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Introduction: Colorectal cancer (CRC) is one of the most deadly and common diseases in the world, accounting for over 881,000 casualties in 2018. The PTPRK-RSPO3 (P:R) fusion is a structural variation in CRC and well known for its ability to activate WNT signaling and tumorigenesis. However, till now, therapeutic targets and actionable drugs are limited in this subtype of cancer., Materials and Method: The purpose of this study is to identify key genes and cancer-related pathways specific for P:R fusion-positive CRC. In addition, we also inferred the actionable drugs in bioinformatics analysis using the Cancer Genome Atlas (TCGA) data., Results: 2,505 genes were altered in RNA expression specific for P:R fusion-positive CRC. By pathway analysis based on the altered genes, ten major cancer-related signaling pathways (Apoptosis, Direct p53, EGFR, ErbB, JAK-STAT, tyrosine kinases, Pathways in Cancer, SCF-KIT, VEGFR, and WNT-related Pathway) were significantly altered in P:R fusion-positive CRC. Among these pathways, the most altered cancer genes (ALK, ACSL3, AXIN, MYC, TP53, GNAQ, ACVR2A, and FAS) specific for P:R fusion and involved in multiple cancer pathways were considered to have a key role in P:R fusion-positive CRC. Based on the drug-target network analysis, crizotinib, alectinib, lorlatinib, brigatinib, ceritinib, erdafitinib, infigratinib and pemigatinib were selected as putative therapeutic candidates, since they were already used in routine clinical practice in other cancer types and target genes of the drugs were involved in multiple cancer-pathways., Competing Interests: The authors have declared that no competing interests exist.

Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/β-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of 'surrogate RSPOs' that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine., Competing Interests: We have read the journal's policy and the authors of this manuscript have the following competing interests: K.C.G. is a founder and scientific advisor of Surrozen, Inc. K.C.G. and V.C.L. are inventors on a patent (WO2018132572A1) applied for by Stanford University describing surrogate RSPOs. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Introduction: Thrombospondin-4 [1] is an extracellular glycoprotein involved in wound healing and tissue remodeling. Although THBS4 is reportedly frequently expressed in solid tumors, there are few reports of the clinicopathological features of carcinomas with THBS4 expression. We evaluated the clinicopathologic significance of THBS4 expression in gastric carcinoma (GC)., Materials and Methods: We retrospectively analyzed the cases of 584 GC patients. The expression of THBS4 in each tumor was evaluated by immunohistochemistry. We then divided the patients into the THBS4-high (n = 223, 38.2%) group and THBS4-low (n = 361, 61.8%) group. THBS4 expression in cancer-associated fibroblasts (CAFs), normal-associated fibroblasts (NFs) and gastric cancer cell lines was examined by western blotting., Results: THBS4 is expressed on stromal cells with αSMA or Podoplanin expression in the GC microenvironment, but not expressed on cancer cells with cytokeratin expression. The western blot analysis results showed that CAFs (but not NFs and cancer cells) expressed THBS4. Compared to the THBS4-low expression status, the THBS4-high expression status was correlated with higher αSMA expression, higher invasion depth, lymph-node metastasis, lymphatic invasion, peritoneal cytology, peritoneal metastasis, larger tumor size, microscopic diffuse type, and the macroscopic diffuse infiltrating type. The THBS4-high group's 5-year overall survival rate was significantly poorer than that of the THBS4-low group. A multivariate analysis revealed that THBS4 expression was an independent prognostic factor., Conclusion: THBS4 is expressed on CAFs in the gastric cancer microenvironment. THBS4 from CAFs is associated with the metastasis of cancer cells, and is a useful prognostic indicator for gastric cancer patients., Competing Interests: The authors have declared that no competing interests exist.

Many studies on retinal injury and repair following elevated intraocular pressure suggest that the survival ratio of retinal neurons has been improved by various measures. However, the visual function recovery is far lower than expected. The homeostasis of retinal synapses in the visual signal pathway is the key structural basis for the delivery of visual signals. Our previous studies found that complicated changes in the synaptic structure between retinal neurons occurred much earlier than obvious degeneration of retinal ganglion cells in rat retinae. The lack of consideration of these earlier retinal synaptic changes in the rescue strategy may be partly responsible for the limited visual function recovery with the types of protective methods for retinal neurons used following elevated intraocular pressure. Thus, research on the modulatory mechanisms of the synaptic changes after elevated intraocular pressure injury may give new light to visual function rescue. In this study, we found that thrombospondin 2, an important regulator of synaptogenesis in central nervous system development, was distributed in retinal macroglia cells, and its receptor α2δ-1 was in retinal neurons. Cell cultures including mixed retinal macroglia cells/neuron cultures and retinal neuron cultures were exposed to elevated hydrostatic pressure for 2 h. The expression levels of glial fibrillary acidic protein (the marker of activated macroglia cells), thrombospondin 2, α2δ-1 and presynaptic proteins were increased following elevated hydrostatic pressure in mixed cultures, but the expression levels of postsynaptic proteins were not changed. SiRNA targeting thrombospondin 2 could decrease the upregulation of presynaptic proteins induced by the elevated hydrostatic pressure. However, in retinal neuron cultures, elevated hydrostatic pressure did not affect the expression of presynaptic or postsynaptic proteins. Rather, the retinal neuron cultures with added recombinant thrombospondin 2 protein upregulated the level of presynaptic proteins. Finally, gabapentin decreased the expression of presynaptic proteins in mixed cultures by blocking the interaction of thrombospondin 2 and α2δ-1. Taken together, these results indicate that activated macroglia cells may participate in alterations of presynaptic proteins of retinal neurons following elevated hydrostatic pressure, and macroglia-derived thrombospondin 2 may modulate these changes via binding to its neuronal receptor α2δ-1.

Background: Nonalcoholic fatty liver disease (NAFLD) is a major public health concern in western societies. Nonalcoholic steatohepatitis (NASH), the progressive form of NAFLD, is characterized by hepatic steatosis, inflammation, oxidative stress and fibrosis. NASH is a risk factor for cirrhosis and hepatocellular carcinoma. NASH is predicted to be the leading cause of liver transplants by 2020. Despite this growing public health concern, there remain no Food and Drug Administration (FDA) approved NASH treatments. Using Ldlr -/- mice as a preclinical model of western diet (WD)-induced NASH, we previously established that dietary supplementation with docosahexaenoic acid (DHA, 22:6,ω3) attenuated WD-induced NASH in a prevention study. Herein, we evaluated the capacity of DHA supplementation of the WD and a low fat diet to fully reverse NASH in mice with pre-existing disease., Methods: Ldlr -/- mice fed the WD for 22 wks developed metabolic syndrome (MetS) and a severe NASH phenotype, including obesity, dyslipidemia, hyperglycemia, hepatic steatosis, inflammation, fibrosis and low hepatic polyunsaturated fatty acid (PUFA) content. These mice were randomized to 5 groups: a baseline group (WDB, sacrificed at 22 wks) and 4 treatments: 1) WD + olive oil (WDO); 2) WD + DHA (WDD); 3) returned to chow + olive oil (WDChO); or 4) returned to chow + DHA (WDChD). The four treatment groups were maintained on their respective diets for 8 wks. An additional group was maintained on standard laboratory chow (Reference Diet, RD) for the 30-wk duration of the study., Results: When compared to the WDB group, the WDO group displayed increased hepatic expression of genes linked to inflammation (Opn, Il1rn, Gdf15), hepatic fibrosis (collagen staining, Col1A1, Thbs2, Lox) reflecting disease progression. Mice in the WDD group, in contrast, had increased hepatic C20-22 ω3 PUFA and no evidence of NASH progression. MetS and NASH markers in the WDChO or WDChD groups were significantly attenuated and marginally different from the RD group, reflecting disease remission., Conclusion: While these studies establish that DHA supplementation of the WD blocks WD-induced NASH progression, DHA alone does not promote full remission of diet-induced MetS or NASH.

Astroglia play key roles in the development of neurons, ranging from regulating neuron survival to promoting synapse formation, yet basic questions remain about whether astrocytes might be involved in forming the dendritic arbor. Here, we used cultured hippocampal neurons as a simple in vitro model that allowed dendritic growth and geometry to be analyzed quantitatively under conditions where the extent of interactions between neurons and astrocytes varied. When astroglia were proximal to neurons, dendrites and dendritic filopodia oriented toward them, but the general presence of astroglia significantly reduced overall dendrite growth. Further, dendritic arbors in partial physical contact with astroglia developed a pronounced pattern of asymmetrical growth, because the dendrites in direct contact were significantly smaller than the portion of the arbor not in contact. Notably, thrombospondin, the astroglial factor shown previously to promote synapse formation, did not inhibit dendritic growth. Thus, while astroglia promoted the formation of presynaptic contacts onto dendrites, dendritic growth was constrained locally within a developing arbor at sites where dendrites contacted astroglia. Taken together, these observations reveal influences on spatial orientation of growth as well as influences on morphogenesis of the dendritic arbor that have not been previously identified., Competing Interests: The authors have declared that no competing interests exist.

R-spondin1 (Rspo1) is a member of a secreted protein family which has pleiotropic functions in development and stem cell growth. Rspo1 knock-out mice are sex-reversed, but some remain sub-fertile, so they fail to nurse their pups. A lack of Rspo1 expression in the mammary gland results in an absence of duct side-branching development and defective alveolar formation. The aim of this study was to characterize the phenotypic and molecular alterations of mammary gland due to Rspo1 knock-out. Using the transcriptional profiling of mammary tissues, we identified misregulated genes in the mammary gland of Rspo1 knock-out mice during pregnancy. A stronger expression of mesenchymal markers was observed, without modifications to the structure of mammary epithelial tissue. Mammary epithelial cell immunohistochemical analysis revealed a persistence of virgin markers, which signify a delay in cell differentiation. Moreover, serial transplantation experiments showed that Rspo1 is associated with a regenerative potential of mammary epithelial cell control. Our finding also highlights the negatively regulated expression of Rspo1's partners, Lgr4 and RNF43, in the mammary gland during pregnancy. Moreover, we offer evidence that Tgf-β signalling is modified in the absence of Rspo1. Taken together, our results show an abrupt halt or delay to mammary development during pregnancy due to the loss of a further differentiated function., Competing Interests: The authors have declared that no competing interests exist.

The polarity of proliferative and differentiated cellular compartments of colonic crypts is believed to be specified by gradients of key mitogens and morphogens. Indirect evidence demonstrates a tight correlation between Wnt- pathway activity and the basal-luminal patterning; however, to date there has been no direct experimental manipulation demonstrating that a chemical gradient of signaling factors can produce similar patterning under controlled conditions. In the current work, colonic organoids (colonoids) derived from cultured, multicellular organoid fragments or single stem cells were exposed in culture to steep linear gradients of two Wnt-signaling ligands, Wnt-3a and R-spondin1. The use of a genetically engineered Sox9-Sox9EGFP:CAGDsRED reporter gene mouse model and EdU-based labeling enabled crypt patterning to be quantified in the developing colonoids. Colonoids derived from multicellular fragments cultured for 5 days under a Wnt-3a or a combined Wnt-3a and R-spondin1 gradient were highly polarized with proliferative cells localizing to the region of the higher morphogen concentration. In a Wnt-3a gradient, Sox9EGFP polarization was 7.3 times greater than that of colonoids cultured in the absence of a gradient; and the extent of EdU polarization was 2.2 times greater than that in the absence of a gradient. Under a Wnt-3a/R-spondin1 gradient, Sox9EGFP polarization was 8.2 times greater than that of colonoids cultured in the absence of a gradient while the extent of EdU polarization was 10 times greater than that in the absence of a gradient. Colonoids derived from single stem cells cultured in Wnt-3a/R-spondin1 gradients were most highly polarized demonstrated by a Sox9EGFP polarization 20 times that of colonoids grown in the absence of a gradient. This data provides direct evidence that a linear gradient of Wnt signaling factors applied to colonic stem cells is sufficient to direct patterning of the colonoid unit in culture.

Exercise decreases adiposity and improves metabolic health; however, the physiological and molecular underpinnings of these phenomena remain unknown. Here, we investigate the effect of endurance training on adipose progenitor lineage commitment. Using mice with genetically labeled adipose progenitors, we show that these cells react to exercise by decreasing their proliferation and differentiation potential. Analyses of mouse models that mimic the skeletal muscle adaptation to exercise indicate that muscle, in a non-autonomous manner, regulates adipose progenitor homeostasis, highlighting a role for muscle-derived secreted factors. These findings support a humoral link between skeletal muscle and adipose progenitors and indicate that manipulation of adipose stem cell function may help address obesity and diabetes.

Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4). In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its presence in the extracellular matrix is required for articular cartilage integrity.

Small arteries are known to develop functional and structural alterations in hypertension. However, the mechanisms of this remodeling are not fully understood. We hypothesized that altered gene expression is associated with the development of hypertension in mesenteric arteries of spontaneously hypertensive rats (SHR). Three sublines of SHR and normotensive Wistar Kyoto rats (WKY) were studied at 6 weeks and 5 months of age. MiRNA and mRNA microarray experiments were performed and analyzed with bioinformatical tools, including Ingenuity Pathway Analysis (IPA). Principal component analysis showed a clear separation in both miRNA and mRNA expression levels between both ages studied, demonstrating strong age-related changes in expression. At the miRNA level, IPA identified differences between SHR and WKY related to metabolic diseases, cellular growth, and proliferation. The mRNAs differentially expressed between SHR and WKY were related to metabolism, cellular movement and proliferation. The most strongly upregulated gene (9.2-fold) was thrombospondin 4 (Thbs4), a protein involved in the endoplasmic reticulum (ER) stress response that activates transcription factor 6α (ATF6α). ATF6α downstream targets were also differentially expressed in SHR vs. WKY. Differential expression of THBS4, the cleaved form of ATF6α, and two of its targets were further confirmed at the protein level by western blot. In summary, these data revealed a number of genes (n = 202) and miRNAs (n = 3) in mesenteric arteries of SHR that had not been related to hypertension previously. The most prominent of these, Thbs4, is related to vascular ER stress that is associated with hypertension.

Testicular vasculogenesis is one of the key processes regulating male gonad morphogenesis. The knowledge of the molecular cues underlining this phenomenon is one of today's most challenging issues and could represent a major contribution toward a better understanding of the onset of testicular morphogenetic disorders. R-spondin 1 has been clearly established as a candidate for mammalian ovary determination. Conversely, very little information is available on the expression and role of R-spondin 1 during testicular morphogenesis. This study aims to clarify the distribution pattern of R-spondin 1 and other partners of its machinery during the entire period of testicular morphogenesis and to indicate the role of this system in testicular development. Our whole mount immunofluorescence results clearly demonstrate that R-spondin 1 is always detectable in the testicular coelomic partition, where testicular vasculature is organized, while Dickkopf-1 is never detectable in this area. Moreover, organ culture experiments of embryonic male UGRs demonstrated that Dickkopf-1 acted as an inhibitor of testis vasculature formation. Consistent with this observation, real-time PCR analyses demonstrated that DKK1 is able to slightly but significantly decrease the expression level of the endothelial marker Pecam1. The latter experiments allowed us to observe that DKK1 administration also perturbs the expression level of the Pdgf-b chain, which is consistent with some authors' observations relating this factor with prenatal testicular patterning and angiogenesis. Interestingly, the DKK1 induced inhibition of testicular angiogenesis was rescued by the co-administration of R-spondin 1. In addition, R-spondin 1 alone was sufficient to enhance, in culture, testicular angiogenesis.

The choroidal circulation plays a central role in maintaining the health of outer retina and photoreceptor function. Alterations in this circulation contribute to pathogenesis of many eye diseases including exudative age-related macular degeneration. Unfortunately, very little is known about the choroidal circulation and its molecular and cellular regulation. This has been further hampered by the lack of methods for routine culturing of choroidal endothelial cells (ChEC), especially from wild type and transgenic mice. Here we describe a method for isolation and culturing of mouse ChEC. We show that expression of thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis and inflammation, has a significant impact on phenotype of ChEC. ChEC from TSP1-deficient (TSP1-/-) mice were less proliferative and more apoptotic, less migratory and less adherent, and failed to undergo capillary morphogenesis in Matrigel. However, re-expression of TSP1 was sufficient to restore TSP1-/- ChEC migration and capillary morphogenesis. TSP1-/- ChEC expressed increased levels of TSP2, phosphorylated endothelial nitric oxide synthase (NOS) and inducible NOS (iNOS), a marker of inflammation, which was associated with significantly higher level of NO and oxidative stress in these cells. Wild type and TSP1-/- ChEC produced similar levels of VEGF, although TSP1-/- ChEC exhibited increased levels of VEGF-R1 and pSTAT3. Other signaling pathways including Src, Akt, and MAPKs were not dramatically affected by the lack of TSP1. Together our results demonstrate an important autocrine role for TSP1 in regulation of ChEC phenotype.

Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4) promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring Lgr4 splice variant encoding only the ectodomain of Lgr4, which was named Lgr4-ED. Lgr4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of Lgr4-ED in mammals. Recombinant Lgr4-ED purified from the conditioned media of transfected 293T cells was found to dose-dependently inhibit the LGR4-mediated Wnt signaling induced by RSPO2 or norrin, suggesting that it is capable of ligand absorption and could have a potential role as an antagonist. Intraperitoneal injection of purified recombinant Lgr4-ED into newborn mice was found to significantly decrease the testicular expression of estrogen receptor alpha and aquaporin 1, which is similar to the phenotype found in Lgr4-null mice. Administration of recombinant Lgr4-ED to superovulated female rats can also decrease the expression of estrogen receptor alpha, aquaporin 1, LH receptor and other key steroidogenic genes as well as bring about the suppression of progesterone production. Thus, these findings suggest that endogenously expressed Lgr4-ED may act as an antagonist molecule and help to fine-tune the R-spondin/norrin-mediated Lgr4-Wnt signaling during gonadal development.

4N1K is a peptide fragment derived from the C-terminal, globular domain of thrombospondin which has been shown to mediate integrin-dependent cell adhesion and promote integrin activation acting via the cell-surface receptor, CD47. However, some studies found that 4N1K could act independently of CD47, putting in question the specificity of 4N1K for CD47. This led us to characterize the cellular and non-cellular effects of 4N1K. We found that 4N1K stimulated a potent increase in binding of a variety of non-specific IgG antibodies to cells in suspension. We also found that these same antibodies, as well as CD47-deficient cells, could bind substrate-immobilized 4N1K significantly better than a control peptide, 4NGG. Furthermore, we found that cells treated with 4N1K at higher concentrations inhibited, while lower concentrations promoted cell adhesion to immobilized fibronectin as an integrin substrate. Importantly, both the stimulatory and the inhibitory activity of 4N1K occurred as efficiently in the CD47-deficient JinB8 cells, as it did in the CD47-expressing parental or in JinB8 cells reconstituted with CD47 expression. Given these results, we suggest that 4N1K interacts non-specifically with epitopes commonly found on the cell surface, and conclude that it is not a suitable peptide for use to study the consequences of CD47 receptor ligation.

Zinc RING finger 3 (ZNRF3) and its homolog RING finger 43 (RNF43) antagonize Wnt signaling in adult stem cells by ubiquitinating Frizzled receptors (FZD), which leads to endocytosis of the Wnt receptor. Conversely, binding of ZNRF3/RNF43 to LGR4-6 - R-spondin blocks Frizzled ubiquitination and enhances Wnt signaling. Here, we present crystal structures of the ZNRF3 ectodomain and its complex with R-spondin 1 (RSPO1). ZNRF3 binds RSPO1 and LGR5-RSPO1 with micromolar affinity via RSPO1 furin-like 1 (Fu1) domain. Anonychia-related mutations in RSPO4 support the importance of the observed interface. The ZNRF3-RSPO1 structure resembles that of LGR5-RSPO1-RNF43, though Fu2 of RSPO1 is variably oriented. The ZNRF3-binding site overlaps with trans-interactions observed in 2:2 LGR5-RSPO1 complexes, thus binding of ZNRF3/RNF43 would disrupt such an arrangement. Sequence conservation suggests a single ligand-binding site on ZNRF3, consistent with the proposed competing binding role of ZNRF3/RNF43 in Wnt signaling.

Neurotrauma or focal brain ischemia are known to trigger molecular and structural responses in the uninjured hemisphere. These responses may have implications for tissue repair processes as well as for the recovery of function. To determine whether the plasticity response in the uninjured hemisphere occurs even after a subtle trauma, we subjected mice to a partial unilateral deafferentation of the hippocampus induced by stereotactically performed entorhinal cortex lesion (ECL). The expression of selected genes was assessed by quantitative real-time PCR in the hippocampal tissue at the injured side and the contralesional side at day 4 and 14 after injury. We observed that expression of genes coding for synaptotagmin 1, ezrin, thrombospondin 4, and C1q proteins, that have all been implicated in the synapse formation, re-arrangement and plasticity, were upregulated both in the injured and the contralesional hippocampus, implying a plasticity response in the uninjured hemisphere. Several of the genes, the expression of which was altered in response to ECL, are known to be expressed in astrocytes. To test whether astrocyte activation plays a role in the observed plasticity response to ECL, we took advantage of mice deficient in two intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP) and vimentin (GFAP(-/-)Vim(-/-) ) and exhibiting attenuated astrocyte activation and reactive gliosis. The absence of GFAP and vimentin reduced the ECL-induced upregulation of thrombospondin 4, indicating that this response to ECL depends on astrocyte activation and reactive gliosis. We conclude that even a very limited focal neurotrauma triggers a distinct response at the contralesional side, which at least to some extent depends on astrocyte activation.

Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human β-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases β-catenin nuclear translocation and β-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human β-cell proliferation while maintaining a β-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human β-cell proliferation ∼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human β-cell proliferation while maintaining the β-cell phenotype.

While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer.

Hematopoietic stem cells (HSCs) are maintained in stem cell niches, which regulate stem cell fate. Extracellular matrix (ECM) molecules, which are an essential part of these niches, can actively modulate cell functions. However, only little is known on the impact of ECM ligands on HSCs in a biomimetic environment defined on the nanometer-scale level. Here, we show that human hematopoietic stem and progenitor cell (HSPC) adhesion depends on the type of ligand, i.e., the type of ECM molecule, and the lateral, nanometer-scaled distance between the ligands (while the ligand type influenced the dependency on the latter). For small fibronectin (FN)-derived peptide ligands such as RGD and LDV the critical adhesive interligand distance for HSPCs was below 45 nm. FN-derived (FN type III 7-10) and osteopontin-derived protein domains also supported cell adhesion at greater distances. We found that the expression of the ECM protein thrombospondin-2 (THBS2) in HSPCs depends on the presence of the ligand type and its nanostructured presentation. Functionally, THBS2 proved to mediate adhesion of HSPCs. In conclusion, the present study shows that HSPCs are sensitive to the nanostructure of their microenvironment and that they are able to actively modulate their environment by secreting ECM factors.

Background: LGR6 (leucine-rich repeat containing, G protein-coupled receptor 6) is a member of the rhodopsin-like seven transmembrane domain receptor superfamily with the highest homology to LGR4 and LGR5. LGR6 was found as one of the novel genes mutated in colon cancer through total exon sequencing and its promoter region is hypermethylated in 20-50% of colon cancer cases. In the skin, LGR6 marks a population of stem cells that can give rise to all cell lineages. Recently, we and others demonstrated that LGR4 and LGR5 function as receptors of R-spondins to potentiate Wnt/β-catenin signaling. However, the binding affinity and functional response of LGR6 to R-spondins, and the activity of colon cancer mutants of LGR6 have not been determined., Principal Findings: We found that LGR6 also binds and responds to R-spondins 1-3 with high affinity to enhance Wnt/β-catenin signaling through increased LRP6 phosphorylation. Similar to LGR4 and LGR5, LGR6 is not coupled to heterotrimeric G proteins or to β-arrestin following R-spondin stimulation. Functional and expression analysis of three somatic mutations identified in colon cancer samples indicates that one mutant fails to bind and respond to R-spondin (loss-of-function), but the other two have no significant effect on receptor function. Overexpression of wild-type LGR6 in HeLa cells leads to increased cell migration following co-treatment with R-spondin1 and Wnt3a when compared to vector control cells or cells overexpressing the loss-of-function mutant., Conclusions: LGR6 is a high affinity receptor for R-spondins 1-3 and potentially functions as a tumor suppressor despite its positive effect on Wnt/β-catenin signaling.

Cytoadherence or sequestration is essential for the pathogenesis of the most virulent human malaria species, Plasmodium falciparum (P. falciparum). Similar to leukocyte-endothelium interaction in response to inflammation, cytoadherence of P. falciparum infected red blood cells (IRBCs) to endothelium occurs under physiological shear stresses in blood vessels and involves an array of molecule complexes which cooperate to form stable binding. Here, we applied single-molecule force spectroscopy technique to quantify the dynamic force spectra and characterize the intrinsic kinetic parameters for specific ligand-receptor interactions involving two endothelial receptor proteins: thrombospondin (TSP) and CD36. It was shown that CD36 mediated interaction was much more stable than that mediated by TSP at single molecule level, although TSP-IRBC interaction appeared stronger than CD36-IRBC interaction in the high pulling rate regime. This suggests that TSP-mediated interaction may initiate cell adhesion by capturing the fast flowing IRBCs whereas CD36 functions as the 'holder' for providing stable binding.

Binocular vision requires intricate control of eye movement to align overlapping visual fields for fusion in the visual cortex, and each eye is controlled by 6 extraocular muscles (EOMs). Disorders of EOMs are an important cause of symptomatic vision loss. Importantly, EOMs represent specialized skeletal muscles with distinct gene expression profile and susceptibility to neuromuscular disorders. We aim to investigate and describe the anatomy of adult zebrafish extraocular muscles (EOMs) to enable comparison with human EOM anatomy and facilitate the use of zebrafish as a model for EOM research. Using differential interference contrast (DIC), epifluorescence microscopy, and precise sectioning techniques, we evaluate the anatomy of zebrafish EOM origin, muscle course, and insertion on the eye. Immunofluorescence is used to identify components of tendons, basement membrane and neuromuscular junctions (NMJs), and to analyze myofiber characteristics. We find that adult zebrafish EOM insertions on the globe parallel the organization of human EOMs, including the close proximity of specific EOM insertions to one another. However, analysis of EOM origins reveals important differences between human and zebrafish, such as the common rostral origin of both oblique muscles and the caudal origin of the lateral rectus muscles. Thrombospondin 4 marks the EOM tendons in regions that are highly innervated, and laminin marks the basement membrane, enabling evaluation of myofiber size and distribution. The NMJs appear to include both en plaque and en grappe synapses, while NMJ density is much higher in EOMs than in somatic muscles. In conclusion, zebrafish and human EOM anatomy are generally homologous, supporting the use of zebrafish for studying EOM biology. However, anatomic differences exist, revealing divergent evolutionary pressures.

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.

Background: Thrombospondin type I domain containing 7A (THSD7A) is a novel neural protein that is known to affect endothelial migration and vascular patterning during development. To further understand the role of THSD7A in angiogenesis, we investigated the post-translational modification scheme of THS7DA and to reveal the underlying mechanisms by which this protein regulates blood vessel growth., Methodology/principal Findings: Full-length THSD7A was overexpressed in human embryonic kidney 293T (HEK293T) cells and was found to be membrane associated and N-glycosylated. The soluble form of THSD7A, which is released into the cultured medium, was harvested for further angiogenic assays. We found that soluble THSD7A promotes human umbilical vein endothelial cell (HUVEC) migration and tube formation. HUVEC sprouts and zebrafish subintestinal vessel (SIV) angiogenic assays further revealed that soluble THSD7A increases the number of branching points of new vessels. Interestingly, we found that soluble THSD7A increased the formation of filopodia in HUVEC. The distribution patterns of vinculin and phosphorylated focal adhesion kinase (FAK) were also affected, which implies a role for THSD7A in focal adhesion assembly. Moreover, soluble THSD7A increased FAK phosphorylation in HUVEC, suggesting that THSD7A is involved in regulating cytoskeleton reorganization., Conclusions/significance: Taken together, our results indicate that THSD7A is a membrane-associated N-glycoprotein with a soluble form. Soluble THSD7A promotes endothelial cell migration during angiogenesis via a FAK-dependent mechanism and thus may be a novel neuroangiogenic factor.