Cellular signaling and biological functions of R-spondins

R-spondins (RSPOs) are a family of cysteine-rich secreted proteins containing a single thrombospondin type I repeat (TSR) domain. A vast amount of information regarding cellular signaling and biological functions of RSPOs has emerged over the last several years, especially with respect to their roles in the activation of the WNT signaling pathway. The identification of several classes of RSPO receptors may indicate that this family of proteins can affect several signaling cascades. Herein, we summarize the current understanding of RSPO signaling and its biological functions, and discuss its potential therapeutic implications to human diseases.     

A WNT/beta-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway.     

Uncoupling the BMP receptor antagonist function from the WNT agonist function of R-spondin 2 using the inhibitory peptide dendrimer RWd

Signaling by bone morphogenetic proteins (BMPs) plays pivotal roles in embryogenesis, adult tissue homeostasis, and disease. Recent studies revealed that the well-established WNT agonist R-spondin 2 (RSPO2) is also a BMP receptor (BMP receptor type 1A) antagonist, with roles in early Xenopus embryogenesis and human acute myeloid leukemia (AML). To uncouple the BMP antagonist function from the WNT agonist function and to promote development of AML therapeutics, here we identified a 10-mer peptide (RW) derived from the thrombospondin 1 domain of RSPO2, which specifically prevents binding between RSPO2 and BMP receptor type 1A without altering WNT signaling. We also show that a corresponding RW dendrimer (RW^d) exhibiting improved half-life relieves inhibition of BMP receptor signaling by RSPO2 in human AML cells, reduces cell growth, and induces differentiation. Moreover, microinjection of RW^d in Xenopus embryos ventralizes the dorsoventral embryonic patterning by upregulating BMP signaling without affecting WNT signaling. Our study corroborates the function of RSPO2 as a BMP receptor antagonist and provides a proof of concept for pharmacologically uncoupling BMP antagonist from WNT agonist functions of RSPO2 using the inhibitor peptide RW^d with enhanced target selectivity and limited side effects.     

Regulation of the Follistatin Gene by RSPO-LGR4 Signaling via Activation of the WNT/β-Catenin Pathway in Skeletal Myogenesis

WNT signaling plays multiple roles in skeletal myogenesis during gestation and postnatal stages. The R-spondin (RSPO) family of secreted proteins and their cognate receptors, members of leucine-rich repeat-containing G protein-coupled receptor (LGR) family, have emerged as new regulatory components of the WNT signaling pathway. We previously showed that RSPO2 promoted myogenic differentiation via activation of WNT/β-catenin signaling in mouse myoblast C2C12 cells in vitro. However, the molecular mechanism by which RSPO2 regulates myogenic differentiation is unknown. Herein, we show that depletion of the LGR4 receptor severely disrupts myogenic differentiation and significantly diminishes the response to RSPO2 in C2C12 cells, showing a requirement of LGR4 in RSPO signaling during myogenic differentiation. We identify the transforming growth factor β (TGF-β) antagonist follistatin (Fst) as a key mediator of RSPO-LGR4 signaling in myogenic differentiation. We further demonstrate that Fst is a direct target of the WNT/β-catenin pathway. Activation and inactivation of β-catenin induced and inhibited Fst expression, respectively, in both C2C12 cells and mouse embryos. Specific TCF/LEF1 binding sites within the promoter and intron 1 region of the Fst gene were required for RSPO2 and WNT/β-catenin-induced Fst expression. This study uncovers a molecular cross talk between WNT/β-catenin and TGF-β signaling pivotal in myogenic differentiation.     

RSPO2 enhances cell invasion and migration via the WNT/β-catenin pathway in human gastric cancer

R-spondins comprise a group of secreted WNT agonists. R-spondin2 (RSPO2) plays a crucial role in the activation of the WNT/β-catenin pathway and oncogenesis, though its specific role in human gastric cancer (GC) remains unclear. In the current study, RSPO2 expression levels were upregulated in cancer specimens and cell lines (AGS and BGC-823). Inhibition of RSPO2 expression levels had distinct effects on cell invasion, migration, and epithelial-mesenchymal transition (EMT) in AGS and BGC-823 cells in vitro. Furthermore, RSPO2 positively correlated with leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), the receptor of RSPO2. Silencing RSPO2 reduced the expression of LGR5 and WNT/β-catenin effector molecule β-catenin together with downstream targets TCF-4 and Cyclin-D1. These observations demonstrate that upregulation of RSPO2 in GC specimens and cell lines is closely related to tumor invasion and migration and that RSPO2 promotes EMT in gastric cancer cells by activating WNT/β-catenin signaling.     

Human RSPO1/R-spondin1 is expressed during early ovary development and augments β-catenin signaling

Human testis development starts from around 42 days post conception with a transient wave of SRY expression followed by up-regulation of testis specific genes and a distinct set of morphological, paracrine and endocrine events. Although anatomical changes in the ovary are less marked, a distinct sub-set of ovary specific genes are also expressed during this time. The furin-domain containing peptide R-spondin1 (RSPO1) has recently emerged as an important regulator of ovary development through up-regulation of the WNT/β-catenin pathway to oppose testis formation. Here, we show that RSPO1 is upregulated in the ovary but not in the testis during critical early stages of gonad development in humans (between 6-9 weeks post conception), whereas the expression of the related genes WNT4 and CTNNB1 (encoding β catenin) is not significantly different between these tissues. Furthermore, reduced R-spondin1 function in the ovotestis of an individual (46,XX) with a RSPO1 mutation leads to reduced β-catenin protein and WNT4 mRNA levels, consistent with down regulation of ovarian pathways. Transfection of wild-type RSPO1 cDNA resulted in weak dose-dependent activation of a β-catenin responsive TOPFLASH reporter (1.8 fold maximum), whereas co-transfection of CTNNB1 (encoding β-catenin) with RSPO1 resulted in dose-dependent synergistic augmentation of this reporter (approximately 10 fold). Furthermore, R-spondin1 showed strong nuclear localization in several different cell lines. Taken together, these data show that R-spondin1 is upregulated during critical stages of early human ovary development and may function as a tissue-specific amplifier of β-catenin signaling to oppose testis determination.     

Receptor tyrosine kinases activate canonical WNT/β-catenin signaling via MAP kinase/LRP6 pathway and direct β-catenin phosphorylation

Receptor tyrosine kinase signaling cooperates with WNT/β-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/β-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate β-catenin at Tyr142, which is known to increase cytoplasmic β-catenin concentration via release of β-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct β-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling.     

RSPO1/β-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary

Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog gene and factors synthesized by the somatic Sertoli cells promote gonocyte differentiation. In the female, ovarian differentiation requires activation of the WNT/β-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/β-catenin signaling pathway in germ cells. In XX Rspo1(-/-) gonads, germ cell proliferation, expression of the early meiotic marker Stra8, and entry into meiosis are all impaired. In these gonads, impaired entry into meiosis and germ cell sex reversal occur prior to detectable Sertoli cell differentiation, suggesting that β-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/β-catenin signaling is involved in meiosis in fetal germ cells and contributes to the cellular decision of germ cells to differentiate into oocyte or sperm.     

R-Spondin potentiates Wnt/β-catenin signaling through orphan receptors LGR4 and LGR5

The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.     

WNT/β-Catenin Signaling Regulates Multiple Steps of Myogenesis by Regulating Step-Specific Targets

Molecules involved in WNT/β-catenin signaling show specific spatiotemporal expression and play vital roles in myogenesis; however, it is still largely unknown how WNT/β-catenin signaling regulates each step of myogenesis. Here, we show that WNT/β-catenin signaling can control diverse biological processes of myogenesis by regulating step-specific molecules. In order to identify the temporally specific roles of WNT/β-catenin signaling molecules in muscle development and homeostasis, we used in vitro culture systems for both primary mouse myoblasts and C2C12 cells, which can differentiate into myofibers. We found that a blockade of WNT/β-catenin signaling in the proliferating cells decreases proliferation activity, but does not induce cell death, through the regulation of genes cyclin A2 (Ccna2) and cell division cycle 25C (Cdc25c). During muscle differentiation, the inhibition of WNT/β-catenin signaling blocks myoblast fusion through the inhibition of the Fermitin family homolog 2 (Fermt2) gene. Blocking WNT/β-catenin signaling in the well-differentiated myofibers results in the failure of maintenance of their structure by disruption of cadherin/β-catenin/actin complex formation, which plays a crucial role in connecting a myofiber''s cytoskeleton to the surrounding extracellular matrix. Thus, our results indicate that WNT/β-catenin signaling can regulate multiple steps of myogenesis, including cell proliferation, myoblast fusion, and homeostasis, by targeting step-specific molecules.     

The PTK7 and ROR2 Protein Receptors Interact in the Vertebrate WNT/Planar Cell Polarity (PCP) Pathway

The non-canonical WNT/planar cell polarity (WNT/PCP) pathway plays important roles in morphogenetic processes in vertebrates. Among WNT/PCP components, protein tyrosine kinase 7 (PTK7) is a tyrosine kinase receptor with poorly defined functions lacking catalytic activity. Here we show that PTK7 associates with receptor tyrosine kinase-like orphan receptor 2 (ROR2) to form a heterodimeric complex in mammalian cells. We demonstrate that PTK7 and ROR2 physically and functionally interact with the non-canonical WNT5A ligand, leading to JNK activation and cell movements. In the Xenopus embryo, Ptk7 functionally interacts with Ror2 to regulate protocadherin papc expression and morphogenesis. Furthermore, we show that Ptk7 is required for papc activation induced by Wnt5a. Interestingly, we find that Wnt5a stimulates the release of the tagged Ptk7 intracellular domain, which can translocate into the nucleus and activate papc expression. This study reveals novel molecular mechanisms of action of PTK7 in non-canonical WNT/PCP signaling that may promote cell and tissue movements.     

Regulatory mechanisms and functions of MAP kinase signaling pathways

Mitogen-activated protein kinase (MAPK) pathways play central roles in controlling diverse cellular functions. They are finely regulated by several mechanisms, including scaffolding of their components, and phosphorylation/dephosphorylation and compartmentalization of MAPKs. A number of molecules have been identified as regulators involved in these mechanisms. They modulate the magnitude and the specificity of MAPK signaling, and thereby regulate the wide variety of signaling outputs. Recent studies have identified novel functions of the MAPK signaling pathways. It is becoming clear that strict regulation of the MAPK pathways underlies their manifold functions in numerous biological processes.     

On how CCN6 suppresses breast cancer growth and invasion

Living cells communicate with their microenvironment and exchange information through signaling pathways in order to carry out most biological processes. The CCN family of proteins has the ability to coordinate the extracellular and intracellular signaling pathways and epithelial-stromal cross-talks. CCN proteins have been shown to play roles in multiple processes including cancer, either as tumor suppressors or oncogenes. Particularly, loss of CCN6 expression has been reported in highly aggressive breast cancer types, especially in inflammatory breast cancer and breast cancer with axillary lymph node metastasis. Recent findings can better explain the biological relevance of CCN6 as a tumor suppressor protein in breast tumorigenesis. CCN6 loss triggers the process of epithelial to mesenchymal transition (EMT), which converts epithelial cells into migratory and invasive mesenchymal-like cells at least in part through modulation of IGF-1 receptor signaling pathway. Emerging data support the hypothesis that CCN6 also exerts growth factor independent functions, especially related to cell survival and anoikis resistance. Thus, our work provides new insights into the functions and mechanisms of tumor suppression exerted by CCN6 in the breast.     

冷诱导RNA结合蛋白的生物学功能与信号通路

冷诱导RNA结合蛋白（ cold-inducible RNA-binding protein, CIRBP）是哺乳动物体内发现的第一个冷诱导蛋白。这种蛋白质在机体内各个组织与器官中均广泛表达,并在正常生理状态或应激条件下,广泛参与多个生物学过程,例如细胞增殖、发展、凋亡、分化和生物节律调节等多个方面。随着研究的深入,发现CIRBP具有一些新的功能,例如在一些炎症的发生和肿瘤的发生过程中,起到促进作用与作为新一代的原癌基因等。CIRBP发挥作用的信号通路,主要有胞外信号调节激酶/丝裂原活化蛋白激酶(extracellular signal-regulated kinases/mitogen-activated protein kinases, ERK/MAPK)、磷脂酰肌醇3激酶/蛋白激酶B(phosphatidylinositol 3-kinase/protein kinase B, PI3K/PKB)、无翅和整合基因(wingless and integration 1,Wnt)、核因子κB(nuclear factor κB, NF-κB)等。本文针对CIRBP的生物学功能和相关信号通路的最新研究进展加以综述,希望能为细胞生物学基础研究与利用该蛋白质进行临床有关疾病的诊治提供新的思路。     

经典WNT信号通路与哺乳动物肺器官发育

肺器官发育是上皮和问充质相互作用的过程,由多条信号通路共同调控。已知经典WNT信号通路对细胞的增殖、凋亡和分化起着重要的调控作用,在小鼠等模式生物上研究发现,它也参与了哺乳动物肺器官发育的调控过程。综述近年来经典WNT信号通路成员在哺乳动物肺器官发育过程中的表达变化、作用功能及表达异常可能诱发的肺部疾病,以期为研究经典WNT信号通路调控人类肺器官发育的分子机制及相关肺部疾病的诊治奠定基础。     

Epigenome-Metabolome-Epigenome signaling cascade in cell biological processes

Cell fate determination as a fundamental question in cell biology has been extensively studied at different regulatory levels for many years. However, the mechanisms of multilevel regulation of cell fate determination remain unclear. Recently, we have proposed an Epigenome-Metabolome-Epigenome (E-M-E) signaling cascade model to describe the cross-over cooperation during mouse somatic cell reprogramming. In this review, we summarize the broad roles of E-M-E signaling cascade in different cell biological processes, including cell differentiation and dedifferentiation, cell specialization, cell proliferation, and cell pathologic processes. Precise E-M-E signaling cascades are critical in these cell biological processes, and it is of worth to explore each step of E-M-E signaling cascade. E-M-E signaling cascade model sheds light on and may open a window to explore the mechanisms of multilevel regulation of cell biological processes.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4-/- and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

Histone deacetylase 10, a potential epigenetic target for therapy

Histone deacetylase (HDAC) 10, a class II family, has been implicated in various tumors and non-tumor diseases, which makes the discovery of biological functions and novel inhibitors a fundamental endeavor. In cancers, HDAC10 plays crucial roles in regulating various cellular processes through its epigenetic functions or targeting some decisive molecular or signaling pathways. It also has potential clinical utility for targeting tumors and non-tumor diseases, such as renal cell carcinoma, prostate cancer, immunoglobulin A nephropathy (IgAN), intracerebral hemorrhage, human immunodeficiency virus (HIV) infection and schizophrenia. To date, relatively few studies have investigated HDAC10-specific inhibitors. Therefore, it is important to study the biological functions of HDAC10 for the future development of specific HDAC10 inhibitors. In this review, we analyzed the biological functions, mechanisms and inhibitors of HDAC10, which makes HDAC10 an appealing therapeutic target.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4+/+ and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.