Connective Tissue Growth Factor Domain 4 Amplifies Fibrotic Kidney

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Connective Tissue Growth Factor Domain 4 Ampliﬁes Fibrotic Kidney Disease through Activation of LDL Receptor–Related Protein 6

†‡ †‡ †‡ †‡ Bryce G. Johnson,* § Shuyu Ren,* § Gamze Karaca,* Ivan G. Gomez,* § Cécile Fligny, Benjamin Smith,* Ayla Ergun,* George Locke,* Benbo Gao,* Sebastian Hayes,* | †‡ Scott MacDonnell, and Jeremy S. Dufﬁeld* §

*Research and Development, Biogen, Cambridge, Massachusetts; Division of Nephrology, Departments of †Medicine and ‡Pathology and §Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington; and |Boehringer Ingelheim, Ridgeﬁeld, Connecticut

ABSTRACT Connective tissue growth factor (CTGF), a matrix-associated protein with four distinct cytokine binding domains, has roles in vasculogenesis, wound healing responses, and fibrogenesis and is upregulated in fibroblasts and myofibroblasts in disease. Here, we investigated the role of CTGF in fibrogenic cells. In mice, tissue-specific inducible overexpression of CTGF by kidney pericytes and fibroblasts had no bearing on nephrogenesis or kidney homeostasis but exacerbated inflammation and fibrosis after ureteral obstruction. These effects required the WNT receptor LDL receptor–related protein 6 (LRP6). Additionally, pericytes isolated from these mice became hypermigratory and hyperproliferative on overexpression of CTGF. CTGF is cleaved in vivo into distinct domains. Treatment with recombinant domain 1, 1+2 (N terminus), or 4 (C terminus) independently activated myofibroblast differentiation and wound healing responses in cultured pericytes, but domain 4 showed the broadest profibrotic activity. Domain 4 exhibited low-affinity binding to LRP6 in in vitro binding assays, and inhibition of LRP6 or critical signaling cascades downstream of LRP6, including JNK and WNT/b-catenin, inhibited the biologic activity of domain 4. Administration of blocking antibodies specifically against CTGF domain 4 or recombinant Dickkopf–related protein-1, an endogenous inhibitor of LRP6, effectively inhibited inflammation and fibrosis associated with ureteral obstruction in vivo. Therefore, domain 4 of CTGF and the WNT signaling pathway are important new targets in fibrosis.

J Am Soc Nephrol 28: 1769–1782, 2017. doi: https://doi.org/10.1681/ASN.2016080826

Fibrosis or scarring of vital organs is a major public of adult disease, they upregulate expression of con- health problem with limited therapeutic options. In nective tissue growth factor (CTGF), also known as CKD, the process of fibrosis contributes to func- CCN2.5 tional compromise and tissue inflammation and promotes progression to organ failure.1,2 Recent Received August 2, 2016. Accepted December 12, 2016. studies have identified adult tissue mesenchymal cells derived from FOXD1+ nephrogenic progeni- B.G.J. and S.R. contributed equally to this work. tors present in the developing kidney as the major Published online ahead of print. Publication date available at source of scar-forming cells in kidney disease.3 The www.jasn.org. FOXD1 lineage of fibrogenic cells is highly overlap- Present address: Dr. Scott MacDonnell, Regeneron, Tarrytown, ping with the populations of P-zero lineage cells New York. 4 and GLI1 lineage cells reported in other studies. Correspondence: Dr. Shuyu Ren or Dr. Jeremy S Duffield, Biogen, FOXD1 lineage cells are located predominantly Building 6, 115 Broadway, Cambridge, MA 02142. Email: alongside the microvasculature, many of which [email protected] or [email protected] are pericytes or pericyte-like.3 During the course Copyright © 2017 by the American Society of Nephrology

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Figure 1. Conditional CTGF overexpression in kidney stromal cells exacerbates fibrotic and inflammatory responses and requires LRP6. (A) Gene map of Foxd1Cre and R26-CIG transgenes. (B) Images of adult kidney showing Foxd1 nephrogenic progenitor–derived resident pericytes (upper panel; arrows) in the medulla and mesangial cells (arrowheads; lower panel) in the glomerulus (g) overexpressing CTGF detected by nGFP and coexpressing PDGFRb. Podocytes also activate Foxd1 during maturation and therefore, overexpress CTGF identified with nGFP (arrows; lower panel). (C) Histologic staining of control and CTGF overexpressing tissues at baseline revealing no significant difference in periodic acid–Schiff (PAS) and picrosirius red stains. (D) Graph showing morphometric

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CTGF is a cysteine-rich, small-secreted, matricellular pro- cell surface integrin-a5b1,30 thereby inducing fibroblast cell tein of 36–38 kD, originally identified in human umbilical vein proliferation and upregulation of collagen type 1.31 endothelial cells and murine fibroblasts, belonging to the CCN Accumulating evidence indicates that Wingless/INT family (CCN1–CCN6).6–8 CTGF has been implicated in vas- (WNT)/b-catenin and noncanonical WNT signaling are acti- cular patterning and angiogenesis and pericyte functions.9–11 vated during the process of human renal fibrosis and may In addition, CTGF has been shown to be an important factor play a dominant role in persistence of fibrogenic cells. Genome- in neoplasia and chronic diseases with fibrosis, including sys- wide association studies have identified single-nucleotide temic sclerosis, interstitial lung disease, and Crohn’s disease, polymorphisms in loci close to WNT pathway genes, where single-nucleotide polymorphisms in the CTGF locus including WNT7A, IGF binding protein 5, SHROOM3, and predispose to these diseases.12–16 CTGF gene expression is DAB2, pointing potentially to the WNT pathway having a caus- upregulated in kidney diseases in fibrotic lesions of diabetic ative role in fibrotic disease.9,32–34 Moreover, CTGF has been nephropathy and GN, and levels of the protein correlate with reported to activate WNT/b-catenin signaling, and WNT/ disease severity.17,18 In an experimental model of kidney dis- b-catenin plays important roles in the progression of ischemic ease induced by unilateral ureteric obstruction (UUO), ad- kidney diseases as well as diabetic nephropathy.9,35,36 We ministration of CTGF antisense oligonucleotides significantly sought to test the role of CTGF in fibrogenic cells and dissect ameliorated interstitial fibrosis induced by UUO.19 Similar its mechanism of action. effects were also found in the other models, including ne- phrectomy (5/6) type 2 diabetic nephropathy model db/ db,20 in which kidney fibrosis was inhibited by anti-CTGF RESULTS treatment. In vitro studies of CTGF indicate that it regulates cell adhesion, migration, proliferation, apoptosis, angiogene- Transgenic Overexpression of CTGF Only in Pericytes, sis, and extracellular matrix production, depending on cellular Fibroblasts, and Podocytes Amplifies Inflammation and context and microenvironment.6 Previous reports in kidney Fibrosis in the Kidney disease focused on the effects of CTGF on epithelial morphol- To study the effect of CTGF on vascular patterning and fibro- ogy and function. However, recent advances in our under- genesis, we generated a transgenic mouse (Rosa26-connective standing of fibrogenesis indicate that pericytes and fibroblasts tissue growth factor-IRES-nuclear green fluorescent protein are more important cellular targets of CTGF activity with re- [R26-CIG]), in which CTGF and nuclear green fluorescent spect to the development and progression of fibrosis.9 protein (nGFP) are conditionally overexpressed at the ubiq- One hypothesis for the profibrotic actions of CTGF is that it uitously active Rosa26 locus only when an upstream loxP presents TGFb to its receptor and is, therefore, necessary for flanked stop sequence is removed from the genomic DNA by TGFb signaling.21,22 Indeed, current clinical trials to limit the activity of Cre recombinase (Figure 1A, Supplemental Fig- pulmonary fibrosis use antibodies that block the binding ure 1, A–D, Supplemental Material). This novel gene targeting area of TGFb to CTGF.23 However, CTGF contains four con- strategy resulted in robust expression of CTGF and nGFP in served domains that bind many different partners. At the N neonatal mouse kidney when the transgene was activated by terminus, domain 1 has homology with IGF binding protein, Cre only in FOXD1 lineage cells and resulted in .98% recom- domain 2 has vWf type C repeats, domain 3 has thrombo- bination in the FOXD1 lineage (Figure 1, A and B, Supple- spondin type 1 repeat, and domain 4 or C-terminal (CT) mental Figure 1, E–G). Transgenic mice were born at Mendelian domain has a cysteine knot motif.6 IGF1 and IGF2 bind to ratios and exhibited normal growth and survival to 6 months in domain 124;BMP4andTGFb bind to domain 225; and LRP1, sterile housing conditions. Kidney development was essentially VEGFA,26,27 and integrin-a5b3and-a6b1 bind to domain normal, with normal glomerular loop formation, normal tu- 3.6,7,28,29 Moreover, CTGF has been reported to have functions bules, no fibrosis (Figure 1, C and D, Supplemental Figure 1, that are independent of TGFb. For example, it promotes ad- H), and no albuminuria. Vascular density in the cortex and hesion and migration of pancreatic stellate cells by binding to medulla appeared normal. Pericytes/fibroblasts in control

results of immunofluorescence labeling of healthy adult kidneys to identify pericytes (PDGFRb), activated fibroblasts/myofibroblasts (aSMA), and endothelial cells (CD31). (E) Transcriptional analysis of healthy adult kidney tissue for genes encoding extracellular matrix components, growth factors, and cytokines normalized to control samples. (F) Schema showing surgical induction of kidney disease and timeline. (G) Immunofluorescence labeling to identify pericytes (PDGFRb) and macrophages (F4/80) and histologic stain for fibrillar collagen (picrosirius red) 7 days after UUO. (H) Graphs showing morphometric analysis for extracellular matrix components, macrophages, interstitial fibroblasts, and fibrillar collagen deposition. (I and J) Transcriptional analysis of kidney tissue of genes encoding extracellular matrix components, growth factors, and cytokines normalized to control samples at baseline and disease conditions. (K) Transcript levels for WNT downstream target genes in kidney tissue. (L and M) Levels of extracellular matrix and inflammatory gene transcription in Foxd1Cre;R26-CIG kidney in response to Lrp6 antisense gapmer oligonucleotide (ASO) treatment. Data are represented as mean6SEM; n=4–7 per group. Scale bars, 50 mm. *P,0.05; **P,0.01; ***P,0.001; ****P,0.001.

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Figure 2. Distinct domains of CTGF activate WNT/b-catenin in pericytes, which is inhibited by DKK1. (A) Schema showing domains of CTGF and potential binding partners. (B) Western blot detecting CTGF and its domains in total kidney (normal kidney/diseased kidney). Note that, in diseased kidneys, the putative cleaved CTGF fragment bands are shown with arrowheads. (C) Images and (D and E) graphs showing that CTGF domains activate WNT signaling in mouse pericytes as detected by nGFP 16 hours after activation in TCF/LEF:H2B-GFP transgenic pericytes. Recombinant DKK1 inhibits distinct CTGF domain–induced canonical WNT responses in mouse pericytes. (F and G) Graphs showing that CTGF DIV induces pericyte migration in a time- and dose-dependent manner. (H and I)

1772 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1769–1782, 2017 www.jasn.org BASIC RESEARCH and R26-CIG mutant kidneys had comparable expression of CTGF overexpression (Figure 1L), and reduced transcription of aSMA (Supplemental Figure 1, F and H). Kidneys from 8- to genes indicative of WNT pathway activation (Figure 1M), pro- 10-week-old mice were similarly normal, showing no evidence viding in vivo evidence that the WNT pathway, via LRP6, is of fibrosis or vascular injury (Figure 1, D and E) or albuminuria important in transducing CTGF extracellular signals. (data not shown), despite preservation of elevated levels of CTGF production (Figure 1, D and E, Supplemental Table 1). CTGF Is Cleaved In Vivo into Multiple Domains That Morphometric quantification of the total population of Independently Activate Pericytes through WNT/ pericytes/fibroblasts, however, consistently showed increased b-Catenin numbers of these cells in the adult kidney (Figure 1D). Trans- Although CTGF is synthesized and exported as the full-length genic mice or controls were subjected to kidney injury by ure- protein, it is subject to cleavage in the extracellular environ- teral obstruction (Figure 1, F–J). In the setting of disease, there ment into multiple domains (Figure 2, A and B), generating was enhanced expansion of PDGFRb+ fibroblasts and recruit- proteins with sizes ranging from 17 to 30 kD. We cloned and ment of F4/80+ macrophages associated with significant in- purified domains 1, 1+2, and 4 and applied them to purified creases in interstitial extracellular matrix protein deposition and cultured mouse pericytes expressing the transgene TCF/ as well as increases in picrosirius red–positive fibrosis (Figure Lef:H2B-GFP, which reports b-catenin transcriptional activity 1, F–H). Consistent with these findings, CTGF transcripts by nGFP expression (Figure 2, C–F). Unexpectedly, pericytes remained increased in the transgenic kidneys along with tran- responded to each domain by activating TCF/Lef signaling. scripts for extracellular matrix proteins (Figure 1I). In addi- Moreover, this signaling was abolished by the application of tion, inflammatory cytokine and chemokine transcripts were recombinant Dickkopf–related protein-1 (DKK1), which in- elevated (Figure 1J). Pericytes were purified from R26-CIG hibits WNT signaling by binding to the receptor LRP6.37 mice. In established culture, they were treated with lentivirus Signaling by CTGF domain 4 was comparable with that stim- to express Cre. Cre expression enables recombination of the ulated by the canonical WNT ligand, Wnt3a. transgene to enable overexpression of CTGF. Seventy-two hours after Cre expression, pericytes became hypermigratory CTGF Domains Independently Stimulate Pericyte and hyperproliferative (Supplemental Figure 2, A and B), Migration, Myofibroblast Differentiation, and Matrix whereas treatment with a control lentivirus had no effect. Accumulation in a WNT/b-Catenin–Dependent Manner These observations are consistent with CTGF overexpression Murine pericyte cultures were treated with CTGF domain 1, 1 in pericytes amplifying cell activation. In the kidney, there was +2, or 4 (Supplemental Material). Each protein showed a dose- evidence of enhanced WNT/b-catenin/LEF1 pathway signaling dependent ability to trigger pericyte migration in a wound- as exemplified by increased transcripts for downstream signaling healing assay (Figure 2, G–K), with domain 4 showing greatest (Figure 1K). In addition, we have previously shown that CTGF potency. Surprisingly, domain 1+2 protein was no more effec- can directly activate the WNT pathway in pericytes.9 To test the tive than domain 1 alone. In each case, although DKK1 had no importance of WNT signaling pathway activation by CTGF in effect on the background level of migration, nevertheless, it vivo, we delivered antisense gapmer oligonucleotides to the kid- abolished migration to each of the domains. Domain 4 and ney via intraperitoneal injection to specifically silence the WNT domain 1+2 stimulated matrix accumulation and aSMA pro- coreceptor, LDL receptor–related protein 6 (LRP6) (Supplemen- tein accumulation as well as accumulation of the matrix sta- tal Figure 2, C–F). Upregulation of Lrp6 in R26-CIG mutant bilizing factor plasminogen activator inhibitor-1 (PAI-1). mice with UUO kidney disease was completely suppressed by Moreover, both stimulated endogenous CTGF synthesis (Fig- the specific antisense gapmer oligonucleotides, whereas tran- ure 2L). These effects were entirely abolished by recombinant scripts of a similar receptor Lrp5 were unaffected, highlighting DKK1, whereas the TGFb receptor inhibitor (SB431542) had a the efficacy and specificity of this approach (Supplemental Fig- much weaker effect on matrix inhibition and no effect on PAI- ure 2, C and D). The specific silencing of LRP6 resulted in 1 or CTGF synthesis. Domain 1 alone did not stimulate matrix marked diminution of receptor expression in the kidney (Sup- production (Figure 2L, Supplemental Figure 3). Pericytes plemental Figures 2, C, E and F). LRP6 silencing blocked the treated with CTGF domains were evaluated for stress fiber upregulation of matrix proteins triggered by CTGF overexpres- formation, a marker of myofibroblast differentiation and con- sion, consistently reduced the inflammatory signature caused by tractility (Figure 2, M–O). Each protein domain stimulated

CTGF DI+II and DI alone stimulate migration with dose dependency. (J) CTGF DIV–induced migration is blocked by recombinant DKK1 (K) CTGF DI+II– or DI–induced migration is blocked by recombinant DKK1. (L) Western blots showing CTGF DIV– and CTGF DI+II–induced matrix proteins, aSMA, and CTGF. DKK1 inhibits all responses to DIV and most responses to DI+II. SB431542 has weak effects on matrix responses only. (M–O) Images and graphs showing the effect of CTGF domains on stress ﬁber formation (arrowheads). DKK1 blocks responses to each domain. Data are mean6SEM; n=6 per group. All blots are representative of three experiments. Scale bars, 25 mm. *P,0.05; **P,0.01; ***P,0.001.

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Figure 3. CTGF domain 4 directly activates the WNT coreceptor LRP6 and stimulates migration predominantly by JNK phosphorylation in pericytes. (A) Western blots detecting pLRP6 or CTGF among mouse pericyte proteins immunoprecipitated by anti-pLRP6 antibodies or control antibodies after stimulation with CTGF DIV with or without DKK1. (B) Western blots of phosphorylated forms of P42/P44 MAPK, JNK, P38 MAPK, and LRP6 in pericytes activated by CTGF DIV or CTGF DIV and DKK1. (C) CTGF DIV–mediated migration is blocked by the JNK inhibitor (SP600125) and WNT export inhibitor (IWP2) but is not blocked by the b-catenin inhibitor (XAV939), TGFbRI inhibitor (SB431542), or P42/44 inhibitor (U0126) in quiescent pericytes at 24 hours. (D) Western blots of phosphorylated forms

1774 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1769–1782, 2017 www.jasn.org BASIC RESEARCH myofibroblast differentiation in pericytes, and in each exper- (SB431542), ERK (U0126), JNK (SP600125), WNT secretion iment, this differentiation was inhibited by DKK1. (IWP2), and b-catenin activation (XAV939) (Figure 3C). Crit- ically, CTGF domain 4–stimulated migration was dependent CTGF Domain 4 Closely Interacts with Pericyte LRP6 to on JNK and the presence of extracellular WNTs. Although we Activate JNK Signaling did not detect specific binding of the N-terminal domain of Application of CTGF CT domain 4 to pericytes results in rapid LRP6 in the binding assay, both domain 1+2 protein (N ter- close association of CTGF with the activated form of LRP6 minus) and domain 1 alone activated LRP6, although less ro- determined by coimmunoprecipitation from cells (Figure 3A). bustly than domain 4. They also activated JNK and ERK (Figure To understand whether this represents a direct receptor-ligand 3D). Similarly to domain 4, migration induced by domain 1+2 interaction, a binding assay was performed using immobilized was highly dependent on JNK activation and the production of recombinant CTGF proteins and soluble LRP6-Fc or control extracellular WNTs (Figure 3, E and F). In keeping with a lack Fc protein. CTGF domain 1+2 showed no binding to LRP6 of efficacy of the TGFbR1 inhibitor, we observed that, although (not shown), and CTGF domain 4 showed only weak affinity CTGF domain 4 weakly activated SMAD2/3, this was indepen- at the highest concentrations (Supplemental Figure 4A), sug- dent of TGFbR1 (Supplemental Figure 3). Next, expression of gesting that a weak direct interaction may exist in vivo,butno LRP6 or b-catenin by pericytes was silenced using siRNA (Fig- affinity constant could be determined. This potential weak ure 3G), and the effect of CTGF domains was evaluated. Loss of direct interaction was further evaluated by expressing LRP6 LRP6 potently inhibited the migration in response to all CTGF in a lymphocyte cell line (Supplemental Figure 4B). Similarly, domains, consistent with the effects seen in vivo (Figure 1), CTGF domain 4 showed a weak interaction only at very high whereas loss of b-catenin did not affect migration (Figure 3, concentrations, but this was unaffected by Wnt3a. The bind- H and I). Silencing of LRP6 reduced levels of MAPK signaling, ing of CTGF domain 4 to lymphocytes was compared with a particularly p42/44 and JNK, but did not affect pSMAD2/3 in mouse fibroblast cell line (Supplemental Figure 4C). The fi- basal (unstimulated) conditions (Figure 3J). Moreover, LRP6 broblast cell line surface showed much higher affinity for silencing markedly attenuated CTGF domain–dependent up- CTGF domain 4 than LRP6-expressing lymphocytes, indicat- regulation of fibrogenic factors, including PAI-1, fibronectin, ing receptor interactions on the fibroblast independent of and CTGF (Figure 3K), implying a positive feedback loop be- LRP6. Therefore, CTGF domain 4 likely binds distinct recep- tween LRP6, JNK, and factors, such as CTGF. Such a feedback tors on pericytes and fibroblasts and then, recruits LRP6 to loop is supported by the preponderance of JNK-responsive form a complex. We performed several rounds of co-IP from transcription factor binding sites in the CTGF locus (Supple- membrane fractions of primary human fibroblasts using both mental Figure 5). Antibodies raised specifically against domain covalent and noncovalent methods to capture CTGF recep- 4 of CTGF (Supplemental Material) exclusively blocked do- tors. We identified several potential interacting proteins by main 4–induced migration selectively and PAI-1 synthesis this method (Supplemental Figure 4D). Only CD109 was a but did not alter the effects mediated by domain 1+2 (Figure cell surface transmembrane receptor. A binding assay for 3, K and L). CTGF and CD109 did not detect any specificbindingaffinity (Supplemental Figure 4E). The absence, therefore, of a detect- Human Kidney Pericytes Also Exhibit Fibrogenic able high-affinity receptor for CTGF supports the hypothesis Responses to CTGF Domains That Are Blocked by that CTGF may have several weak receptor interactions, in- DKK1 cluding the interaction with LRP6. Consistent with such a Human pericytes purified from kidneys as a PDGFRb+, NG2+ model, CTGF domain 4 rapidly stimulates LRP6 phosphory- population, when expanded in vitro, rapidly lose NG2 expres- lation as well as downstream effectors c-Jun N-terminal kinase sion, maintain PDGFRb, and weakly express aSMA (Figure (JNK) and extracellular signaling–related kinase (ERK) acti- 4A, Supplemental Material). Treatment with CTGF domains vation. These induced effects are all diminished by DKK1 (Figure 1, 1+2, and 4 all induced pericyte migration that was inhibited 3B). To test the relative importance of these activated intracel- by DKK1 (Figure 4B). Formation of stress fibers indicative of lular signaling pathways, migratory responses to domain myofibroblast differentiation was induced by each of the 4 were evaluated in the presence of inhibitors of TGFbR1 CTGF domains, and all were inhibited by DKK1 (Figure 4,

of P42/P44, JNK, P38, and LRP6 in pericytes activated by CTGF DIV, DI, or DI+II. (E and F) Graphs showing that migration in response to both CTGF DI+II and CTGF DI alone is selectively blocked by the JNK inhibitor (SP600125) and WNT export inhibitor (IWP2) in pericytes after 24 hours. (G–I) Western blots and graphs showing the effect of siRNA against LRP6 or b-catenin or scrambled sequence on expression of LRP6 and b-catenin and the subsequence effect on migration triggered by CTGF domains. (J, K) Blots showing the effect of signaling responses to CTGF domains after silencing of LRP6 or mock silencing in basal/unstimulated conditions (J) or in conditions stimulated by different CTGF domains (K). (L) The effect of blocking antibodies against CTGF DIV on migration induced by CTGF domains. (M) The effect of antibodies against CTGF DIV on DIV-induced PAI-1 expression. Data are expressed as mean6SEM; n=6 per group. Blots are representative of three independent experiments. *P,0.05; **P,0.01; ***P,0.001.

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Figure 4. Domain 4 of CTGF causes migration and stress fiber formation and dominantly induces profibrotic responses in human kidney pericytes, which are blocked by DKK1. (A) Flow cytometric plot and immunofluorescence images indicating selection of NG2+ PDGFRb+ cells from human fetal kidney results in a population of cells that are PDGFRb+, transiently NG2+, and weakly aSMA+. (B) Migratory responses to CTGF domains at 24 hours in human are all inhibited by DKK1. (C and D) Stress fibers form in response to CTGF domains and are all blocked by DKK1. (E) Quantitative PCR indicates that CTGF domain 4 alone upregulates transcripts associated with fibrogenesis 48 hours after stimulation. (F) Western blots showing COL1, PAI-1, and CTGF production in response to a number of stimuli

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C and D). CT domain 4 effectively stimulated increased independent cell signaling process from TGFbR signaling that COL1A1, CTGF,andACTA2 transcripts (Figure 4E) and is controlled by CTGF. Finally, confirmation of CTGF signal- strongly increased synthesis of PAI-1, CTGF, and COL1 pro- ing in human fibrotic kidneys is evidenced by highly upregu- teins (Figure 4F), whereas other CTGF domains had a much lated gene expression for CTGF-specificinducedgenes weaker effect on human pericytes. DKK1 inhibited PAI-1 and FBLN1, COL14A1,andSERPINF1 (Supplemental Figure 7). CTGF induced by CTGF domain 4 and had modest effects on COL1 accumulation in this timeframe. The effect of different CTGF-Induced Inflammation and Fibrosis Are Blocked CTGF domains on cell signaling was evaluated in human per- In Vivo by Administration of Recombinant DKK1 or icytes (Figure 4, G and H). CTGF domains all stimulated a Antibodies against Domain 4 of CTGF short-lived increase in active p42/44 MAPK, whereas domains The study of CTGF activity in the kidney (Figure 1) shows that 4 and 1+2 stimulated a sustained increase in active JNK in CTGF production by pericytes/fibroblasts is sufficient to am- pericytes, activation of LRP6, activation of SMAD2/3, and ac- plify fibrogenic and inflammatory signaling. In vivo and in cumulation of b-catenin. All of these effects were markedly vitro studies suggest CTGF domain 4 may play a dominant blunted by silencing of the LRP6 receptor (Supplemental role in fibrogenic signaling by interacting with LRP6, whereas Figure 6A), confirming the critical role for LRP6 in CTGF- domain 1/1+2 also stimulates fibrogenic signaling via aWNT/ mediated cell signaling in human cells. The pattern of signal- LRP6-dependent mechanism. To evaluate the significance of ing in human pericytes differed from mouse cells in important these observations, we generated recombinant DKK1 and ways. CTGF domains activated p38 as an early response more blocking antibodies against domain 4 of CTGF and tested their robustly, domain 1 alone had the strongest effect on p42/44 ability to block fibrogenesis in a model of kidney disease (Fig- activation, and domain 1+2 also enhanced collagen 1 produc- ure 5A, Supplemental Material). Recombinant DKK1 was tion (Figure 4, F and G). Nevertheless, despite these differ- readily detected in the kidney parenchyma after parenteral ences, CTGF domain 4 had the broadest overall fibrogenic administration and blocked WNT signaling (Figure 5, B and effects on human pericytes, which were inhibited by DKK1. C). It effectively inhibited myofibroblast activation as detected Unlike mouse pericytes, the human cell response to CTGF by aSMA expression and deposition of fibrous matrix proteins domain 4 showed some dependence on TGFbR1 as exhibited as detected by Sirius red stain or immunoreactivity for matrix by the effect of its inhibitor, SB431542 (Figure 4F). Unexpect- proteins, including laminin and fibronectin (Figure 5, E–G). edly, all CTGF domains rapidly and persistently upregulated CTGF-responsive effector proteins, including PAI-1 and TGFbR1 expression in human pericytes, an effect that was CTGF, were also downregulated, consistent with rDKK1 independent on LRP6 and blocked by antibodies against hibiting CTGF-mediated fibrogenic signaling (Figure 5, D–G). CTGF, confirming specificity of the observation (Supplemen- In addition, expression of inflammatory cytokines, including tal Figure 6, B–D) and suggesting that CTGF domain 4 signal- Il1b and Tnfa, was reduced in the tissue (Figure 5G) as was the ing in human cells may secondarily activate TGFb signaling recruitment of macrophages (Figure 5H). Therefore, rDKK1 via LRP6-dependent upregulation of TGFbR1. Toexamine the effectively blocked myofibroblast activation and showed evi- potential interdependence of CTGF signaling via LRP6 on dence of inhibiting WNT signaling and CTGF-mediated sig- secondarily activating TGFbR1 signaling, we compared tran- naling by reduction in PAI-1, laminin, and CTGF. DKK1 scriptional responses of human pericyte cultures to CTGF do- reduced CTGF-induced genes as well as TGFb-induced genes, main 4 and the canonical TGFbR1 ligand, TGFb,byRNA suggesting that DKK1 blocks additional profibrotic signals sequencing (Supplemental Figure 7, Supplemental Material). (Supplemental Figure 7, G and H). To clarify the significance The two cytokines share ,50% overlap in induced transcrip- of CTGF domain 4 in vivo specifically, anti-CTGF domain 4 tional changes. Notably, CTGF uniquely activates a combina- polyclonal antibodies that effectively block CTGF domain 4– tion of cell division, ECM accumulation, and inflammatory induced migration and myofibroblast differentiation (Figure responses, and a network analysis of those genes with a fold 3) were administered intraperitoneally during the course of change .2.0 induced by CTGF identified a core node of ma- kidney disease induced by ureteral obstruction. These anti- trix factor gene upregulation, including COL1, -3, -5, -11, and bodies also inhibited fibrogenesis and inflammation in this -14; periostin; SPARC; matrix gla protein; and sulfatase-1, as kidney disease model (Figure 5, I–O). Anti-domain 4 anti- well as an inflammatory response pathway that was not found bodies blunted WNT/b-catenin/LEF1 signaling (Figure 5J) in TGFb-stimulated genes (Supplemental Dataset 1, Supple- and inhibited pathologic matrix deposition (Figure 5, K mental Figure 7). Together, these findings further support an and L). This was associated with a reduction in PDGFRb+

at 24 hours. Note that DIV protein markedly increases these proteins, whereas DI+II is less effective, and DI alone produces no response. DKK1 blocks these responses, and SB431542 also inhibits PAI-1 and CTGF responses. (G and H) Blots showing signaling responses of the different CTGF domains in human pericytes. Data are mean6SEM; n=6 per group. Blots are representative of three independent experiments. *P,0.05; **P,0.01; ***P,0.001.

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Figure 5. DKK1 or anti-CTGF domain 4 antibodies inhibit fibrogenic signaling in models of kidney disease. (A) Schema showing the timeline for UUO-mediated disease induction and the delivery of rDKK1 or vehicle. (B) Western blot showing total DKK1 levels in kidney tissue after disease induction and delivery of vehicle or rDKK1. (C) Graph showing the effect of rDKK1 on expression of WNT/b-catenin responses in kidney after UUO. (D) Representative images and (E) morphometric quantification of myofibroblasts and interstitial fibrosis 7 days after UUO. (F) Western blots showing CTGF-responsive protein levels in the kidney in response to rDKK1. (G) Quantitative PCR for levels for transcripts of matrix proteins, fibrogenic factors, and inflammatory response factors. (H) Quantification of F4/80+ macrophages

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fibroblasts and myofibroblasts as well as a larger reduction in CTGF-overexpressing transgenic mice. Its activity is depen- macrophage recruitment (Figure 5M) and supported by re- dent on a close interaction with and activation of the WNT duction in matrix protein and aSMA accumulation detected receptor LRP6. Despite extensive studies to identify a high- by immunoblotting (Figure 5N). Transcripts for matrix com- affinity receptor for CTGF domain 4, we did not identify such ponents, including fibronectin and Col1a1, were reduced as an interacting protein. We consistently detected a direct, low- were transcripts for inflammatory cytokines, including Tnfa. affinity interaction between CTGF domain 4 and LRP6. We (Figure 5O). found evidence of specific binding activity of CTGF domain 4 to fibroblast cell surface in culture, suggesting that fibroblasts have several low-affinity receptors for domain 4, and LRP6 is DISCUSSION one of these. Moreover, the dependence on LRP6 for signal transduction both in vitro and in vivo supports the interaction Although CTGF is widely held to cause profibrotic signaling, it studies in proposing LRP6 as an important coreceptor that is expressed in healthy tissue, where its function has not been mediates intracellular signaling (Supplemental Figure 8). well understood. In addition, the contribution of CTGF to However, we cannot exclude the possibility that LRP6 is acti- fibrogenesis in the kidney has not been rigorously evaluated vated by CTGF domain 4 through an indirect mechanism. previously, but CTGF is consistently upregulated in human Consistent with the central role for LRP6 as a receptor for kidney diseases. Recent advances in our understanding of CTGF domain 4 is the observation that the WNT/LRP6 in- the fibrogenic process have identified critical fibrogenic cells hibitor DKK1 completely abrogates all activity of CTGF do- in the kidney with close association to the vasculature, cells that main 4, which is recapitulated by silencing of LRP6 both in respond to receptor activation in distinct ways from epithelial vivo and in vitro. Although canonical WNT signaling, as ex- cells of the nephron.3,5,38 The studies here show rigorously that emplified by b-catenin activity, is stimulated in fibrogenic cells CTGF alone is not fibrogenic and has very little bioactivity in in response to each of the domains, inhibitor and gene silenc- healthy tissues. This is in keeping with other studies that show ing studies indicate that b-catenin signaling is less important that mutation of CTGF has minor effects only on vascular than JNK activation in response to CTGF activity. This is con- patterning39 and makes CTGF an attractive therapeutic target, sistent with previous studies9 and emphasizes a critical role for because disrupting its function in healthy adult tissues is un- noncanonical WNT pathway signaling from LRP6. The obser- likely to cause deleterious effects. The absence of fibrogenic vation that the WNT ligand inhibitor IWP2 blocks CTGF sig- activity by CTGF alone may be due to low-level expression of naling suggests that a WNT ligand molecule must be present at the low-affinity receptors, such as LRP6 (Supplemental Figure the LRP6 receptor to enable CTGF domain signaling and that 8), in healthy tissues that are then highly upregulated in dis- LRP6 functions as a node for fibrogenic signaling. Because ease. In addition, the necessity of WNT ligands to be permis- CTGF can bind WNT ligands,40 it is possible that CTGF pre- sive for CTGF signaling may also contribute to the lack of sents WNTs to LRP6 as a molecular complex, and this be- effect of CTGF in healthy kidney, because WNT ligands are comes an effector ligand. This is consistent with our finding highly upregulated in disease. Nevertheless, in the setting of that CTGF domain 4 alone shows a reproducible weak binding disease, CTGF overproduction by pericytes and fibroblasts activity for LRP6. Although weak ligand-receptor affinities are alone enhances both inflammation and fibrosis, indicating the exception, there are a number of biologically important that CTGF signaling amplifies existing disease, and this en- weak interactions documented, including receptors for nerve hancement requires LRP6. These studies identify that CTGF is growth factor and adenosine. cleaved into separate protein domains in vivo and show that, Although domain 1+2 has fibrogenic activity in pericytes, contrary to earlier studies that focused on epithelial cell bi- these activities are less effective than domain 4, particularly ology and TGFb signaling, CTGF signals predominantly by with regard to matrix deposition, and domain 1 alone has no activating WNT receptors in fibrogenic cells of the kidney effect on matrix deposition in the assays evaluated. Unexpect- rather than enhancing TGFbR1 signaling. edly, the biologic actions of the N-terminal domain 1 and The CT (domain 4) of CTGF exhibits the most potent and domain 1+2 combined are also dependent on LRP6 and comprehensive fibrogenic activity on kidney pericytes. This broadly blocked by DKK1. Although we did not find evidence cleaved protein is upregulated in disease and enhanced in the for direct binding of the N-terminal domain to LRP6 in invitro

after 7 days of UUO. (I) Schema showing the timeline for UUO-mediated disease induction and the delivery of anti-CTGF domain 4 antibodies or vehicle. (J) Effect of anti-CTGF DIV on WNT/b-catenin responses in kidney after UUO. (K) Representative images and (L) quantification of laminin accumulation and interstitial fibrosis. (M) Graphs showing morphometric quantification of protein markers in kidney tissue sections in response to disease and anti-CTGF DIV antibodies. (N) Western blots showing CTGF-responsive protein levels in the kidney in response to anti-CTGF DIV. (O) Quantitative PCR for transcript levels of matrix proteins and inflammatory factors in response to (L) anti-CTGF DIV. Blots are representative of two or more independent experiments; n=6 per group. Scale bar, 50 mm. *P,0.05; **P,0.01; ***P,0.001; ****P,0.0001.

J Am Soc Nephrol 28: 1769–1782, 2017 Connective Tissue Growth Factor Amplifies Fibrosis 1779 BASIC RESEARCH www.jasn.org binding assays, there are two possible explanations. (1)Onlywhen fibroblast activation results in a reduction of inflammatory bound to other ligands can the N-terminal domain be an effective cytokines. ligand for LRP6. (2) LRP6 may stabilize other binding partners for We conclude that fibrogenic signaling by CTGF in the domain 1+2, such as the TGF superfamily receptors, at the cell kidneyoccurs through an LRP6- and JNK-dependent mech- surface. Additional studies to understand the dynamic role of anism and that domain 4 of CTGF existsasadistinctprotein LRP6 in regulating cell surface receptor expression, stabilization, in vivo,carriessignificant independent fibrogenic signaling and clustering will be required. properties, and mediates these effects via adirectbutweak Current human clinical trials are using blocking antibodies interaction with LRP6. Blockade of LRP6 by either re- against domain 2 of CTGF (PMC2924405); our findings, how- combinant DKK1 or specific antibodies against domain 4 ever, in the kidney implicate domain 4of CTGF as an important of CTGF is a novel strategy to counteract fibrogenesis in domain with fibrogenic activity. In addition, because of cleav- kidney disease. age in vivo, CTGF domain 4 can function independently of other domains, potentially rendering antibodies against do- CONCISE METHODS main 2 alone ineffective. The studies on human fibrogenic cells suggest that domain 4 has greater fibrogenic efficacy than domain 1+2. The in vivo studies presented here indicate Conditional Allele Gene Targeting The conditional R26-CIG allele was generated by homologous re- that antibodies that selectively target domain 4 effectively in- combination in V6.5 Hybrid ES cells of 129/B6 background.42 To hibit inflammation and fibrogenesis, providing independent generate the transgene, the mouse CTGF CDS sequence was used verification that domain 4 is a critical target in fibrogenesis, (Consensus CDS Gene CCDS23751.1). Specific recombination at and suggest that a revised approach to targeting CTGF in hu- the Rs26 locus was confirmed by long PCR using the following con- man disease is warranted. In addition, a soluble recombinant ditions: 59-CTCGGCATGGACGAGCTGTACAAG-39 and 59- form of DKK1, an endogenous inhibitor of LRP6 activity, po- AGCAGGACCAAATGTGGTGCAGTGTTGA-39; LPCR conditions: tently blocks fibrogenic activity in the kidney and blocks 94°C for 2 minutes; 13 cycles at 94°C for 10 seconds and 65°C for CTGF-dependent biology. This recombinant molecule effectively 40 seconds with 21°C per cycle; 30 cycles at 94°C for 10 seconds, 50°C delivered to the kidney parenchyma by intraperitoneal adminis- for 30 seconds, and 68°C for 4 minutes and 45 seconds + 10 seconds tration blocks WNT signaling and inhibits not only fibrosis but per cycle; and 68°C for 5 minutes. Founder males were crossed to also, inflammation. Therefore, recombinant DKK1 has effective albino C57BL6 females (B6[Cg]-Tyrc-2J/J; Jackson) and tested drug-like properties and may be a more effective inhibitor of for germline transmission using the following PCR primer se- CTGF-stimulated fibrogenesis than a single antibody. In addition quences: 59-CTCGGCATGGACGAGCTGTACAAG-39 and 59- to the described effects of domain 1+2, domain 1 alone also ac- CATACTGTAGTAAGGATCTCAAGCAGG-39; PCR conditions: 94°C tivates LRP6; it has fibrogenic activity, stimulating promigratory for 2 minutes; 34 cycles at 94°C for 15 seconds, 54°C for 30 seconds, and contractile functions, and therefore, it has independent activ- and 72°C for 45 seconds; and 72°C for 3 minutes. ity in the N terminus of the protein. Because domain 1 also can exist as a separate protein in vivo,thisdistinctfunctioninthe N-terminal domain may play distinct roles in disease. Statistical Analyses Error bars are SEMs. Statistical analyses were carried out using GraphPad Human and mouse pericytes behaved similarly in our assays Prizm (GraphPad Software). The statistical significance was evaluated by and showed a robust response to CTGF DIV. Nevertheless, one-way ANOVA, with Dunnets post hoc testing for two groups. there was one notable difference: human pericytes exhibited some dependence on TGFbR1 signaling in response to CTGF DIV, whereas mouse cells did not. We previously made the ACKNOWLEDGMENTS observation that TGFbR1 closely interacts with LRP6.9 Here, we showed that CTGF DIV rapidly results in TGFbR1 We thank Kim Muczinski (University of Washington), Bill Stallcup accumulation, but this accumulation is prevented by LRP6 (Burnham Institute), Allan Capili (Biogen), and Roel Goldschmeding silencing. This finding occurs even in the absence of exoge- (University Medical Center, Utrecht) for help or advice. nous CTGF. One explanation is that LRP6 may play a role in These studies were funded by Biogen, Sanofi Nephrology Trainee TGFbR1 stabilization in pericytes, and therefore, CTGF DIV Fellowships awarded to S.R. and C.F., American Heart Association signaling through LRP6 results in rapid recruitment of grant 12GRNT12040023 (to J.S.D.), a research agreement between TGFbR1 to the surface, which amplifies cell signaling. One Boehringer Ingelheim and the University of Washington (J.S.D.), and intriguing possibility is that, in human fibrogenic cells, CTGF National Institutes of Health grant DK087389 (to J.S.D.). DIV may secondarily activate ligand-independent TGFbR1 activation through receptor upregulation, which has been described previously.41 Finally, blocking CTGF signaling or LRP6 signaling has additional anti-inflammatory effects. Re- DISCLOSURES cent studies point to the fibroblast as an important source of All employees of Biogen (Cambridge, MA) have stock in the company. J.S.D. chemokines and cytokines. We predict that inhibition of has served on the scientific advisory boards for Promedior Inc. (Lexington,

1780 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1769–1782, 2017 www.jasn.org BASIC RESEARCH

MA) and Regulus Therapeutics (San Diego, CA), has stock options with Prom- 16. Sonnylal S, Shi-Wen X, Leoni P, Naff K, Van Pelt CS, Nakamura H, edior Inc., is a cofounder of Muregen LLC (Brookline, MA), and has patents Leask A, Abraham D, Bou-Gharios G, de Crombrugghe B: Selective for the use of agents to treat kidney disease. expression of connective tissue growth factor in fibroblasts in vivo promotes systemic tissue fibrosis. Arthritis Rheum 62: 1523–1532, 2010 17. Wahab NA, Schaefer L, Weston BS, Yiannikouris O, Wright A, Babelova REFERENCES A, Schaefer R, Mason RM: Glomerular expression of thrombospondin- 1, transforming growth factor beta and connective tissue growth factor 1. Lupher ML Jr., Gallatin WM: Regulation of fibrosis by the immune at different stages of diabetic nephropathy and their interdependent system. Adv Immunol 89: 245–288, 2006 roles in mesangial response to diabetic stimuli. Diabetologia 48: 2650– 2. Duffield JS: Cellular and molecular mechanisms in kidney fibrosis. JClin 2660, 2005 Invest 124: 2299–2306, 2014 18. Kanemoto K, Usui J, Nitta K, Horita S, Harada A, Koyama A, Aten J, 3. Humphreys BD, Lin SL, Kobayashi A, Hudson TE, Nowlin BT, Bonventre Nagata M: In situ expression of connective tissue growth factor in hu- JV, Valerius MT, McMahon AP, Duffield JS: Fate tracing reveals the man crescentic glomerulonephritis. Virchows Arch 444: 257–263, 2004 pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. 19.YokoiH,MukoyamaM,NagaeT,MoriK,SuganamiT,SawaiK, Am J Pathol 176: 85–97, 2010 Yoshioka T, Koshikawa M, Nishida T, Takigawa M, Sugawara A, Nakao 4. Kramann R, Schneider RK, DiRocco DP, Machado F, Fleig S, Bondzie K: Reduction in connective tissue growth factor by antisense treatment PA, Henderson JM, Ebert BL, Humphreys BD: Perivascular Gli1+ pro- ameliorates renal tubulointerstitial fibrosis. J Am Soc Nephrol 15: genitors are key contributors to injury-induced organ fibrosis. Cell Stem 1430–1440, 2004 Cell 16: 51–66, 2015 20. Okada H, Inoue T, Kanno Y, Kobayashi T, Watanabe Y, Kopp JB, Carey 5. Schrimpf C, Xin C, Campanholle G, Gill SE, Stallcup W, Lin SL, Davis GE, RM, Suzuki H: Interstitial fibroblast-like cells express renin-angiotensin Gharib SA, Humphreys BD, Duffield JS: Pericyte TIMP3 and ADAMTS1 system components in a fibrosing murine kidney. Am J Pathol 160: modulate vascular stability after kidney injury. J Am Soc Nephrol 23: 765–772, 2002 868–883, 2012 21. Grotendorst GR: Connective tissue growth factor: A mediator of TGF- 6. Mason RM: Fell-Muir lecture: Connective tissue growth factor (CCN2) beta action on fibroblasts. Cytokine Growth Factor Rev 8: 171–179, – a pernicious and pleiotropic player in the development of kidney fi- 1997 brosis. Int J Exp Pathol 94: 1–16, 2013 22. Leask A, Denton CP, Abraham DJ: Insights into the molecular mecha- 7. Lipson KE, Wong C, Teng Y, Spong S: CTGF is a central mediator of nism of chronic fibrosis: The role of connective tissue growth factor in tissue remodeling and fibrosis and its inhibition can reverse the process scleroderma. J Invest Dermatol 122: 1– 6, 2004 of fibrosis. Fibrogenesis Tissue Repair 5[Suppl 1]: S24, 2012 23. Adler SG, Schwartz S, Williams ME, Arauz-Pacheco C, Bolton WK, Lee 8. Bradham DM, Igarashi A, Potter RL, Grotendorst GR: Connective tissue T, Li D, Neff TB, Urquilla PR, Sewell KL: Phase 1 study of anti-CTGF growth factor: A cysteine-rich mitogen secreted by human vascular monoclonal antibody in patients with diabetes and microalbuminuria. endothelial cells is related to the SRC-induced immediate early gene Clin J Am Soc Nephrol 5: 1420–1428, 2010 product CEF-10. JCellBiol114: 1285–1294, 1991 24. Vorwerk P, Hohmann B, Oh Y, Rosenfeld RG, Shymko RM: Binding 9. Ren S, Johnson BG, Kida Y, Ip C, Davidson KC, Lin SL, Kobayashi A, properties of insulin-like growth factor binding protein-3 (IGFBP-3), Lang RA, Hadjantonakis AK, Moon RT, Duffield JS: LRP-6 is a co- IGFBP-3 N- and C-terminal fragments, and structurally related proteins receptor for multiple fibrogenic signaling pathways in pericytes and mac25 and connective tissue growth factor measured using a bio- myofibroblasts that are inhibited by DKK-1. Proc Natl Acad Sci USA sensor. Endocrinology 143: 1677–1685, 2002 110: 1440–1445, 2013 25. Abreu JG, Ketpura NI, Reversade B, De Robertis EM: Connective-tissue 10. Hall-Glenn F, De Young RA, Huang BL, van Handel B, Hofmann JJ, growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Chen TT, Choi A, Ong JR, Benya PD, Mikkola H, Iruela-Arispe ML, Lyons Nat Cell Biol 4: 599–604, 2002 KM: CCN2/connective tissue growth factor is essential for pericyte 26. Gao R, Brigstock DR: Low density lipoprotein receptor-related protein adhesion and endothelial basement membrane formation during an- (LRP) is a heparin-dependent adhesion receptor for connective tissue giogenesis. PLoS One 7: e30562, 2012 growth factor (CTGF) in rat activated hepatic stellate cells. Hepatol Res 11. Suzuma K, Naruse K, Suzuma I, Takahara N, Ueki K, Aiello LP, King GL: 27: 214–220, 2003 Vascular endothelial growth factor induces expression of connective 27. Hashimoto G, Inoki I, Fujii Y, Aoki T, Ikeda E, Okada Y: Matrix metal- tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase- loproteinases cleave connective tissue growth factor and reactivate akt-dependent pathways in retinal vascular cells. JBiolChem275: angiogenic activity of vascular endothelial growth factor 165. JBiol 40725–40731, 2000 Chem 277: 36288–36295, 2002 12. Fonseca C, Lindahl GE, Ponticos M, Sestini P, Renzoni EA, Holmes AM, 28. Babic AM, Chen CC, Lau LF: Fisp12/mouse connective tissue growth Spagnolo P, Pantelidis P, Leoni P, McHugh N, Stock CJ, Shi-Wen X, factor mediates endothelial cell adhesion and migration through in- Denton CP, Black CM, Welsh KI, du Bois RM, Abraham DJ: A poly- tegrin alphavbeta3, promotes endothelial cell survival, and induces morphism in the CTGF promoter region associated with systemic angiogenesis in vivo. Mol Cell Biol 19: 2958–2966, 1999 sclerosis. NEnglJMed357: 1210–1220, 2007 29. Gao R, Brigstock DR: Connective tissue growth factor (CCN2) induces 13. Kawaguchi Y, Ota Y, Kawamoto M, Ito I, Tsuchiya N, Sugiura T, adhesion of rat activated hepatic stellate cells by binding of its C-terminal Katsumata Y, Soejima M, Sato S, Hasegawa M, Fujimoto M, Takehara K, domain to integrin alpha(v)beta(3) and heparan sulfate proteoglycan. Kuwana M, Yamanaka H, Hara M: Association study of a polymorphism J Biol Chem 279: 8848–8855, 2004 of the CTGF gene and susceptibility to systemic sclerosis in the Japa- 30. Weston BS, Wahab NA, Mason RM: CTGF mediates TGF-beta-induced nese population. Ann Rheum Dis 68: 1921–1924, 2009 fibronectin matrix deposition by upregulating active alpha5beta1 in- 14. Igarashi A, Nashiro K, Kikuchi K, Sato S, Ihn H, Grotendorst GR, tegrin in human mesangial cells. JAmSocNephrol14: 601–610, 2003 Takehara K: Significant correlation between connective tissue 31. Grotendorst GR, Rahmanie H, Duncan MR: Combinatorial signaling growth factor gene expression and skin sclerosis in tissue sections pathways determine fibroblast proliferation and myofibroblast differ- from patients with systemic sclerosis. J Invest Dermatol 105: 280– entiation. FASEB J 18: 469–479, 2004 284, 1995 32. Pattaro C, Teumer A, Gorski M, Chu AY, Li M, Mijatovic V, Garnaas M, 15. Liu S, Shi-wen X, Abraham DJ, Leask A: CCN2 is required for bleomycin- Tin A, Sorice R, Li Y, Taliun D, Olden M, Foster M, Yang Q, Chen MH, induced skin fibrosis in mice. Arthritis Rheum 63: 239–246, 2011 Pers TH, Johnson AD, Ko YA, Fuchsberger C, Tayo B, Nalls M, Feitosa

J Am Soc Nephrol 28: 1769–1782, 2017 Connective Tissue Growth Factor Ampliﬁes Fibrosis 1781 BASIC RESEARCH www.jasn.org

MF, Isaacs A, Dehghan A, d’Adamo P, Adeyemo A, Dieffenbach AK, 38. Kida Y, Ieronimakis N, Schrimpf C, Reyes M, Duffield JS: EphrinB2 re- Zonderman AB, Nolte IM, van der Most PJ, Wright AF, Shuldiner AR, verse signaling protects against capillary rarefaction and fibrosis after Morrison AC, Hofman A, Smith AV, Dreisbach AW, Franke A, kidney injury. J Am Soc Nephrol 24: 559–572, 2013 Uitterlinden AG, Metspalu A, Tonjes A, Lupo A, Robino A, Johansson A, 39. Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, Demirkan A, Kollerits B, Freedman BI, Ponte B, Oostra BA, Paulweber Daluiski A, Lyons KM: Connective tissue growth factor coordinates B, Kramer BK, Mitchell BD, Buckley BM, Peralta CA, Hayward C, Helmer chondrogenesis and angiogenesis during skeletal development. De- C, Rotimi CN, Shaffer CM, Muller C, Sala C, van Duijn CM, Saint-Pierre velopment 130: 2779–2791, 2003 A, Ackermann D, Shriner D, Ruggiero D, Toniolo D, Lu Y, Cusi D, 40. Mercurio S, Latinkic B, Itasaki N, Krumlauf R, Smith JC: Connective- Czamara D, Ellinghaus D, Siscovick DS, Ruderfer D, Gieger C, Grallert tissue growth factor modulates WNT signalling and interacts with the H, Rochtchina E, Atkinson EJ, Holliday EG, Boerwinkle E, Salvi E, WNT receptor complex. Development 131: 2137–2147, 2004 Bottinger EP, Murgia F, Rivadeneira F, Ernst F, Kronenberg F, Hu FB, 41. Feng XH, Derynck R: Ligand-independent activation of transforming Navis GJ, Curhan GC, Ehret GB, Homuth G, Coassin S, Thun GA, Pistis growth factor (TGF) beta signaling pathways by heteromeric cytoplas- G, Gambaro G, Malerba G, Montgomery GW, Eiriksdottir G, Jacobs G, mic domains of TGF-beta receptors. JBiolChem271: 13123–13129, Li G, Wichmann HE, Campbell H, Schmidt H, Wallaschofski H, Volzke H, 1996 Brenner H, Kroemer HK, Kramer H, Lin H, Leach IM, Ford I, Guessous I, 42. Eggan K, Akutsu H, Loring J, Jackson-Grusby L, Klemm M, Rideout Rudan I, Prokopenko I, Borecki I, Heid IM, Kolcic I, Persico I, Jukema WM, Yanagimachi R, Jaenisch R 3rd: Hybrid vigor, fetal overgrowth, JW, Wilson JF, Felix JF, Divers J, Lambert JC, Stafford JM, Gaspoz JM, and viability of mice derived by nuclear cloning and tetraploid embryo Smith JA, Faul JD, Wang JJ, Ding J, Hirschhorn JN, Attia J, Whitfield JB, complementation. Proc Natl Acad Sci USA 98: 6209–6214, 2001 Chalmers J, Viikari J, Coresh J, Denny JC, Karjalainen J, Fernandes JK, 43. Hung C, Linn G, Chow YH, Kobayashi A, Mittelsteadt K, Altemeier WA, Endlich K, Butterbach K, Keene KL, Lohman K, Portas L, Launer LJ, Gharib SA, Schnapp LM, Duffield JS: Role of lung pericytes and resi- Lyytikainen LP, Yengo L, Franke L, Ferrucci L, Rose LM, Kedenko L, Rao dent fibroblasts in the pathogenesis of pulmonary fibrosis. Am J Respir M, Struchalin M, Kleber ME, Cavalieri M, Haun M, Cornelis MC, Ciullo Crit Care Med 188: 820–830, 2013 M, Pirastu M, de Andrade M, McEvoy MA, Woodward M, Adam M, 44. Gomez IG, MacKenna DA, Johnson BG, Kaimal V, Roach AM, Ren S, Cocca M, Nauck M, Imboden M, Waldenberger M, Pruijm M, Metzger Nakagawa N, Xin C, Newitt R, Pandya S, Xia TH, Liu X, Borza DB, Grafals M, Stumvoll M, Evans MK, Sale MM, Kahonen M, Boban M, Bochud M, M, Shankland SJ, Himmelfarb J, Portilla D, Liu S, Chau BN, Duffield JS: Rheinberger M, Verweij N, Bouatia-Naji N, Martin NG, Hastie N, Anti-microRNA-21 oligonucleotides prevent Alport nephropathy pro- Probst-Hensch N, Soranzo N, Devuyst O, Raitakari O, Gottesman O, gression by stimulating metabolic pathways. JClinInvest125: 141– Franco OH, Polasek O, Gasparini P, Munroe PB, Ridker PM, Mitchell P, 156, 2015 Muntner P, Meisinger C, Smit JH, ICBP Consortium; AGEN Consor- 45. Nakagawa N, Xin C, Roach AM, Naiman N, Shankland SJ, Ligresti G, tium; CARDIOGRAM; CHARGe-Heart Failure Group; ECHOGen RenS,SzakS,GomezIG,Duffield JS: Dicer1 activity in the stromal Consortium, Kovacs P, Wild PS, Froguel P, Rettig R, Magi R, Biffar R, compartment regulates nephron differentiation and vascular pattern- Schmidt R, Middelberg RP, Carroll RJ, Penninx BW, Scott RJ, Katz R, ing during mammalian kidney organogenesis. Kidney Int 87: 1125– Sedaghat S, Wild SH, Kardia SL, Ulivi S, Hwang SJ, Enroth S, Kloiber S, 1140, 2015 Trompet S, Stengel B, Hancock SJ, Turner ST, Rosas SE, Stracke S, Harris 46. Chau BN, Xin C, Hartner J, Ren S, Castano AP, Linn G, Li J, Tran PT, TB, Zeller T, Zemunik T, Lehtimaki T, Illig T, Aspelund T, Nikopensius T, Kaimal V, Huang X, Chang AN, Li S, Kalra A, Grafals M, Portilla D, Esko T, Tanaka T, Gyllensten U, Volker U, Emilsson V, Vitart V, Aalto V, MacKenna DA, Orkin SH, Duffield JS: MicroRNA-21 promotes fibrosis Gudnason V, Chouraki V, Chen WM, Igl W, Marz W, Koenig W, Lieb W, of the kidney by silencing metabolic pathways. Sci Transl Med 4: Loos RJ, Liu Y, Snieder H, Pramstaller PP, Parsa A, O’Connell JR, Susztak 121ra18, 2012 K, Hamet P, Tremblay J, de Boer IH, Boger CA, Goessling W, Chasman 47. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, DI, Kottgen A, Kao WH, Fox CS: Genetic associations at 53 loci highlight Chaisson M, Gingeras TR: STAR: Ultrafast universal RNA-seq aligner. cell types and biological pathways relevant for kidney function. Nat Bioinformatics 29: 15–21, 2013 Commun 7: 10023, 2016 48. Li B, Dewey CN: RSEM: Accurate transcript quantification from RNA- 33. He W, Dai C, Li Y, Zeng G, Monga SP, Liu Y: Wnt/beta-catenin signaling Seq data with or without a reference genome. BMC Bioinformatics 12: promotes renal interstitial fibrosis. J Am Soc Nephrol 20: 765–776, 2009 323, 2011 34. Woroniecka KI, Park AS, Mohtat D, Thomas DB, Pullman JM, Susztak K: 49. Love MI, Huber W, Anders S: Moderated estimation of fold change Transcriptome analysis of human diabetic kidney disease. Diabetes 60: and dispersion for RNA-seq data with DESeq2. Genome Biol 15: 550, 2354–2369, 2011 2014 35. Rooney B, O’Donovan H, Gaffney A, Browne M, Faherty N, Curran SP, 50. Cox J, Mann M: MaxQuant enables high peptide identification rates, Sadlier D, Godson C, Brazil DP, Crean J: CTGF/CCN2 activates canonical individualized p.p.b.-range mass accuracies and proteome-wide pro- Wnt signalling in mesangial cells through LRP6: Implications for the patho- tein quantification. Nat Biotechnol 26: 1367–1372, 2008 genesis of diabetic nephropathy. FEBS Lett 585: 531–538, 2011 51. Krogh A, Larsson B, von Heijne G, Sonnhammer EL: Predicting trans- 36. Lin SL, Li B, Rao S, Yeo EJ, Hudson TE, Nowlin BT, Pei H, Chen L, Zheng membrane protein topology with a hidden Markov model: Application JJ, Carroll TJ, Pollard JW, McMahon AP, Lang RA, Duffield JS: Mac- to complete genomes. JMolBiol305: 567–580, 2001 rophage Wnt7b is critical for kidney repair and regeneration. Proc Natl Acad Sci USA 107: 4194–4199, 2010 37. Semënov MV, Tamai K, Brott BK, Kühl M, Sokol S, He X: Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr Biol 11: 951–961, This article contains supplemental material online at http://jasn.asnjournals. 2001 org/lookup/suppl/doi:10.1681/ASN.2016080826/-/DCSupplemental.

1782 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1769–1782, 2017