Slit/Robo signaling regulates Leydig cell steroidogenesis

Emmanuelle Martinot,Derek Boerboom

doi:10.1186/s12964-020-00696-6

Abstract

BackgroundFirst identified as a regulator of neuronal axon guidance, Slit/Robo signaling has since been implicated in additional physiologic and pathologic processes, such as angiogenesis, organogenesis and cancer progression. However, its roles in the regulation of testis function have been little explored.MethodsImmunohistochemistry and RT-qPCR analyses were performed to detect the expression of Slit/Robo signaling effectors in the adult mouse testis. To identify the roles and mechanisms of Slit/Robo signaling in the regulation of steroidogenesis, RT-qPCR, immunoblotting and hormone measurements were carried out using Leydig cells (primary cultures and the MA10 cell line) treated with exogenous SLIT ligands, and testes from Robo1-null mice.ResultsSlit1, -2 and -3 and Robo1 and -2 expression was detected in the adult mouse testis, particularly in Leydig cells. In vitro treatment of Leydig cells with exogenous SLIT ligands led to a decrease in the expression of the steroidogenic genes Star, Cyp11a1, and Cyp17a1. SLIT2 treatment decreased the phosphorylation of the key steroidogenic gene regulator CREB, possibly in part by suppressing AKT activity. Furthermore, SLIT2 treatment reduced the responsiveness of MA10 cells to luteinizing hormone by decreasing the expression of Lhcgr. Consistent with these in vitro results, an increase in testicular Star mRNA levels and intra-testicular testosterone concentrations were found in Robo1-null mice. Finally, we showed that the expression of the Slit and Robo genes in Leydig cells is enhanced by testosterone treatment in vitro, by an AR-independent mechanism.ConclusionTaken together, these results suggest that Slit/Robo signaling represents a novel mechanism that regulates Leydig cell steroidogenesis. It may act in an autocrine/paracrine manner to mediate negative feedback by testosterone on its own synthesis.4VdBjKyUK_zz54UEEeomAMVideo

Highlights

First identified as a regulator of neuronal axon guidance, Slit/Robo signaling has since been implicated in additional physiologic and pathologic processes, such as angiogenesis, organogenesis and cancer progression
Slit and Robo genes are expressed in the Leydig cells of the mouse testis We first determined which Slit ligands and Robo receptors are expressed in the adult mouse testis by RT-qPCR, using a brain sample as a positive control
The specificity of the staining was validated using negative and positive controls (Additional file 1: Fig. S1). These results were confirmed at the mRNA level, as all aforementioned Slit/Robo family members were detected in a Leydig cell-enriched sample (Fig. 1a)

Summary

Introduction

First identified as a regulator of neuronal axon guidance, Slit/Robo signaling has since been implicated in additional physiologic and pathologic processes, such as angiogenesis, organogenesis and cancer progression. In breast and lung cancer cells, Slit expression is frequently lost, and overexpression of Slit was associated with an increase in cell adhesion and a decrease in cell proliferation [21]. In this context, SLIT2 appears to signal via AKT/GSK3β, thereby modulating the stability and the transcriptional regulatory activity of the proto-oncogene CTNNB1 (β-catenin) [22, 23]

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