Neuronal Chemorepellent Slit2 Inhibits Vascular Smooth Muscle Cell Migration by Suppressing Small GTPase Rac1 Activation

Abstract

The Slits are secreted proteins with roles in axonal guidance and leukocyte migration. On binding to Robo receptors, Slit2 repels developing axons and inhibits leukocyte chemotaxis. Slit2 is cleaved into Slit2-N, a protein tightly binding to cell membranes, and Slit2-C, a diffusible fragment. In the present study, we characterized the functional role of Slit2-N in vascular smooth muscle cells (VSMCs) and the cell association properties of 2 truncated versions of Slit2-N. Here, we document for the first time that Slit2-N is a chemorepellent of VSMCs. Intact blood vessels expressed Slit2 and Robo receptors as demonstrated by immunohistochemistry and quantitative real time PCR. Recombinant Slit2-N prevented the platelet-derived growth factor (PDGF)-stimulated migration of VSMCs. Slit2-N also abrogated PDGF-mediated activation of small guanosine triphosphatase (GTPase) Rac1, a member of the Rho GTPase superfamily of proteins involved in regulating the actin cytoskeleton. Furthermore, Slit2-N inhibited the PDGF-induced formation of lamellipodia, a crucial cytoskeletal reorganization event for cell motility. Slit2-N had no effect on the PDGF-mediated increase in DNA synthesis determined by [3H]thymidine uptake, suggesting that VSMC growth is unaffected by Slit2. Analysis of 2 engineered Slit2-N fragments (Slit2-N/1118 and Slit2-N/1121) indicated that 3 amino acids upstream of the putative cleavage site (Arg1121, Thr1122) are involved in the association of Slit2-N to the cell membrane. Our data assign a novel functional role to Slit2 in vascular function and show that cell guidance mechanisms that operate in the developing central nervous system are conserved in VSMCs.

Repulsive axon guidance molecule Slit3 is a novel angiogenic factor

“…This observation agrees with a recent report examining Slit3 expression in human aorta VSMCs 24 and suggests that Slit3 may function similarly through the autocrine and paracrine mechanisms to regulate VSMC function. In angiogenesis, ECs recruit VSMCs/ pericytes to form mature vascular structures.…”

“…The Slit3 Ϫ/Ϫ mice exhibit a congenital diaphragmatic hernia (CDH) phenotype, and Slit3 and Robo1/4 are expressed in wild-type developing diaphragm ( Figure 1A). 24,25 We examined the vasculature of developing diaphragms by whole-mount staining with an antimouse PECAM-1 antibody on embryonic day 15.5, a stage when the diaphragm is just formed and before the occurrence of CDH in the Slit3 Ϫ/Ϫ mice. As shown in Figure 7O-T, the phenotypically normal Slit3 heterozygous (Slit3 ϩ/Ϫ ) diaphragms had an uniform vessel architecture with a regular branching pattern and density, whereas the Slit3 Ϫ/Ϫ diaphragms showed tortuous vascular patterns with dramatically reduced density and branches.…”

“…15,[20][21][22] Slit2 was found to interact with Robo1 and Robo4, which are expressed by endothelial cells (ECs) to modulate EC migration and to participate in tumor angiogenesis, thus demonstrating that Slit2 directly modulates angiogenesis. Slit3 was reported to be expressed in vessels 23,24 ; however, the role of Slit3 in angiogenesis is not known. In this study, we examined the angiogenic effect of Slit3 and demonstrated that Slit3 is a novel and potent angiogenic factor.…”

Intraislet SLIT–ROBO signaling is required for beta-cell survival and potentiates insulin secretion

“…It was not surprising to find that SLITs potentiate glucose-stimulated insulin secretion given that Cdc42, Rac1, and RhoA play important roles in Slit-induced cellular migration (33,47,48). The down-regulation of cortical F-actin staining observed upon SLIT treatment is in agreement with the requirement for Cdc42 and RhoA inactivation via increasing Rho GTPase-activating proteins for the repulsive effects of SLITs in neurons and olfactory ensheathing cells (33,47).…”

Expression, clinical significance and mechanism of Slit2 in papillary thyroid cancer

“…Slit refers to a family of related genes which encode a corresponding set of secreted proteins, also collectively referred to as Slit (7). The classical function of Slit in proteins is to act as midline repellents, preventing the crossing of longitudinal axons through the midline of the central nervous system of most bilaterian animal species.…”