SLIT2, a secreted protein known for its role of repulsion in axon guidance and neuronal migration, can also inhibit leukocyte chemotaxis induced by chemotactic factors. It was found recently that Slit 2 has tumor suppressor activity and is frequently inactivated in lung and breast cancers./////////A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function. Svensson KJ et al. (2016) Activation of brown and beige fat can reduce obesity and improve glucose homeostasis through nonshivering thermogenesis. Whether brown or beige fat also secretes paracrine or endocrine factors to promote and amplify adaptive thermogenesis is not fully explored. Here we identify Slit2, a 180 kDa member of the Slit extracellular protein family, as a PRDM16-regulated secreted factor from beige fat cells. In isolated cells and in mice, full-length Slit2 is cleaved to generate several smaller fragments, and we identify an active thermogenic moiety as the C-terminal fragment. This Slit2-C fragment of 50 kDa promotes adipose thermogenesis, augments energy expenditure, and improves glucose homeostasis in vivo. Mechanistically, Slit2 induces a robust activation of PKA signaling, which is required for its prothermogenic activity. Our findings establish a previously unknown peripheral role for Slit2 as a beige fat secreted factor that has therapeutic potential for the treatment of obesity and related metabolic disorders.//////////////////
NCBI Summary:
This gene encodes a member of the slit family of secreted glycoproteins, which are ligands for the Robo family of immunoglobulin receptors. Slit proteins play highly conserved roles in axon guidance and neuronal migration and may also have functions during other cell migration processes including leukocyte migration. Members of the slit family are characterized by an N-terminal signal peptide, four leucine-rich repeats, nine epidermal growth factor repeats, and a C-terminal cysteine knot. Proteolytic processing of this protein gives rise to an N-terminal fragment that contains the four leucine-rich repeats and five epidermal growth factor repeats and a C-terminal fragment that contains four epidermal growth factor repeats and the cysteine knot. Both full length and cleaved proteins are secreted extracellularly and can function in axon repulsion as well as other specific processes. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Sep 2015]
General function
Ligand
Comment
Cellular localization
Secreted
Comment
Ovarian function
Luteolysis
Comment
Slit2/Robo4 Signaling: Potential Role of a VEGF-Antagonist Pathway to Regulate Luteal Permeability. Bekes I et al. (2017) Introduction The corpus luteum (CL) is dependent on luteal vascular permeability, which is controlled by human chorionic gonadotropin (hCG) via vascular endothelial growth factor (VEGF). In this study we investigated the role of a potential VEGF antagonist pathway - Slit2/Robo4 - and its influence on endothelial cell adhesion. Materials and Methods Luteinized granulosa cells (LGCs) were stimulated with hCG in the absence or presence of a VEGF inhibitor. The expression of VEGF and Slit2 were measured. Human umbilical vein endothelial cells (HUVECs) were stimulated with Slit2 or VEGF, and gene expressions of cadherin 5 (CDH5) and claudin 5 (CLDN5) were measured. Following Robo4 knockdown, CDH5, CLDN5 and endothelial permeability were measured. Results Stimulation of human LGCs with hCG significantly increased VEGF while Slit2 expression was significantly suppressed. Inhibition of VEGF action after hCG stimulation did not change Slit2 suppression. Slit2 knockdown did not affect VEGF expression. While VEGF stimulation of HUVECs significantly suppressed CDH5 and CLDN5 gene expression, stimulation of HUVECs with Slit2 resulted in a significant increase in CDH5 and CLDN5. Robo4 knockdown was done, leading to downregulation of CDH5 and CLDN5 which resulted in significantly increased permeability. Conclusions Our results indicate the existence of a VEGF-antagonist pathway in the CL that decreases vascular permeability. During the functional life of the CL the pathway is suppressed by hCG. It is possible that stimulation of this pathway could be used to treat ovarian hyperstimulation syndrome. Einleitung Voraussetzung für die regelrechte Funktion des Corpus luteum ist ein durchlässiges Gefäßsystem. Diese vaskuläre Durchlässigkeit wird durch die Einwirkung des humanen Choriogonadotropins (hCG) auf den Vascular Endothelial Growth Factor (VEGF) kontrolliert. In dieser Studie untersuchten wir ein potenzielles VEGF-antagonistisches System – das Slit2/Robo4-System – und dessen Auswirkung auf die endotheliale Zelladhäsion. Material und Methoden Luteinisierte Granulosazellen (LGC) wurden mit hCG stimuliert mit oder ohne Beigabe eines VEGF-Hemmers. Es wurde die VEGF- und Slit2-Expression gemessen. Aus der menschlichen Nabelschnur gewonnene venöse Endothelzellen (HUVECs) wurden mit Slit2 oder VEGF stimuliert. Danach wurde die Genexpression von Cadherin 5 (CDH5) und Claudin 5 (CLDN5) gemessen. Es wurden ein Robo4-Knockdown durchgeführt und die nachfolgende CDH5- und CLDN5-Expression sowie die endotheliale Durchlässigkeit gemessen. Ergebnisse Die Stimulation von menschlichen LGCs mit dem hCG führte zu einer wesentlichen Steigerung von VEGF, während die Slit2-Expression signifikant unterdrückt wurde. Die Hemmung der VEGF-Aktivität nach der hCG-Stimulation wirkte sich nicht auf die Unterdrückung der Slit2-Expression aus. Der Slit2-Knockdown hatte keine Auswirkungen auf die VEGF-Expression. Während die VEGF-Stimulation von HUVECs die Genexpression von CDH5 und CLDN5 signifikant unterdrückte, führte die Slit2-Stimulation von HUVECs zu einer signifikanten Steigerung der CDH5- und CLDN5-Expression. Es wurde ein Robo4-Knockdown durchgeführt, was zu einer Herabregulation von CDH5 und CLDN5 führte und die Durchlässigkeit signifikant steigerte. Schlussfolgerung Unsere Ergebnisse weisen auf das Vorhandensein eines VEGF-antagonistischen Systems im Corpus luteum hin, das die vaskuläre Gefäßdurchlässigkeit mindert. Dieses System wird während der Funktionsdauer des Corpus luteum durch das hCG unterdrückt. Es kann daher angenommen werden, dass eine Stimulation dieses Systems zur Behandlung beispielsweise des ovariellen Hyperstimulationssyndroms eingesetzt werden könnte.//////////////////
Prostaglandin F2a up-regulates the Slit/Robo expression in mouse corpus luteum during luteolysis. Zhang X 2013 et al.
Prostaglandin F2a (PGF2a) is a key factor in triggering the regression of the corpus luteum (CL). In addition, it has been reported that Slit/Robo signaling is involved in regulating luteolysis. However, the interactions between PGF2a and Slit/Robo in luteolysis progress remain to be established. The present study was designed to identify whether the luteolysis is regulated by the interactions of PGF2a and Slit/Robo in mouse CL. Real-time PCR and immunohistochemistry results showed that Slit2 and its receptor Robo1 are highly and specifically co-expressed in mouse CL. Functional studies showed that Slit/Robo participates in mouse luteolysis by enhancing cell apoptosis and upregulating caspase3 expression. Both in vitro and in vivo studies showed that PGF2a significantly increases Slit2 and Robo1 expression during luteolysis through PKC-dependent ERK and P38 signaling pathways, whereas an inhibitor of Slit/Robo signaling significantly decreases the stimulating effect of PGF2a on CL. These findings indicate that Slit/Robo signaling plays important roles in PGF2a-induced luteolysis by mediating the PGF2a signaling pathway in CL.
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Expression regulated by
Steroids
Comment
Progesterone down-regulates SLIT/ROBO expression in mouse corpus luteum. Zhang X et al. (2017) Progesterone produced by the corpus luteum (CL) is essential for preparation, implantation and maintenance of gestation. Furthermore, progesterone plays a protective role against luteolysis in rodents. It has been reported that Slit/Robo family members expressed in the CL and involved in prostaglandin F2α (PGF2α) induced luteolysis. However, the interactions between progesterone and Slits/Robos in CL are not clear. This study was designed to examine whether or not luteolysis is regulated by the interaction of progesterone and Slits/Robos in mouse CL. In the current study, we used Real-time PCR to identify the effect of progesterone on Slit2/Robo1 expression in cultured luteal cells in vitro, and the exogenous progesterone injection on mouse luteolysis and Slit/Robo expression in vivo was studied via Real-time PCR and Western bolt. Our in vitro experiment revealed that 1μM progesterone significantly decreased Slit2/Robo1 mRNA levels at 6h, 12h and 24h. Our in vivo experiment showed that the mRNA and protein levels of Slit2 and Robo1 decreased significantly 7days after progesterone supplement. These findings indicate that progesterone maintains CL function and resists luteolysis possibly through down-regulating Slit/Robo signaling pathway in the CL.//////////////////
Ovarian localization
Oocyte, Luteal cells, Surface epithelium
Comment
Glucocorticoid Regulation of SLIT/ROBO Tumour Suppressor Genes in the Ovarian Surface Epithelium and Ovarian Cancer Cells. Dickinson RE et al. The three SLIT ligands and their four ROBO receptors have fundamental roles in mammalian development by promoting apoptosis and repulsing aberrant cell migration. SLITs and ROBOs have emerged as candidate tumour suppressor genes whose expression is inhibited in a variety of epithelial tumours. We demonstrated that their expression could be negatively regulated by cortisol in normal ovarian luteal cells. We hypothesised that after ovulation the locally produced cortisol would inhibit SLIT/ROBO expression in the ovarian surface epithelium (OSE) to facilitate its repair and that this regulatory pathway was still present, and could be manipulated, in ovarian epithelial cancer cells. Here we examined the expression and regulation of the SLIT/ROBO pathway in OSE, ovarian cancer epithelial cells and ovarian tumour cell lines. Basal SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 expression was lower in primary cultures of ovarian cancer epithelial cells when compared to normal OSE (P<0.05) and in poorly differentiated SKOV-3 cells compared to the more differentiated PEO-14 cells (P<0.05). Cortisol reduced the expression of certain SLITs and ROBOs in normal OSE and PEO-14 cells (P<0.05). Furthermore blocking SLIT/ROBO activity reduced apoptosis in both PEO-14 and SKOV-3 tumour cells (P<0.05). Interestingly SLIT/ROBO expression could be increased by reducing the expression of the glucocorticoid receptor using siRNA (P<0.05). Overall our findings indicate that in the post-ovulatory phase one role of cortisol may be to temporarily inhibit SLIT/ROBO expression to facilitate regeneration of the OSE. Therefore this pathway may be a target to develop strategies to manipulate the SLIT/ROBO system in ovarian cancer.
This gene was found highly expressed in mouse ovary by RT-PCR.
Novel regulated expression of the SLIT/ROBO pathway in the ovary: possible role during luteolysis in women. Dickinson RE et al. The human corpus luteum (CL) undergoes luteolysis, associated with marked tissue and vascular remodeling, unless conception occurs and the gland is 'rescued' by human chorionic gonadotropin (hCG). In Drosophila the Slit gene product, a secreted glycoprotein, acts as a ligand for the roundabout (robo) transmembrane receptor. Together they influence the guidance and migration of neuronal and non-neuronal cells. In vertebrates three Slit (Slit1, Slit2, Slit3) and four Robo (Robo1, Robo2, Robo3/Rig-1, Robo4/Magic Robo) genes have been identified. ROBO1, SLIT2 and SLIT3 are also inactivated in human cancers and may regulate apoptosis and metastasis. Since processes such as apoptosis and tissue remodeling occur during the regression of the CL, the aim of this study was to investigate the expression, regulation and effects of the SLIT and ROBO genes in human luteal cells. Immunohistochemistry and RT-PCR revealed that SLIT2, SLIT3, ROBO1 and ROBO2 are expressed in luteal steroidogenic cells and fibroblast-like cells of the human CL. Furthermore, using real-time quantitative PCR, expression of SLIT2, SLIT3, and ROBO2 was maximal in the late-luteal phase and significantly reduced after luteal 'rescue' in vivo with exogenous hCG (P<0.05). Additionally, hCG significantly inhibited SLIT2, SLIT3 and ROBO2 expression in cultured luteinized granulosa cells (P<0.05). Blocking SLIT-ROBO activity increased migration and significantly decreased levels of apoptosis in primary cultures of luteal cells (P<0.05). Overall these results suggest the SLIT/ROBO pathway could play an important role in luteolysis in women.
Follicle stages
Primordial, Corpus luteum
Comment
Involvement of the SLIT/ROBO pathway in follicle development in the fetal ovary. Dickinson RE et al. In humans and domestic mammals pivotal processes in ovary development, including primordial follicle assembly, occur prenatally. These events are essential for determining fertility in adult life however they remain poorly understood at the mechanistic level. In mammals the SLITs (SLIT1, SLIT2, SLIT3) and their ROBO (ROBO1, ROBO2, ROBO3/RIG-1, ROBO4/MAGIC ROBO) receptors regulate neural, leukocyte, vascular smooth muscle cell and endothelial cell migration. In addition the SLIT/ROBO pathway has functional roles in embryonic development and in the adult ovary by inhibiting cell migration and promoting apoptosis. We therefore characterised follicle formation and investigated the expression and localisation of the ROBO/SLIT pathway in the ovine fetal ovary. Using RT-PCR, we identified SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 in sheep ovaries harvested across gestation. The real-time quantitative PCR results implied that ROBO2 and ROBO4 expression were elevated during the early stages of follicle formation and stayed abundant during primordial follicle maturation (P<0.05). Immunohistochemistry examination demonstrated that ROBO1 was localised to the pre-granulosa cells while ROBO2, ROBO4 and SLIT2 were expressed in the oocytes of the developing primordial follicle. This indicates that in the fetal ovary SLIT-ROBO signalling may require an autocrine and paracrine interaction. Furthermore at the time of increased SLIT-ROBO expression there was a significant reduction in the number of proliferating oocytes in the developing ovary (P<0.0001). Overall these results suggest, for the first time, that the SLIT-ROBO pathway is expressed at the time of follicle formation during fetal ovary development.