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Connective Tissue Growth Factor Domain 4 Amplifies Fibrotic Kidney

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Connective Tissue Growth Factor Domain 4 Amplifies Fibrotic Kidney BASIC RESEARCH www.jasn.org 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, inhibi- ted 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 J Am Soc Nephrol 28: 1769–1782, 2017 ISSN : 1046-6673/2806-1769 1769 BASIC RESEARCH www.jasn.org 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) over- expressing 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 1770 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1769–1782, 2017 www.jasn.org BASIC RESEARCH 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
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