The Advantages and Disadvantages of Sfrp1 and Sfrp2 Expression in Pathological Events

Tohoku J. Exp. Med., 2010, 221, 11-17 Sfrp1/2 in Deseases 11

Invited Review for the 90th Anniversary

Pilar Esteve1 and Paola Bovolenta1

1Departamento de Neurobiología Molecular, Celular y del Desarrollo, Instituto Cajal (CSIC) and CIBER de Enfermedades Raras (CIBERER), Madrid, Spain

Secreted Frizzled Related Proteins (Sfrps) are a family of secreted proteins that can bind both to Wnt ligands and Frizzled receptors, thereby modulating the Wnt signalling cascades. Recent studies have shown that Sfrps can also interact with Wnt unrelated molecules such as RANKL, a member of the tumor necrosis factor family, Tolloid metalloproteinases or integrin-ﬁbronectin complexes. Alterations in the levels of Sfrp expression have been recently associated with different pathological conditions, including tumor formation and bone and myocardial disorders. Here, we summarise the evidence that relates Sfrps with these diseases and discuss how the proposed multiple Sfrp interactions with Wnt related and unrelated pathways may explain their implication in such diverse pathologies.

Secreted Frizzled Related Proteins (Sfrps) were tissue homeostasis (Nusse 2005). Its wide variety of actions initially and independently identified as soluble factors is achieved by the combinatorial use of 19 distinct Wnt implicated in early embryonic development (Leyns et al. ligands and of different classes of receptors and coreceptors, 1997; Wang et al. 1997) and as modulators of apoptotic which activate at least three partially independent transduc- events (Melkonyan et al. 1997). This protein family, which tion cascades (van Amerongen and Nusse 2009). It seems counts with five members in mammals, received this name thus reasonable that modulators of Wnt signaling, such as because their N-terminal region shows a remarkable Sfrps (Bovolenta et al. 2008), should have similar intricate sequence homology with the extracellular Wnt binding interactions with both Wnt and Fz. However, to make domain of Frizzled receptors (Hoang et al. 1996; Leyns et matters worse, Sfrps have been shown to interact also with al. 1997; Lin et al. 1997; Wang et al. 1997). Initial studies, molecules unrelated to Wnts. For example, Sfrp1 binds to using Xenopus embryos as a model, demonstrated that frzb and inhibits the activity of RANKL, a member of the TNF (Sfrp3), the first isolated member of the family, bound Wnt family involved in osteoclast formation (Hausler et al. ligands and prevented canonical Wnt signalling activation, 2004). Sfrp2 specifically binds to Tolloid metalloproteinases pointing to a Wnt antagonist function based on ligand and thereby regulates procollagen processing (Kobayashi et sequestration (Kawano and Kypta 2003). This relatively al. 2009) or interacts with an integrin-fibronectin complex simple scenario was complicated by subsequent studies modulating apoptosis (Lee et al. 2004), while Sfrp3 binds to showing that Sfrps can inhibit non-canonical signalling EGF blocking its function in different cell contexts (LaMonica et al. 2009; Matsuyama et al. 2009; Sugiyama et (Scardigli et al. 2008). al. 2010) either by binding to Fz receptors, activating an A possible explanation for this variety of interactions intracellular signal (Rodriguez et al. 2005; Dufourcq et al. may reside in the structure of these proteins, which are 2008; Kawano et al. 2009) or by enhancing Wnt diffusion in composed of two independently folded domains. The the extracellular space, consequently expanding their signal- Cysteine Rich Domain (CRD) at the N-terminus, shares ling range (Mii and Taira 2009). Thus, context-dependent similarities with the extracellular domain of transmembrane interactions among different Wnt, Sfrps and Fz molecules proteins including, as mentioned above, Fz receptors, the seem to modulate the intricate Wnt signalling cascade in Shh transducer Smoothened and the Wnt receptor tyrosine both a positive and negative way (Fig. 1). kinases RORs (Roszmusz et al. 2001). The C-terminal Wnt signaling is probably one of the most extensively domain contains a Netrin-Related motif (NTR) defined, in used cell-cell communication pathway in development and the most closely related Sfrp1/2/5, by six cysteine residues

Received March 4, 2010; revision accepted for publication March 26, 2010. doi:10.1620/tjem.221.11 Correspondence: Pilar Esteve and Paola Bovolenta, Departamento de Neurobiología Molecular, Celular y del Desarrollo, Instituto Cajal (CSIC) and CIBER de Enfermedades Raras (CIBERER), Avda. Dr. Arce 37, Madrid 28002, Spain. e-mail: [email protected] or [email protected]

11 12 P. Esteve and P. Bovolenta

Fig. 1. Multifunctional molecular interactions of Sfrp1/2 and their implication in pathological events. The drawing depicts possible mechanisms of action of Sfrp1/2 and their implications in pathological events. A) Sfrp1 and Sfrp2 sequester Wnts, thereby antagonize Wnt/βcatenin signaling. B) Frzb (Sfrp3) and Crescent favor the diffusion of Wnts, enhancing their signaling range. C) Sfrp1 and Sfrp2 bind to Fz receptors activating intracellular signals. D) Sfrp1 binds to RANKL preventing interaction with the RANK receptor. E) Sfrp2 binds to and enhances the procollagen proteinase activity of BMP1/ Tolloid-like metallo-proteinases, accelerating the processing of pro-collagen. F) Sizzled binds to and inhibits the Chordinase activity of BMP1/ Tolloid-like metalloproteinases. Unprocessed Chordin binds and sequesters BMPs. that form three disulfide bridges. This motif characterises a of CpG islands causes gene silencing and is considered one number of unrelated proteins including Netrin-1, tissue of the most frequent epigenetic alterations associated with inhibitors of metalloproteinases (TIMPs), complement cancer in humans (Esteller 2005a, 2005b). Numerous genes proteins and TypeI procollagen C-proteinase enhancer became hypermethylated and silenced in different type of proteins (PCOLCEs) (Banyai and Patthy 1999). cancers but Sfrp1 and Sfrp2 hypermethylation seems to Although it was initially shown that the CRD domain occur from the very beginning in basically all tumour types, of Sfrps is necessary and sufficient for Wnt binding (Lin et including breast (Zhou et al. 1998; Turashvili et al. 2006; al. 1997; Bafico et al. 1999), subsequent studies demon- Suzuki et al. 2008), gastric (To et al. 2001), cervix (Ko et al. strated that the CRD domain also interact with Fz and 2002), hepatocellular (Huang et al. 2007), pancreas (Bu et Tolloid-like metalloproteases (Lee et al. 2006; Lopez-Rios al. 2008), lung (Fukui et al. 2005), ovary (Takada et al. et al. 2008; Kobayashi et al. 2009) while the NTR domain 2004), renal (Dahl et al. 2007; Gumz et al. 2007), prostate can tightly bind to Wnt ligands independently of the (Lodygin et al. 2005) and colon (Suzuki et al. 2002; presence of the CRD domain (Uren et al. 2000; Lopez-Rios Caldwell et al. 2004; Suzuki et al. 2004) carcinomas, as et al. 2008), supporting the context dependent functions well as in non-solid tumours like myeloid leukaemias (Jost observed for Sfrps. et al., 2008) or myelomas (Jost et al. 2009). The correlation Below we discuss how this variety of possible Sfrps between cancer and loss of Sfrp1 expression is so strong interactions may explain their implication in unrelated that, at least for bladder carcinomas (Stoehr et al. 2004; pathological processes such as cancer, bone or myocardial Urakami et al. 2006), it has been proposed that the status of diseases. We will focus on Sfrp1 and Sfrp2, since these are Sfrp1 promoter methylation could serve as an epigenetic the family members, which received most attention. biomarker for cancer detection and progression, where hypermethylation will be associated with unfavourable Sfrp1 and Sfrp2 generally protect prognosis (Lee et al. 2004; Veeck et al. 2006). from carcinogenesis In line with these epigenetic studies, a large-scale anal- The suppression or down-regulation of Sfrp1 and Sfrp2 ysis in tumours derived from a total of 36 organs demon- expression seems to be a key event in the onset of tumour strated a complete loss of Sfrp1 expression in 82% of the formation. In addition to somatic mutations, epigenetic analysed samples (Dahl et al. 2007). Deletion of the chro- DNA alterations play crucial roles in tumorigenesis (Baylin mosomal region 8p12-p11.1 comprising the SFRP1 locus and Herman 2000; Feinberg and Tycko 2004). Methylation has been also reported in a few cancer cases (Ugolini et al. of gene promoter regions enriched in CpG dinucleotides 1999; Stoehr et al. 2004; Dahl et al. 2007). A tempting (CpG islands) is among the epigenetic modifications that speculation from these studies is that Sfrp1, and possibly controls the levels of gene transcription. Hypermethylation the related Sfrp2, act as tumour suppressor genes (Lee et al. Sfrp1/2 in Deseases 13

2004; Veeck et al. 2006) and their silencing may predispose together with the local availability of Fz, Wnts and down- to neoplastic progression (Wong et al. 2002). Supporting stream signalling components- including those of the non this possibility, forced Sfrp1 re-expression in breast (Suzuki canonical pathway (Katoh 2005)- should be major determi- et al. 2008; Matsuda et al. 2009), hepatocellular (Shih et al. nants in the onset and progression of carcinogenesis (Uren 2007; Jiang et al. 2009), prostate (Lodygin et al. 2005) and et al. 2000; Bovolenta et al. 2008; Mii and Taira 2009). It cervical tumours (Chung et al. 2009), where Sfrp1 had been still needs to be determined whether other molecular path- silenced, induces apoptosis, suppresses cell proliferation, ways important for carcinogenesis are directly affected by transformation and invasion both in vitro and in vivo. the changing Sfrp expression levels. Despite these observations, the incidence of spontaneous tumour formation is not increased in Sfrp1 null mice Sfrp1 prevents bone repair (Trevant et al. 2008), suggesting that additional genetic Sfrp1 activity affects many aspects of bone and carti- alterations are necessary for the tumour onset and/or that lage formation and consequently alterations in its expres- Sfrp1suppression is relevant for tumour progression more sion or function cause bone and cartilage disorders. Sfrp1 than for its genesis. is expressed in cartilage (Hoang et al. 1996) and in osteo- Given the variety of Sfrps’ functions described at the blasts and osteoclasts. In vitro assays demonstrated that beginning, the obvious question is what are the molecular Sfrp1 inhibits osteoclast formation (Hausler et al. 2004) mechanisms by which Sfrp1 inhibits tumorigenesis. while genetic inactivation of Sfrp1 accelerates chondrocyte Aberrant activation of Wnt signalling, due either to genetic differentiation and endochondral ossification (Gaur et al. mutations or to the altered expression of its pathway com- 2006) as well as trabecular bone formation in aged animals ponents, is a common event in the onset and progression of (Bodine et al. 2004, 2005). This last defect is the result of cancer (Nusse 2005; Polakis 2007). As mentioned above, an increased osteoblast proliferation and differentiation Sfrps are thought to act as Wnt signalling inhibitors. From associated with a slower lost of bone mass caused by a the initial description of a direct link between the epigenetic decrease of the physiological apoptosis of osteoblasts and inactivation of Sfrp1 (Suzuki et al. 2002) and the upregula- osteocyts (Bodine et al. 2004, 2005). tion of Wnt/βcatenin cascade in colon cancer (Suzuki et al. Consistent with these results, Sfrp1 has been found to 2004), it has been generally accepted that Wnt/β catenin be over-expressed in bones after prolonged glucocorticoids repression is the mechanism by which Sfrp1 inhibits tumour treatment, which is known to induce osteoporosis likely cell growth and prevents metastatic invasion. This mecha- through Sfrp1 activity (Wang et al. 2005). Similarly, Sfrp1 nism was proven in some cases when re-expression of Sfrp1 expression level may be an etiological factor of post- inhibited Wnt/β catenin activation (Suzuki et al. 2004; menopausal (Sims et al. 2008) and senile osteoporosis Nojima et al. 2007; Shih et al. 2007). However, there are (Ohnaka et al. 2009) because different polymorphisms in many examples of Wnt/βcatenin-independent carcinogene- the 3´ untranslated region of Sfrp1 have been associated sis. In these tumour cells, Sfrp1 is similarly silenced and its with variations in bone mineral density in women. On the restoration is sufficient to counteract their phenotype, indi- contrary, genetic inactivation of Sfrp1 in mice improves cating that the loss of Sfrp1 expression contributes to acti- bone fracture repair promoting osteogenesis and matrix vate other oncogenic signalling pathways (Fukui et al. 2005; mineralization (Gaur et al. 2009), two critical steps of bone Nojima et al. 2007; Chung et al. 2009) as proposed for pros- repair. tate cancer (Lodygin et al. 2005; Kawano et al. 2009). In conclusion, Sfrp1 is involved in metabolic bone Among the alternative pathways it is worth considering disorders, osteoporosis and aging. What the molecular those linked to metalloproteases, which are heavily involved consequences of Sfrp1 dysfunction in bone diseases are is in metastatic events. Indeed, members of the Sfrp family still unclear but they may involve both the antagonism of seem to control the activity of Tolloid-like metalloproteases Wnt/βcatenin pathway (Bodine et al. 2004) and the binding (Lee et al. 2006; Muraoka et al. 2006; Kobayashi et al. and inhibition of RANKL, which together with M-CSF 2009). stimulates osteoclast formation (Hausler et al. 2004). An In contrast to the anti-tumorigenic and anti-metastatic additional mechanism may involve the control of Bone effects described for Sfrp1, there are a number of reports Morphogenetic Protein (BMP) signaling, which is a potent suggesting that Sfrp1 can also act as an oncogene that con- inducer of bone morphogenesis. Indeed, a non mammalian fers metastatic properties to renal carcinoma (Saini et al. Sfrp family member, Sizzled, binds to BMP1/Tolloid, a 2009) and promotes prostate carcinogenesis (Joesting et al. metalloprotease that normally degrades the BMP antagonist, 2005). This dual function-as an oncogene or a tumour sup- chordin thereby indirectly inhibiting BMP signaling (Lee et pressor gene- occasionally observed even in the same type al. 2006; Muraoka et al. 2006). A similar role in inhibition of cancer (Joesting et al. 2005; Kawano et al. 2009), sug- of Bmp signaling has been reported for Sfrp1 and Sfrp2 gests that the cellular context is an important determinant of during caudal neural tube closure in mice (Misra and Matise Sfrp1 activity. This, again, should not be a surprise given 2010). the various forms in which Sfrp1 can influence Wnt or other Whether Sfrp1 independently affects all these signalling pathways. Therefore, Sfrp1 concentration pathways or there is cross talk among them still needs to be 14 P. Esteve and P. Bovolenta determined. Notably, it has been recently demonstrated that to coronary artery ligation. As a consequence, their systolic Wnt signaling, through GSK3 activity, enhances BMP sig- function was improved as compared to that of wild type naling (Fuentealba et al. 2007). animals. This result is explained by the observations that Independently of the mechanism of actions it is normally the appearance of collagen fibres in the injured obvious that inhibitors of Sfrp1 could be useful area of the heart coincides with the onset of Sfrp2 pharmacological agents in fracture repair as well as to expression, suggesting that Sfrp2 favours collagen alleviate osteoporosis. Two classes of molecules have been deposition and fibrosis (Kobayashi et al. 2009). Most so far tested. The first is a diphenylsulfone sulphonamide, notably, Sfrp2 simultaneously binds the metalloprotease which binds specifically to Sfrp1, suppresses osteocyte Tolloid/BMP1 and the procollagen, enhancing BMP1 apoptosis and stimulates ex vivo murine bone formation in proteinase activity, thereby accelerating the processing of an organ culture assay (Bodine et al. 2009; Moore et al. the procollagen precursor in mature collagen (Kobayashi et 2009). The second one is a minooxothiazolidine, which al. 2009) also binds to Sfrp1. Its addition in an ex vivo mouse Thus, Sfrp2 seem to have a dual effect on myocardial calvaria assay increases total bone area and the number of repair likely mediated by two unrelated mechanisms of osteoblasts (Shi et al. 2009), clearly suggesting that Sfrp1 is action. In the initial stages of recovery from infarction, a promising therapeutic target in bone degenerative Sfrp2 reduces the size of the damaged tissue, likely through diseases. the inhibition of Wnt/canonical signalling. Soon after however, it adopts the function of a TypeI procollagen Dual function of Sfrp2 in heart recovery C-proteinase enhancer protein, leading to fibrosis. Thus, The heart has a limited regenerative capacity and the although Sfrp2 has been proposed as a useful agent for heart development of novel regenerative therapies is one of the repair (Barandon et al. 2003; Mirotsou et al. 2007), we need goals of current cardiovascular research. Stem cell based a better understanding of its mechanisms of action to design approaches are quite promising especially in cases of therapeutic strategies that can separate its beneficial from its myocardial infarction (Rameshwar et al. 2010). In fact, harmful effects on damaged myocardial tissue. transplantation of bone marrow-derived stem cells following heart ischemia promotes local recovery and improves the Conclusions general function of the heart (Abdel-Latif et al. 2007). This Here we have focused on the roles of Sfrp1 and 2 in recovery seems in large part due to paracrine factors tumour formation, bone and myocardial disorders. secreted by transplanted stem cells (Gnecchi et al. 2005, Nevertheless, there are several other pathological conditions 2008). Recent studies have demonstrated that Sfrp2 is one in which these molecules have been implicated. For of the most relevant of these factors. Intra-cardiac example, Sfrp1 has been suggested to be among the factors transplantation of mesenchymal stem cells genetically responsible for elevated intraocular pressure in glaucoma engineered to over-express the serine/threonine kinase AKT (Wang et al. 2008). Similarly, inhibition of the elevated led to a dramatic reduction of the infarction size and to the Sfrp1 levels alleviates the inflammation and destruction of recovery of cardiac function in injured rodent hearts (Mangi periodontal tissue that occurs in periodontitis (Li and Amar et al. 2003). Microarray analysis revealed that Sfrp2 is 2007). Furthermore, there are also reports linking other among the upregulated proteins in Akt-mesechymal cells. Sfrp family members to diseases. This is the case, for Sfrp2 seems to be responsible for most of the beneficial example, of Sfrp4 and osteomalacia, a condition in which effects of the transplanted stem cells during early stages of the bones becomes soft and flexible often due to the lack of infarction. It promotes myocardial survival and repair, vitamin D. Unexpectedly, Sfrp4 plays an important role in reduces the infracted size (Mirotsou et al. 2007; Alfaro et al. homeostasis of phosphorus and inorganic phosphate (Pi), 2008) and favor neovascularisation (Blankesteijn et al. inhibiting vitamin D synthesis and thus intestinal Pi absorp- 2001). The beneficial effects in cardiac repair of Sfrp2 are tion (Berndt and Kumar 2007). How this occurs is unclear, shared by Sfrp1. In transgenic mice overexpressing Sfrp1, but it certainly offers an additional example of the pleiotro- infarct size was reduced and cardiac function preserved pic activities of Sfrps. Furthermore, Sfrp3 seems upregulat- (Barandon et al. 2003). Although still unclear, these effects ed in metastatic renal cell cancer, which suggests that inhi- are probably related to the control of Wnt/β catenin bition of Sfrp signaling in this context would not be signalling (Zhang et al. 2009). beneficial to patients (Hirata et al. 2010). After myocardial infarction, dead myocytes are It is thus evident that basic studies of the interactions removed and replaced by a fibrotic scar formed by collagen between the different Sfrp molecules and Wnt-related and deposits. This cardiac fibrosis reduces elasticity and unrelated proteins will be of key importance to understand proportionally decreases the contractile function of the the role of this family of soluble factors in a wide variety of heart. Somewhat in contrast with the idea that Sfrp2 has a diseases. These studies will also help to define whether, beneficial effect on heart recovery, a recent study how and in which conditions Sfrps can be useful therapeutic demonstrated that collagen deposition and fibrosis were targets or therapeutic agents themselves. reduced in healed cardiac tissue of Sfrp2 null mice subjected Sfrp1/2 in Deseases 15

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