay (Invitrogen) ferences between two groups were determined using Student’s unpaired using manufacturer instructions using a multiwell plate reader (BMG t test with Welch’s correction. For comparisons between more than two FluoStar Optima). groups, one-way or two-way ANOVA was used, as indicated in figure leg- end. Significance was considered as P < 0.05. Flow Cytometry. Cells were trypsinized with 2.5% trypsin–EDTA, resuspended in PBS with 0.2% BSA, and then fixed with cold 70% ethanol. Cell suspensions ACKNOWLEDGMENTS. This work was supported by NIH/National Heart, Lung, were then stained with propidium iodide containing RNase A (BD Pharmingen). and Blood Institute Grants R01HL133761 and R01HL120919 (to E.M.S.), Flow cytometry was accomplished using a three-laser, 12-color BD LSR-II with T32HL007937 and F32HL136066 (to R.M.B.), T32HL066988 (to J.K.L.), and excitation at 610/20 nm (BD Biosciences). Data were analyzed using FlowJo. T32HL066988 and F32HL134206 (to P.Q.); American Heart Association Grant 15POST25550114 (to J.K.L.); and a Pilot Study Grant from the Aab Cardiovascular Cellular Fractionation. Separation of the nuclear and nonnuclear cellular Research Institute at the University of Rochester School of Medicine and Den- fractions was performed as described in Materials and Methods. tistry. E.M.S. was supported in part by a grant from Novartis Pharmaceuticals.

1. Hill JA, Olson EN (2008) Cardiac plasticity. N Engl J Med 358:1370–1380. 19. Candi E, et al. (1999) Transglutaminase cross-linking properties of the small proline- 2. Mozaffarian D, et al.; Writing Group Members; American Heart Association Statistics rich 1 family of cornified cell envelope proteins. Integration with loricrin. J Biol Chem Committee; Stroke Statistics Subcommittee (2016) Heart disease and stroke statistics— 274:7226–7237. 2016 update: A report from the American Heart Association. Circulation 133:e38–e360. 20. Steinert PM, Candi E, Kartasova T, Marekov L (1998) Small proline-rich proteins are 3. Hinz B, et al. (2007) The myofibroblast: One function, multiple origins. Am J Pathol cross-bridging proteins in the cornified cell envelopes of stratified squamous epi- 170:1807–1816. thelia. J Struct Biol 122:76–85. 4. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA (2002) Myofibroblasts and 21. Tarcsa E, et al. (1998) Structural and transglutaminase substrate properties of the mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 3: small proline-rich 2 family of cornified cell envelope proteins. J Biol Chem 273: – 349 363. 23297–23303. – 5. Kalluri R, Zeisberg M (2006) Fibroblasts in cancer. Nat Rev Cancer 6:392 401. 22. Dinkel H, et al. (2016) ELM 2016—data update and new functionality of the eu- 6. Ubil E, et al. (2014) Mesenchymal-endothelial transition contributes to cardiac neo- karyotic linear motif resource. Nucleic Acids Res 44:D294–D300. – vascularization. Nature 514:585 590. 23. Hornbeck PV, et al. (2012) PhosphoSitePlus: A comprehensive resource for investigating 7. Tamaki Y, et al. (2013) Metastasis-associated protein, S100A4 mediates cardiac fibrosis the structure and function of experimentally determined post-translational modifica- potentially through the modulation of p53 in cardiac fibroblasts. J Mol Cell Cardiol 57: tions in man and mouse. Nucleic Acids Res 40:D261–D270. 72–81. 24. Mizuguchi Y, et al. (2012) SPRR2A enhances p53 deacetylation through HDAC1 and 8. Vousden KH, Lu X (2002) Live or let die: The cell’s response to p53. Nat Rev Cancer 2: down regulates p21 promoter activity. BMC Mol Biol 13:20. 594–604. 25. Demetris AJ, et al. (2008) Small proline-rich proteins (SPRR) function as SH3 domain 9. Faesen AC, Luna-Vargas MP, Sixma TK (2012) The role of UBL domains in ubiquitin- ligands, increase resistance to injury and are associated with epithelial-mesenchymal specific proteases. Biochem Soc Trans 40:539–545. transition (EMT) in cholangiocytes. J Hepatol 48:276–288. 10. Kaplan ML, et al. (1994) Cardiac adaptations to chronic exercise in mice. Am J Physiol 26. Eckhart L, Lippens S, Tschachler E, Declercq W (2013) Cell death by cornification. 267:H1167–H1173. Biochim Biophys Acta 1833:3471–3480. 11. Braitsch CM, Kanisicak O, van Berlo JH, Molkentin JD, Yutzey KE (2013) Differential 27. Martinet N, et al. (1988) Epidermal and hair follicle transglutaminases. Partial char- expression of embryonic epicardial progenitor markers and localization of cardiac acterization of soluble enzymes in newborn mouse skin. J Biol Chem 263:4236–4241. fibrosis in adult ischemic injury and hypertensive heart disease. J Mol Cell Cardiol 65: 28. Candi E, et al. (1995) Biochemical, structural, and transglutaminase substrate prop- 108–119. erties of human loricrin, the major epidermal cornified cell envelope protein. J Biol 12. McMullen JR, et al. (2007) Protective effects of exercise and phosphoinositide – 3-kinase(p110alpha) signaling in dilated and hypertrophic cardiomyopathy. Proc Chem 270:26382 26390. Natl Acad Sci USA 104:612–617. 29. Furtado MB, et al. (2014) Cardiogenic genes expressed in cardiac fibroblasts con- – 13. McMullen JR, et al. (2004) The insulin-like growth factor 1 receptor induces physio- tribute to heart development and repair. Circ Res 114:1422 1434. logical heart growth via the phosphoinositide 3-kinase(p110alpha) pathway. J Biol 30. Epping MT, et al. (2011) TSPYL5 suppresses p53 levels and function by physical in- – Chem 279:4782–4793. teraction with USP7. Nat Cell Biol 13:102 108. 14. McMullen JR, et al. (2003) Phosphoinositide 3-kinase(p110alpha) plays a critical role 31. Kanisicak O, et al. (2016) Genetic lineage tracing defines myofibroblast origin and for the induction of physiological, but not pathological, cardiac hypertrophy. Proc function in the injured heart. Nat Commun 7:12260. Natl Acad Sci USA 100:12355–12360. 32. Das A, et al. (2013) Stromal-epithelial crosstalk regulates kidney progenitor cell dif- 15. Ayeni JO, et al. (2014) Dual phosphorylation of Cdk1 coordinates cell proliferation ferentiation. Nat Cell Biol 15:1035–1044. with key developmental processes in Drosophila. Genetics 196:197–210. 33. Wang Z, et al. (2004) Myocardin and ternary complex factors compete for SRF to 16. Steinert PM, Marekov LN (1999) Initiation of assembly of the cell envelope barrier control smooth muscle gene expression. Nature 428:185–189. structure of stratified squamous epithelia. Mol Biol Cell 10:4247–4261. 34. Nguyen AT, et al. (2013) Smooth muscle cell plasticity: Fact or fiction? Circ Res 112: 17. Steinert PM, et al. (1999) Transglutaminase crosslinking and structural studies of the 17–22. human small proline rich 3 protein. Cell Death Differ 6:916–930. 35. Takeda N, et al. (2010) Cardiac fibroblasts are essential for the adaptive response of 18. Candi E, et al. (1999) Acquisition of ordered conformation by the N-terminal domain the murine heart to pressure overload. J Clin Invest 120:254–265. of the human small proline rich 2 protein. Biochem Biophys Res Commun 262: 36. Frangogiannis NG (2014) The inflammatory response in myocardial injury, repair, and 395–400. remodelling. Nat Rev Cardiol 11:255–265. CELL BIOLOGY

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