Kobayashi K , Luo M , Zhang Y , Wilkes DC , Ge G , Grieskamp T , Yamada C , Liu TC , Huang G , Basson CT , Kispert A , Greenspan DS , Sato TN .

R01 GM071679-02 NIGMS NIH HHS , R01 GM071679-03 NIGMS NIH HHS , R01 GM071679-04 NIGMS NIH HHS , R01 GM071679 NIGMS NIH HHS

	Figure 2. sFRP2 binds BMP1 with a KD in the physiological range, predominantly via its Fzl domain. (a) Immunoblots show that His-tagged sFRP2 pulls down FLAG-tagged BMP1, upon precipitation with Ni-NTA agarose. (b) BMP1 binds His-tagged sFRP2 on a BIAcore sensor chip with KD = 7.13 nM. (c) Immunoblots show that the Fc-tagged sFRP2 Fzl domain pulls down FLAG-tagged BMP1 about as effectively as full-length sFRP2-Fc. (d) Fc-tagged Fzl domain of sFRP2 on a Biacore sensor chip binds BMP1 with a KD (KD = 8.68 nM) similar to that of full-length sFRP2-Fc (KD = 6.01 nM), whereas binding of Fc-tagged sFRP2 NTR domain to BMP1 is negligible. (e) Schematic diagram of sFRP2-Fc constructs used for pull-down and BIAcore assays. For full scans of a and c, see Supplementary Information, Fig. S6.
	Figure 3. sFRP2 and Szl bind Tolloid-like proteinases via non-protease domain sequences and BMP1/procollagen binding is enhanced by sFRP2. (a) Left, an immunoblot shows input amounts of sFRP2-Fc, FLAG-tagged BMP1 and a deleted form of BMP1 containing all domains except the protease domain (BMP1-D) and isolated BMP1 protease domain (BMP1-P). Right, an immunoblot shows that protein G agarose pull down of sFRP2-Fc pulls down full-length BMP1 and BMP1-D, but not BMP1-P. (b) Left, an immunoblot shows input amounts of sRFP2-Fc and FLAG-tagged mTLL1, or isolated mTLL1 protease domain (mTLL1-P). Right, an immunoblot shows that sFRP2-Fc pulls down full-length mTLL1, but not mTLL1-P. (a and b) arrowheads indicate sFRP2-Fc, and asterisks denote sFRP2 degradation products which bind non-specifically to anti-FLAG antibodies. (c) Left, an immunoblot shows input amounts of His-tagged Szl, and FLAG-tagged BMP1, BMP1-D and BMP1-P. Right, immunoblot shows that Ni-NTA agarose pull down of His-tagged Szl pulls down full-length BMP1 and BMP1-D, but not BMP1-P. (d) Left, an immunoblot shows input amounts of His-tagged Szl, mTLL1, and mTLL1-P. Right, an immunoblot shows that Szl pulls down mTLL1, but not mTLL1-P. (e) Protein domain structures of full-length and truncated forms of BMP1 and mTLL1. Pro, prodomain; Prot, protease domain; CB, CUB domain; E, EGF motif; CT, C-terminal domain. (f) Immunoblots show that procollagen binds sFRP2, and that sFRP2 enhances binding of procollagen to BMP1. Samples were immunoprecipitated with antibody (LF-67) directed against the pro-α1(I) C-telopeptide region. LF-67 is shown to pull down a procollagen/sFRP2-His complex and the presence of sFRP2-His enhances co-immunoprecipitation of procollagen and BMP1*, a form of BMP1 designed to bind but not cleave or release procollagen31 (Quantification of signals on the blots indicate approximately 4-fold enhancement of BMP1* binding by procollagen in the presence of sFRP2). For full scans of a–d and f, see Supplementary Information, Fig. S6.
	Figure 4. Reduced processing of type I procollagen and deposition of collagen into ECM by Sfrp2−/− fibroblasts. Western blot analysis with antibody against the proα1(I) C-telopeptide domain (a small globular region retained on mature α chains, between the triple-helical domain and BMP1 cleavage site) demonstrates decreased processing of procollagen in conditioned media and decreased deposition of collagen into the ECM associated with cell layers of Sfrp2−/− MEF cultures (a) and heart fibroblasts (b) compared to wild-type (sFRP2+/+) cells. Due to the dramatic difference in collagen levels in the cell layer samples of wild-type and Sfrp2−/− heart fibroblasts, samples were re-probed with anti-α tubulin antibody to demonstrate equal loading in the two lanes. Densitometric quantification is shown of the relative amounts of the various collagen forms in conditioned media of MEFs (c) and heart fibroblasts (d). Pro forms are precursors that retain both N- and C-propeptides; α1(I) forms are mature chains, capable of forming fibrils, from which both N- and C-propeptides have been proteolytically removed; pN forms are processing intermediates of proα1(I) chains from which the C- but not N-propeptide has been removed by BMP1-like proteinases; pC forms are processing intermediates from which the N- but not C-propeptide has been removed by non-BMP1-like proteinases. pC and pN forms were not separated by SDS-PAGE in these experiments. Densitometric quantification is shown of the relative amounts of mature α1(I) chains incorporated into cell layer-associated ECM of wild type (+/+) and sFRP2-null (−/−) MEFs (e) and heart fibroblasts (f). For full scans of a and b, see Supplementary Information, Fig. S6.
	Figure 5. Induction of Sfrp2 and Bmp1 during the fibrosis phase in the infarcted heart. (a) β-galactosidase staining of the infarcted sFRP2lacZ/+ left-ventricle at days 1, 4, 7, and 14 following coronary artery ligation. Higher magnifications of the boxed areas in the top panels are in bottom panels. At day one, the sirius-red counterstaining (orange) identifies normal collagens in the interstitial space. At this stage, no sFRP2 expression was detected. At day four, the slightly wider areas are stained by sirius-read (orange), which is accompanied by scattered distribution of β-galactosidase-stained sFRP2-expressing fibroblastic cells (blue). By day seven, extensive collagenous fibrosis stained by sirius-red (orange) is detected. Within the fibrotic area, many fibroblastic cells express sFRP2 (blue). At day 14, more extensive sirius-red staining, denoting collagenous scar tissue that has replaced dead myocytes, is clearly detected, and many fibroblastic cells are seen to express sFRP2 (blue). Upregulation of sFRP2 expression in the infarcted area was also confirmed by in situ hybridization staining with a riboprobe for sFRP2 (Supplementary Information: Fig. S3). Scale bars are shown in each panel. (b) Quantitative RT-PCR analysis of sFRP2, BMP1 and mTLL1. Whole ventricle tissue RNA was isolated at days four, seven and 14 following coronary artery ligation. The amounts of sFRP2, BMP1 and mTLL1 were compared to those of non-operated normal ventricle tissue and fold-increases are shown. Duplicate PCR reactions for each sample were carried out and the standard deviations were calculated from the results obtained from two independent experiments. Marked increases of sFRP2 and BMP1 transcripts were detected by day four and their transcript levels continued to increase until day seven. By day 14, their transcript levels decreased, but still remained relatively elevated. The mTLL1 transcript level increase was much less that those of sFRP2 and BMP1. The absolute amounts of sFRP2 transcripts at day 0, day4, day7, day14 were quantified and determined as 6.8×102, 1.5×104, 6.5×104, 1.3×104 molecules per 20ng total RNA, respectively. Expression analysis of all Wnt ligands and Wnt antagonists genes indicates that the expression of Sfrp2 gene exhibits the highest upregulation within the fibrotic area of infarcted hearts (Supplementary Information: Figs. S3&S4).
	Figure 6. Reduced fibrosis in the infarcted sFRP2-deficient heart. (a) Sirius-red stained cross sectioned infarcted left ventricle of wild-type (WT) and sFRP2-deficient (Sfrp2lacZ/lacZ) hearts at day 14 after coronary artery ligation. The infarcted wild-type ventricle shows dilatation and extensive sirius-red stained fibrosis (orange). The infarcted Sfrp2lacZ/lacZ ventricle shows much less dilation, and the sirius-red stained area (orange) is reduced. Scale bars are shown in each panel. (b) Quantification of the sirius-red stained fibrotic area. Sirius-red stained areas, determined using AxioVision image analysis software (Carl Zeiss), were compared to whole ventricular areas and are shown as percentages. A representative section from each of a total of nine (n=9) wild type and five (n=5) sFRP2 null hearts was evaluated and the standard deviations were calculated. The sirius-red stained areas in wild type and Sfrp2lacZ/lacZ left ventricles were ∼25 – 35% and ∼15 – 20% of the entire ventricle areas, respectively. (c) Quantification of total hydroxyproline (HP) amounts. Samples from four independent experiments (n=4) for each group were assayed and shown here. The standard deviations and P values are shown. In sham operated control wild type and sFRP2-null hearts, approximately 2 – 5µg of hydroxyproline was present per mg dry weight left-ventricle. In the infarcted wild type heart, approximately 10 – 16µg of hydroxyproline was detected per mg dry weight left-ventricle. In contrast, only 3 – 8µg of hydroxyproline per dry weight left ventricle was detected in the sFRP2-null heart.
	Figure 7. Improved cardiac function of the infarcted sFRP2-deficient heart. (a) Ejection fraction (EF) of wild-type (WT) and sFRP2-deficient (Sfrp2lacZ/lacZ) hearts at day seven following coronary artery ligation. Each dot represents an individual mouse (i.e., WT sham: n=3; WT MI: n=7; Sfrp2lacZ/lacZ sham: n=3; Sfrp2lacZ/lacZ MI: n=6). At day seven, the infarcted (MI) hearts of both wild type (WT) and sFRP2-deficient (Sfrp2lacZ/lacZ ) mice showed wide ranges of EF (10% – 55%) and no significant (NS) difference. The EF of sham operated mice showed a normal range of EF. (b) EF of wild-type (WT) and sFRP2-deficient (sFRP2lacZ/lacZ) hearts at day 14 following coronary artery ligation. Each dot represents an individual mouse (i.e., WT sham: n=3; WT MI: n=9; Sfrp2lacZ/lacZ sham: n=3; Sfrp2lacZ/lacZ MI: n=7). At day 14, The infarcted (MI) hearts of all wild type (WT) mice showed consistently low EF (10 – 20%). In contrast, the infarcted (MI) hearts of sFRP2-deficient (Sfrp2lacZ/lacZ) mice showed higher EFs (25 – 60%) and two of the null mice showed EFs (60 –70%) equivalent to those of sham operated mice. The EF of sham operated mice showed a normal range of EF. (c) Change of EFs over time. The EFs of the infarcted wild-type (WT) hearts continued to decline over time, indicating functional failure. In contrast, the EFs of the infarcted sFRP2-deficient (sFRP2lacZ/lacZ) hearts in general remained approximately the same or in some cases slightly improved over time. The data shown in a&b are used to generate this plot. (d) M-mode images of representative wild-type (WT) and sFRP2-deficient (sFRP2lacZ/lacZ) hearts at day 14 following coronary artery ligation. The wild-type heart showed virtually no pumping. In contrast, the sFRP2-deficient heart showed some pumping activity. The time-lapse movie of each heart can be viewed in the (supplementary information Movies 1 & 2).

Bányai, The NTR module: domains of netrins, secreted frizzled related proteins, and type I procollagen C-proteinase enhancer protein are homologous with tissue inhibitors of metalloproteases. 1999, Pubmed

Bányai, The NTR module: domains of netrins, secreted frizzled related proteins, and type I procollagen C-proteinase enhancer protein are homologous with tissue inhibitors of metalloproteases. 1999, Pubmed
Barandon, Involvement of FrzA/sFRP-1 and the Wnt/frizzled pathway in ischemic preconditioning. 2005, Pubmed
Barandon, Reduction of infarct size and prevention of cardiac rupture in transgenic mice overexpressing FrzA. 2003, Pubmed
Berg, Determination of 3- and 4-hydroxyproline. 1982, Pubmed
Bodine, The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice. 2004, Pubmed
Cleutjens, The infarcted myocardium: simply dead tissue, or a lively target for therapeutic interventions. 1999, Pubmed
De Robertis, Dorsal-ventral patterning and neural induction in Xenopus embryos. 2004, Pubmed , Xenbase
Dufourcq, FrzA, a secreted frizzled related protein, induced angiogenic response. 2002, Pubmed
Finch, Purification and molecular cloning of a secreted, Frizzled-related antagonist of Wnt action. 1997, Pubmed , Xenbase
Fisher, Antisera and cDNA probes to human and certain animal model bone matrix noncollagenous proteins. 1995, Pubmed
Garrigue-Antar, Deletion of epidermal growth factor-like domains converts mammalian tolloid into a chordinase and effective procollagen C-proteinase. 2004, Pubmed
Ge, Mammalian tolloid-like 1 binds procollagen C-proteinase enhancer protein 1 and differs from bone morphogenetic protein 1 in the functional roles of homologous protein domains. 2006, Pubmed
Ge, Bone morphogenetic protein 1 processes prolactin to a 17-kDa antiangiogenic factor. 2007, Pubmed
Ge, BMP1 controls TGFbeta1 activation via cleavage of latent TGFbeta-binding protein. 2006, Pubmed
Hopkins, The bone morphogenetic protein 1/Tolloid-like metalloproteinases. 2007, Pubmed
Jugdutt, Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough? 2003, Pubmed
Jugdutt, Remodeling of the myocardium and potential targets in the collagen degradation and synthesis pathways. 2003, Pubmed
Kawano, Secreted antagonists of the Wnt signalling pathway. 2003, Pubmed , Xenbase
Kessler, Type I procollagen C-proteinase from mouse fibroblasts. Purification and demonstration of a 55-kDa enhancer glycoprotein. 1989, Pubmed
Kim, The stomach mesenchymal transcription factor Barx1 specifies gastric epithelial identity through inhibition of transient Wnt signaling. 2005, Pubmed
Kim, Pax-6 regulates expression of SFRP-2 and Wnt-7b in the developing CNS. 2001, Pubmed
Lee, Embryonic dorsal-ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases. 2006, Pubmed , Xenbase
Lee, Autocrine/paracrine secreted Frizzled-related protein 2 induces cellular resistance to apoptosis: a possible mechanism of mammary tumorigenesis. 2004, Pubmed
Lee, SHH-N upregulates Sfrp2 to mediate its competitive interaction with WNT1 and WNT4 in the somitic mesoderm. 2000, Pubmed
Lei, Wnt signaling inhibitors regulate the transcriptional response to morphogenetic Shh-Gli signaling in the neural tube. 2006, Pubmed
Martyn, The ventralized ogon mutant phenotype is caused by a mutation in the zebrafish homologue of Sizzled, a secreted Frizzled-related protein. 2003, Pubmed
Mayr, Fritz: a secreted frizzled-related protein that inhibits Wnt activity. 1997, Pubmed , Xenbase
Melkonyan, SARPs: a family of secreted apoptosis-related proteins. 1997, Pubmed
Mirotsou, Secreted frizzled related protein 2 (Sfrp2) is the key Akt-mesenchymal stem cell-released paracrine factor mediating myocardial survival and repair. 2007, Pubmed
Moali, Substrate-specific modulation of a multisubstrate proteinase. C-terminal processing of fibrillar procollagens is the only BMP-1-dependent activity to be enhanced by PCPE-1. 2005, Pubmed
Muraoka, Sizzled controls dorso-ventral polarity by repressing cleavage of the Chordin protein. 2006, Pubmed
Nathan, sFRPs: a declaration of (Wnt) independence. 2009, Pubmed
Oshima, Myeloma cells suppress bone formation by secreting a soluble Wnt inhibitor, sFRP-2. 2005, Pubmed
Pappano, Use of Bmp1/Tll1 doubly homozygous null mice and proteomics to identify and validate in vivo substrates of bone morphogenetic protein 1/tolloid-like metalloproteinases. 2003, Pubmed
Polesskaya, Wnt signaling induces the myogenic specification of resident CD45+ adult stem cells during muscle regeneration. 2003, Pubmed
Rattner, A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors. 1997, Pubmed
Roth, Secreted Frizzled-related proteins inhibit motility and promote growth of human malignant glioma cells. 2000, Pubmed
Salic, Sizzled: a secreted Xwnt8 antagonist expressed in the ventral marginal zone of Xenopus embryos. 1997, Pubmed , Xenbase
Satoh, Sfrp1 and Sfrp2 regulate anteroposterior axis elongation and somite segmentation during mouse embryogenesis. 2006, Pubmed
Schier, Molecular genetics of axis formation in zebrafish. 2005, Pubmed
Scott, Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis. 1999, Pubmed , Xenbase
Sun, Infarct scar: a dynamic tissue. 2000, Pubmed
Suzuki, Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer. 2004, Pubmed
Swynghedauw, Molecular mechanisms of myocardial remodeling. 1999, Pubmed
Takahara, Type I procollagen COOH-terminal proteinase enhancer protein: identification, primary structure, and chromosomal localization of the cognate human gene (PCOLCE). 1994, Pubmed
Wodarz, Mechanisms of Wnt signaling in development. 1998, Pubmed , Xenbase
WOESSNER, The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. 1961, Pubmed
Yabe, Ogon/Secreted Frizzled functions as a negative feedback regulator of Bmp signaling. 2003, Pubmed , Xenbase
Zi, Expression of Frzb/secreted Frizzled-related protein 3, a secreted Wnt antagonist, in human androgen-independent prostate cancer PC-3 cells suppresses tumor growth and cellular invasiveness. 2005, Pubmed
Zou, Aberrant methylation of secreted frizzled-related protein genes in esophageal adenocarcinoma and Barrett's esophagus. 2005, Pubmed