Isthmin: A multifunctional secretion protein.

Isthmin is a polypeptide secreted by adipocytes that was first detected in Xenopus gastrula embryos. Recent studies have focused on the biological functions of isthmin in growth and development, angiogenesis, and metabolism. Distinct spatiotemporal expression of isthmin-1 (ISM-1) was observed during growth and development. ISM-1 plays an important role in the occurrence and development of cancer by regulating cell proliferation, migration, angiogenesis, and immune microenvironments. Moreover, ISM-1, as a newly identified insulin-like adipokine, increases adipocyte glucose uptake and inhibits hepatic lipid synthesis. However, the biological function of ISM-1 remains largely unknown. In this review, we highlight the structure and physiological functions of isthmin and explore its application potential, contributing to a better understanding of its function and providing prevention and treatment strategies for various diseases.

Identification of adhesion-associated extracellular matrix component thrombospondin 3 as a prognostic signature for clear cell renal cell carcinoma.

PURPOSE: Clear cell renal cell carcinoma (ccRCC) is a highly aggressive disease, and approximately 30% of patients are diagnosed at the metastatic stage. Even with targeted therapies, the prognosis of advanced ccRCC is poor. The aim of this study was to investigate clinical prognosis signatures by analyzing the ccRCC datasets in The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the function of thrombospondin 3 (THBS3) in ccRCC. MATERIALS AND METHODS: We analyzed the ccRCC datasets in TCGA and CPTAC to search for extracellular matrix (ECM)-related and adhesion-associated genes, and conducted overall survival, Cox, and receiver operating characteristic analyses. We also performed CCK8, colony formation, and transwell assays to compared the proliferation and migration ability of THBS3 knockout cells with those of cells without THBS3 knockout. RESULTS: Comprehensive bioinformatics analysis revealed that THBS3 is a novel candidate oncogene that is overexpressed in ccRCC tumor tissue and that its elevated expression indicates poor prognosis. Our study also showed that knockdown of THBS3 inhibits proliferation, colony formation, and migration of ccRCC cells. CONCLUSIONS: In summary, our data have revealed that THBS3 is upregulated in cancer tissues and could be used as a novel prognostic marker for ccRCC. Our findings thus offer theoretical support with bioinformatics analyses to the study of ECM and adhesion proteins in ccRCC, which may provide a new perspective for the clinical management of ccRCC.

Functions of Matricellular Proteins in Dental Tissues and Their Emerging Roles in Orofacial Tissue Development, Maintenance, and Disease.

Matricellular proteins (MCPs) are defined as extracellular matrix (ECM) associated proteins that are important regulators and integrators of microenvironmental signals, contributing to the dynamic nature of ECM signalling. There is a growing understanding of the role of matricellular proteins in cellular processes governing tissue development as well as in disease pathogenesis. In this review, the expression and functions of different MP family members (periostin, CCNs, TSPs, SIBLINGs and others) are presented, specifically in relation to craniofacial development and the maintenance of orofacial tissues, including bone, gingiva, oral mucosa, palate and the dental pulp. As will be discussed, each MP family member has been shown to have non-redundant roles in development, tissue homeostasis, wound healing, pathology and tumorigenesis of orofacial and dental tissues.

Contribution of Thrombospondin-1 and -2 to Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome.

Thrombospondin (TSP) proteins have been shown to impact T-cell adhesion, migration, differentiation, and apoptosis. Thrombospondin-1 (TSP-1) is specifically upregulated in several inflammatory diseases and can effectively promote lipopolysaccharide- (LPS-) induced inflammation. In contrast, thrombospondin-2 (TSP-2) has been associated with activation of "anti-inflammatory" T-regulatory cells (Tregs). In this study, we investigated the effects of both TSP-1 and TSP-2 overexpression on macrophage polarization and activation in vitro and in vivo. We analyzed the effects of TSP-1 and TSP-2 on inflammation, vascular endothelial permeability, edema, ultrastructural morphology, and apoptosis in lung tissues of an ARDS mouse model and cultured macrophages. Our results demonstrated that TSP-2 overexpression effectively attenuated LPS-induced ARDS in vivo and promoted M2 macrophage phenotype polarization in vitro. Furthermore, TSP-2 played a role in regulating pulmonary vascular barrier leakage by activating the PI3K/Akt pathway. Overall, our findings indicate that TSP-2 can modulate inflammation and could therefore be a potential therapeutic target against LPS-induced ARDS.

Targeting the Wnt signaling pathway through R-spondin 3 identifies an anti-fibrosis treatment strategy for multiple organs.

The Wnt/ß-catenin signaling pathway has been implicated in human proliferative diseases such as cancer and fibrosis. The functions of ß-catenin and several other components of this pathway have been investigated in fibrosis. However, the potential role of R-spondin proteins (RSPOs), enhancers of the Wnt/ß-catenin signaling, has not been described. A specific interventional strategy targeting this pathway for fibrosis remains to be defined. We developed monoclonal antibodies against members of the RSPO family (RSPO1, 2, and 3) and probed their potential function in fibrosis in vivo. We demonstrated that RSPO3 plays a critical role in the development of fibrosis in multiple organs. Specifically, an anti-RSPO3 antibody, OMP-131R10, when dosed therapeutically, attenuated fibrosis in carbon tetrachloride (CCl4)-induced liver fibrosis, bleomycin-induced pulmonary and skin fibrosis models. Mechanistically, we showed that RSPO3 induces multiple pro-fibrotic chemokines and cytokines in Kupffer cells and hepatocytes. We found that the anti-fibrotic activity of OMP-131R10 is associated with its inhibition of ß-catenin activation in vivo. Finally, RSPO3 was found to be highly elevated in the active lesions of fibrotic tissues in mouse models of fibrosis and in patients with idiopathic pulmonary fibrosis (IPF) and nonalcoholic steatohepatitis (NASH). Together these data provide an anti-fibrotic strategy for targeting the Wnt/ß-catenin pathway through RSPO3 blockade and support that OMP-131R10 could be an important therapeutic agent for fibrosis.

Extracellular matrix: the gatekeeper of tumor angiogenesis.

The extracellular matrix is a network of secreted macromolecules that provides a harmonious meshwork for the growth and homeostatic development of organisms. It conveys multiple signaling cascades affecting specific surface receptors that impact cell behavior. During cancer growth, this bioactive meshwork is remodeled and enriched in newly formed blood vessels, which provide nutrients and oxygen to the growing tumor cells. Remodeling of the tumor microenvironment leads to the formation of bioactive fragments that may have a distinct function from their parent molecules, and the balance among these factors directly influence cell viability and metastatic progression. Indeed, the matrix acts as a gatekeeper by regulating the access of cancer cells to nutrients. Here, we will critically evaluate the role of selected matrix constituents in regulating tumor angiogenesis and provide up-to-date information concerning their primary mechanisms of action.

Molecular classification of membranous nephropathy.

PURPOSE OF REVIEW: Continuous expansion of our knowledge in the pathogenesis of membranous nephropathy possible by the identification of antibodies recognized specific podocytes antigens results in unprecedent patient management strategy. RECENT FINDINGS: Circulating anti-phospholipase A2 receptor (PLA2R) and anti-thrombospondin domain 7A (THSD7A) antibodies strongly relate with the modifications of podocytes biology leading to the new molecular diagnosis of membranous nephropathy. Immunization against THSD7A involves extra-renal mechanism. However, the pathway of anti-PLA2R immunization still remains unresolved. Experimental data highlight the crucial role of THSD7A in the attachment of podocytes to the glomerular basement membrane, rewarding the THSD7A pathogenicity, whereas the third of Koch's postulates is still not fulfilled for anti-PLA2R antibodies. The anti-PLA2R antibodies epitope spreading will possibly be even more specific marker improving the molecular classification of membranous nephropathy. Two immune epitopes have been identified in the N-terminal tail of THSD7A but without evidence of epitope spreading as for anti-PLA2R. SUMMARY: In 2019, the Kidney Diseases: Improving Global Outcomes guidelines recognized anti-PLA2R antibodies (but not anti-THSD7A antibodies) as a valuable molecular risk factor for the pejorative evolution of kidney function and recommended their monitoring for the diagnosis and the assessment of membranous nephropathy immune activity. Screening for malignancy is particularly advised in THSD7A-mediated membranous nephropathy.

MiR-222 inhibits apoptosis in porcine follicular granulosa cells by targeting the THBS1 gene.

Apoptosis of granulosa cells affects follicular atresia and reproduction and is regulated by miRNAs and the expression of certain genes. For the present study, we investigated the regulatory relationship between microRNA-222 (miR-222) and THBS1 in porcine follicular granulosa cells (pGCs) and its effects on apoptosis to provide empirical data for developing methods to improve pig fecundity. Results revealed that miR-222 promotes the proliferation of pGCs. MiRNA mimics and luciferase reporter assays revealed that miR-222 functions as an anti-apoptotic factor in pGCs. MiR-222 mimics in pGCs result in the upregulation of the anti-apoptotic BCL-2 gene, down-regulation of the proapoptotic caspase-3 gene, and inhibition of apoptosis. MiR-222 inhibitors reduced BCL-2 and had no significant effect on caspase-3. MiR-222 mimics promoted estrogen levels. Inhibition of THBS1 inhibited pGC apoptosis. Transfection of THBS1-siRNA reduced the proapoptotic BAX gene. MiR-222 can directly target the 3'-untranslated region of the THBS1 gene. MiR-222 mimics suppressed THBS1 mRNA and proteins, but these were upregulated by the miR-222 inhibitor. Transfection of THBS1-siRNA resulted in the inhibition of the miR-222 inhibitor, which suggests that miR-222 inhibits pGC apoptosis by targeting THBS1. These findings suggest that miR-222 and THBS1 play important roles in follicular atresia, ovarian development, and female reproduction.

LGR4 is essential for R-spondin1-mediated suppression of food intake via pro-opiomelanocortin.

Leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) suppresses food intake after its activation by binding of its ligands, R-spondins. We investigated the mechanism of food intake suppression by R-spondin1 in a region-specific Lgr4 gene knockout (LGR4 cKO) mouse model, generated by deletion of the Lgr4 gene in arcuate nucleus (ARC) using Lgr4fx/fx mice combined with infection of an AAV-Cre vector. After R-spondin1 administration, LGR4 cKO mice didn't exhibit a suppressed appetite, compared to that in control mice, which received a vehicle. In ARC of LGR4 cKO mice, Pomc mRNA expression was reduced, leading to suppressed food intake. On the other hand, neurons-specific LGR4 KO mice exhibited no differences in Pomc expression, and no structural differences were observed in the ARC of mutant mice. These results suggest that LGR4 is an essential part of the mechanism, inducing Pomc gene expression with R-spondin1 in ARC neurons in mice, thereby regulating feeding behavior. Abbreviations: LGR4: Leucine-rich repeat-containing G-protein coupled receptor 4; RSPOs: roof plate-specific spondins; ARC: arcuate nucleus; AAV: adeno associated virus; POMC: pro-opiomelanocortin; CART: cocaine and amphetamine-regulated transcript; NPY: neuropeptide Y; AgRP: agouti-related peptide; Axin2: axis inhibition protein 2; Lef1: lymphoid enhancer binding factor 1; ccnd1: cyclin D1.

Recent advances in understanding the roles of matrix metalloproteinases in tumour invasion and metastasis.

This review aims to provide an overview of recent developments regarding the roles of MMPs in tumour invasion and metastasis. Much of the mortality burden belonging to cancer relates to its ability to invade adjacent tissue and form metastases at distant sites. This would not be possible without remodelling of the ECM, a process which is enabled by the functions of MMPs. Recent studies provide a better understanding of the importance of the biophysical nature of the ECM, how this influences cancer cell motility, and how MMPs act to modify matrix stiffness. The regulation of MMPs and the role of immune cell generated MMPs has also become better understood. All of this provides a framework for the therapeutic targeting of MMPs and recent advances in the development of selective MMPs inhibitors are also reviewed. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The role of R-spondin 1 through activating Wnt/ß-catenin in the growth, survival and migration of ovarian cancer cells.

Aberrant activation of the Wnt/ß-catenin has been shown to promote progression in various cancers, including ovarian cancer. However, the molecular mechanisms involved in Wnt/ß-catenin activation are not well elucidated. In the work, we identify that R-spondin 1 is an upstream regulator in Wnt/ß-catenin pathway to promote growth, survival and migration in ovarian cancer cells. We observe the upregulation of transcript and protein levels of R-spondin 1 in ovarian cancer cell lines and tissues compared to normal counterparts. R-spondin 1 upregulation via genetic (overexpression) and pharmacological (recombinant protein) approaches facilitates growth and migration of normal ovarian cells. R-spondin 1 downregulation via siRNA knockdown decreases proliferation and migration, and induces apoptosis in ovarian cancer cells. In addition, recombinant R-spondin 1 protects ovarian cancer cell against chemotherapy whereas R-spondin 1 knockdown sensitizes ovarian cancer cell response to chemotherapy. Importantly, increased ß-catenin activities and mRNA expression levels of Wnt/ß-catenin-targeted genes are detected in normal ovarian cells overexpressing R-spondin 1. In contrast, R-spondin 1 inhibition suppresses Wnt/ß-catenin signaling in ovarian cancer cells. We further identify that R-spondin 1 regulates ovarian cancer biological activities via activating Wnt/ß-catenin. Our work is the first to highlight the critical roles of R-spondin 1 in ovarian cancer progression and chemoresistance. Our work also provides a proper understanding on the regulation of Wnt/ß-catenin pathway in ovarian cancer.

Human Properdin Modulates Macrophage: Mycobacterium bovis BCG Interaction via Thrombospondin Repeats 4 and 5.

Mycobacterium tuberculosis can proficiently enter macrophages and diminish complement activation on its cell surface. Within macrophages, the mycobacterium can suppress macrophage apoptosis and survive within the intracellular environment. Previously, we have shown that complement regulatory proteins such as factor H may interfere with pathogen-macrophage interactions during tuberculosis infection. In this study, we show that Mycobacterium bovis BCG binds properdin, an upregulator of the complement alternative pathway. TSR4+5, a recombinant form of thrombospondin repeats 4 and 5 of human properdin expressed in tandem, which is an inhibitor of the alternative pathway, was also able to bind to M. bovis BCG. Properdin and TSR4+5 were found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Quantitative real-time PCR revealed elevated pro-inflammatory responses (TNF-α, IL-1ß, and IL-6) in the presence of properdin or TSR4+5, which gradually decreased over 6 h. Correspondingly, anti-inflammatory responses (IL-10 and TGF-ß) showed suppressed levels of expression in the presence of properdin, which gradually increased over 6 h. Multiplex cytokine array analysis also revealed that properdin and TSR4+5 significantly enhanced the pro-inflammatory response (TNF-α, IL-1ß, and IL-1α) at 24 h, which declined at 48 h, whereas the anti-inflammatory response (IL-10) was suppressed. Our results suggest that properdin may interfere with mycobacterial entry into macrophages via TSR4 and TSR5, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. This study offers novel insights into the non-complement related functions of properdin during host-pathogen interactions in tuberculosis.

Negative regulators of angiogenesis: important targets for treatment of exudative AMD.

Angiogenesis contributes to the pathogenesis of many diseases including exudative age-related macular degeneration (AMD). It is normally kept in check by a tightly balanced production of pro- and anti-angiogenic factors. The up-regulation of the pro-angiogenic factor, vascular endothelial growth factor (VEGF), is intimately linked to the pathogenesis of exudative AMD, and its antagonism has been effectively targeted for treatment. However, very little is known about potential changes in expression of anti-angiogenic factors and the role they play in choroidal vascular homeostasis and neovascularization associated with AMD. Here, we will discuss the important role of thrombospondins and pigment epithelium-derived factor, two major endogenous inhibitors of angiogenesis, in retinal and choroidal vascular homeostasis and their potential alterations during AMD and choroidal neovascularization (CNV). We will review the cell autonomous function of these proteins in retinal and choroidal vascular cells. We will also discuss the potential targeting of these molecules and use of their mimetic peptides for therapeutic development for exudative AMD.

Thrombospondin-4 mediates TGF-ß-induced angiogenesis.

TGF-ß is a multifunctional cytokine affecting many cell types and implicated in tissue remodeling processes. Due to its many functions and cell-specific effects, the consequences of TGF-ß signaling are process-and stage-dependent, and it is not uncommon that TGF-ß exerts distinct and sometimes opposing effects on a disease progression depending on the stage and on the pathological changes associated with the stage. The mechanisms underlying cell- and process-specific effects of TGF-ß are poorly understood. We are describing a novel pathway that mediates induction of angiogenesis in response to TGF-ß1. We found that in endothelial cells (EC) thrombospondin-4 (TSP-4), a secreted extracellular matrix (ECM) protein, is upregulated in response to TGF-ß1 and mediates the effects of TGF-ß1 on angiogenesis. Upregulation of TSP-4 does not require the synthesis of new protein, is not caused by decreased secretion of TSP-4, and is mediated by activation of SMAD3. Using Thbs4-/- mice and TSP-4 shRNA, we found that TSP-4 mediated pro-angiogenic functions in cultured EC and angiogenesis in vivo in response to TGF-ß1. We observed~3-fold increases in tumor mass and levels of angiogenesis markers in animals injected with TGF-ß1, and these effects did not occur in Thbs4-/- animals. Injections of an inhibitor of TGF-ß1 signaling SB-431542 also decreased the weights of tumors and cancer angiogenesis. Our results from in vivo angiogenesis models and cultured EC document that TSP-4 mediates upregulation of angiogenesis by TGF-ß1. Upregulation of pro-angiogenic TSP-4 and selective effects of TSP-4 on EC may contribute to stimulation of tumor growth by TGF-ß despite the inhibition of cancer cell proliferation.

Thrombospondin-2 promotes prostate cancer bone metastasis by the up-regulation of matrix metalloproteinase-2 through down-regulating miR-376c expression.

BACKGROUND: Thrombospondin-2 (TSP-2) is a secreted matricellular glycoprotein that is found to mediate cell-to-extracellular matrix attachment and participates in many physiological and pathological processes. The expression profile of TSP-2 on tumors is controversial, and it up-regulates in some cancers, whereas it down-regulates in others, suggesting that the functional role of TSP-2 on tumors is still uncertain. METHODS: The expression of TSP-2 on prostate cancer progression was determined in the tissue array by the immunohistochemistry. The molecular mechanism of TSP-2 on prostate cancer (PCa) metastasis was investigated through pharmaceutical inhibitors, siRNAs, and miRNAs analyses. The role of TSP-2 on PCa metastasis in vivo was verified through xenograft in vivo imaging system. RESULTS: Based on the gene expression omnibus database and immunohistochemistry, we found that TSP-2 increased with the progression of PCa, especially in metastatic PCa and is correlated with the matrix metalloproteinase-2 (MMP-2) expression. Additionally, through binding to CD36 and integrin ανß3, TSP-2 increased cell migration and MMP-2 expression. With inhibition of p38, ERK, and JNK, the TSP-2-induced cell migration and MMP-2 expression were abolished, indicating that the TSP-2's effect on PCa is MAPK dependent. Moreover, the microRNA-376c (miR-376c) was significantly decreased by the TSP-2 treatment. Furthermore, the TSP-2-induced MMP-2 expression and the subsequent cell motility were suppressed upon miR-376c mimic stimulation. On the other hand, the animal studies revealed that the bone metastasis was abolished when TSP-2 was stably knocked down in PCa cells. CONCLUSIONS: Taken together, our results indicate that TSP-2 enhances the migration of PCa cells by increasing MMP-2 expression through down-regulation of miR-376c expression. Therefore, TSP-2 may represent a promising new target for treating PCa.

Nkx2-5 regulates cardiac growth through modulation of Wnt signaling by R-spondin3.

A complex regulatory network of morphogens and transcription factors is essential for normal cardiac development. Nkx2-5 is among the earliest known markers of cardiac mesoderm that is central to the regulatory pathways mediating second heart field (SHF) development. Here, we have examined the specific requirements for Nkx2-5 in the SHF progenitors. We show that Nkx2-5 potentiates Wnt signaling by regulating the expression of the R-spondin3 (Rspo3) gene during cardiogenesis. R-spondins are secreted factors and potent Wnt agonists that in part regulate stem cell proliferation. Our data show that Rspo3 is markedly downregulated in Nkx2-5 mutants and that Rspo3 expression is regulated by Nkx2-5. Conditional inactivation of Rspo3 in the Isl1 lineage resulted in embryonic lethality secondary to impaired development of SHF. More importantly, we find that Wnt signaling is significantly attenuated in Nkx2-5 mutants and that enhancing Wnt/ß-catenin signaling by pharmacological treatment or by transgenic expression of Rspo3 rescues the SHF defects in the conditional Nkx2-5(+/-) mutants. We have identified a previously unrecognized genetic link between Nkx2-5 and Wnt signaling that supports continued cardiac growth and proliferation during development. Identification of Rspo3 in cardiac development provides a new paradigm in temporal regulation of Wnt signaling by cardiac-specific transcription factors.

Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

Thrombospondin-2 secreted by human umbilical cord blood-derived mesenchymal stem cells promotes chondrogenic differentiation.

Increasing evidence indicates that the secretome of mesenchymal stem cells (MSCs) has therapeutic potential for the treatment of various diseases, including cartilage disorders. However, the paracrine mechanisms underlying cartilage repair by MSCs are poorly understood. Here, we show that human umbilical cord blood-derived MSCs (hUCB-MSCs) promoted differentiation of chondroprogenitor cells by paracrine action. This paracrine effect of hUCB-MSCs on chondroprogenitor cells was increased by treatment with synovial fluid (SF) obtained from osteoarthritis (OA) patients but was decreased by SF of fracture patients, compared to that of an untreated group. To identify paracrine factors underlying the chondrogenic effect of hUCB-MSCs, the secretomes of hUCB-MSCs stimulated by OA SF or fracture SF were analyzed using a biotin label-based antibody array. Among the proteins increased in response to these two kinds of SF, thrombospondin-2 (TSP-2) was specifically increased in only OA SF-treated hUCB-MSCs. In order to determine the role of TSP-2, exogenous TSP-2 was added to a micromass culture of chondroprogenitor cells. We found that TSP-2 had chondrogenic effects on chondroprogenitor cells via PKCα, ERK, p38/MAPK, and Notch signaling pathways. Knockdown of TSP-2 expression on hUCB-MSCs using small interfering RNA abolished the chondrogenic effects of hUCB-MSCs on chondroprogenitor cells. In parallel with in vitro analysis, the cartilage regenerating effect of hUCB-MSCs and TSP-2 was also demonstrated using a rabbit full-thickness osteochondral-defect model. Our findings suggested that hUCB-MSCs can stimulate the differentiation of locally presented endogenous chondroprogenitor cells by TSP-2, which finally leads to cartilage regeneration.

Thrombospondin-2 facilitates assembly of a type-I collagen-rich matrix in marrow stromal cells undergoing osteoblastic differentiation.

We examined the effects of Thrombospondin-2 (TSP2) deficiency on assembly of collagenous extracellular matrix (ECM) by primary marrow-derived mesenchymal stromal cells (MSC) undergoing osteoblast differentiation in culture. After 30 d, wild-type cells had accumulated and mineralized a collagen-rich insoluble matrix, whereas the TSP2-null cultures contained markedly lower amounts of matrix collagen and displayed reduced mineral. Differences in matrix collagen were seen as early as day 9, at which time wild-type cultures contained more total collagen per cell than did TSP2-null cells. Collagen was unevenly distributed amongst different extracellular compartments in the two cell-types. Collagen levels in conditioned medium of wild-type cells were higher than those of TSP2-null cells, but were roughly equivalent in the acid-soluble, newly cross-linked matrixes. Conversely, the mature, cross-linked acid-insoluble matrix layer of wild-type cells contained about twice as much collagen as TSP2-null cell-derived matrix. Western blot analysis of type-I collagen in detergent-soluble and insoluble matrix fractions supported the premise that matrix collagen levels were reduced in TSP2-null MSC undergoing osteoblastic differentiation in vitro. Western blot and immunofluorescent analysis suggested that assembly of fibronectin into matrix was not affected by TSP2 deficiency. Instead, western blots of conditioned medium demonstrated a marked reduction in mature, fully processed type-I collagen in the absence of TSP2. Our data suggest that in the context of osteoblast differentiation, TSP2 promotes the assembly of a type-I collagen-rich matrix by facilitating pro-collagen processing.

Oocyte-derived R-spondin2 promotes ovarian follicle development.

R-spondin proteins are adult stem cell growth factors capable of stimulating gut development by activating LGR4, 5, and 6 receptors to promote Wnt signaling. Although multiple Wnt ligands and cognate Frizzled receptors are expressed in the ovary, their physiological roles are unclear. Based on bioinformatic and in situ hybridization analyses, we demonstrated the exclusive expression of R-spondin2 in oocytes of ovarian follicles. In cultured somatic cells from preantral follicles, R-spondin2 treatment (ED50: 3 ng/ml) synergized with Wnt3a to stimulate Wnt signaling. In cultured ovarian explants from prepubertal mice containing preantral follicles, treatment with R-spondin2, similar to follicle stimulating hormone, promoted the development of primary follicles to the secondary stage. In vivo administration of an R-spondin agonist stimulated the development of primary follicles to the antral stage in both immature mice and gonadotropin releasing hormone antagonist-treated adult mice. Subsequent treatment with gonadotropins allowed the generation of mature oocytes capable of undergoing early embryonic development and successful pregnancy. Furthermore, R-spondin agonist treatment of immune-deficient mice grafted with human cortical fragments stimulated the development of primary follicles to the secondary stage. Thus, oocyte-derived R-spondin2 is a paracrine factor essential for primary follicle development, and R-spondin agonists could provide a new treatment regimen for infertile women with low responses to the traditional gonadotropin therapy.