ST2 regulates bone loss in a site-dependent and estrogen-dependent manner.

Interleukin-33 (IL-33) and its receptor, ST2, are implicated in bone remodeling. The lack of estrogen after menopause results in an accelerated bone loss. Here we investigated the role of ST2 in the bone loss induced by estrogen deficiency. ST2-deficient mice (ST2-/- ) and their littermates (wildtype [WT]) were ovariectomized (OVX), while ovary-intact mice were used as controls. Bone sites were analyzed by microcomputed tomography, histomorphometry, and quantitative real-time polymerase chain reaction (qPCR). Deletion of IL-33 or ST2 resulted in a similar bone loss in the femur and maxilla. Ovariectomy in WT mice caused bone loss in the same areas. The lack of ST2 in OVX mice did not alter bone remodeling in the femur but prevented bone loss in the maxilla. Consistently, ovariectomy increased the IL-33 messenger RNA (mRNA) levels in the maxilla but not in the femur. Under mechanical stimulation, ovariectomy and ST2 deletion independently increased bone remodeling induced by orthodontic tooth movement, which was also associated with a greater number of osteoclasts and a reduced number of osteoblasts in the maxillary bone. ST2-/- OVX mice, however, displayed twice as many osteoblasts as that of WT OVX mice. Ovariectomy and ST2 deletion differently altered the cytokine mRNA levels in the maxilla. Remarkably, interleukin-10 expression was decreased in both WT OVX and ST2-/- mice, and this reduction was completely restored in ST2-/- OVX mice. The results demonstrate that estrogen and IL33/ST2 independently protect against bone loss. However, the ovariectomy-induced bone loss is IL-33/ST2-dependent in the maxilla but not in the femur, indicating a bimodal and site-specific role of ST2 in bone remodeling.

Expression of semaphorin class 3 is higher in the proliferative phase on the human endometrium.

PURPOSE: The semaphorins are related to angiogenesis and cell proliferation depending on the tissue. The purpose of this study was to assess gene expression of class 3 semaphorin (SEMA3A-F) and protein expression of semaphorin 3A (SEMA3A) within human endometrium throughout the menstrual cycle. METHODS: Gene expression of SEMA3A-F was analyzed by real-time PCR (qRT-PCR) and protein expression of SEMA3A was analyzed by ELISA in endometrial biopsies in the proliferative and secretory phase of the menstrual cycle. RESULTS: Gene expression of SEMA3A, SEMA3C, SEMA3D, and SEMA3E was statistically significant decreased in secretory compared to proliferative phase endometrium (p < 0.05). Accordingly, SEMA3A protein expression in the secretory phase was lower than protein expression in proliferative phase endometrium (p ≤ 0.05). CONCLUSION: SEMA3A, 3C, 3D, and 3E are possibly related to cell proliferation in the endometrium, being more expressed in the proliferative phase of the cycle. This finding may stimulate studies of class 3 semaphorins as a possible target for treatment of endometrial pathologies.

SEMA3A partially reverses VEGF effects through binding to neuropilin-1.

Cross-talk between hematopoietic stem cells (HSCs) and bone marrow stromal cells (BMSCs) is essential for HSCs regulation and leukemogenesis. Studying bone marrow of myelodysplasia patients, a pre-leukemic condition, we found mRNA overexpression of vascular endothelial growth factor A (VEGFA) in CD34+ HSCs and semaphorin 3A (SEMA3A) in BMSCs. To better understand the role of VEGFA and SEMA3A in leukemogenesis, we recruited 30 myelodysplastic syndrome (MDS) patients, 29 acute myeloid leukemia (6 secondary to MDS) patients and 12 controls. We found higher VEGFA expression in de novo AML patients (without prior MDS) group (p=0.0073) and higher SEMA3A expression in all BMSCs patient's samples compared to control group. We then overexpressed VEGFA in an acute myelogenous leukemia cell line, KG1 cells, and in normal CD34+ cells. This overexpression increased KG1 (p=0.045) and CD34+ cell (p=0.042) viability and KG1 (p=0.042) and CD34+ cell (p=0.047) proliferation. Moreover, KG1 and CD34+ cells overexpressing VEGFA also had increased proliferation when co-cultured with human marrow stromal HS5 cells (p=0.045 and p=0.02, respectively). However, co-culture of these transformed cells with HS5 cells overexpressing SEMA3A reduced KG1 (p=0.004) and CD34+ (p=0.009) proliferation. Co-culture of KG1 transformed cells with HS27 cells overexpressing SEMA3A reduced KG1 proliferation as well (p=0.01). To investigate whether the dominant SEMA3A effect over VEGFA could be due to competition for neuropilin1 receptor (NRP1), we performed immunoprecipitation with anti-NRP1 antibody of cell extracts of co-cultured KG1 and HS5 cells, induced or not by VEGFA and SEMA3A recombinant proteins. Results showed a preferential association of NRP1 with SEMA3A, suggesting that SEMA3A can partially reverse the effects caused by the VEGFA preventing its binding with the NRP1 receptor. Since both hematopoietic cells, leukemic and normal, showed similar behavior, we suppose that the attempt to reversion of VEGF effects by SEMA3A is a homeostatic phenomenon in the hematopoietic niche. Finally, we conclude that VEGFA overexpression confers AML cell advantages and SEMA3A may partially reverse this effect; thus, SEMA3A protein combined with VEGFA inhibitors could be beneficial for AML treatment.

Glycan-dependent binding of galectin-1 to neuropilin-1 promotes axonal regeneration after spinal cord injury.

Following spinal cord injury (SCI), semaphorin 3A (Sema3A) prevents axonal regeneration through binding to the neuropilin-1 (NRP-1)/PlexinA4 receptor complex. Here, we show that galectin-1 (Gal-1), an endogenous glycan-binding protein, selectively bound to the NRP-1/PlexinA4 receptor complex in injured neurons through a glycan-dependent mechanism, interrupts the Sema3A pathway and contributes to axonal regeneration and locomotor recovery after SCI. Although both Gal-1 and its monomeric variant contribute to de-activation of microglia, only high concentrations of wild-type Gal-1 (which co-exists in a monomer-dimer equilibrium) bind to the NRP-1/PlexinA4 receptor complex and promote axonal regeneration. Our results show that Gal-1, mainly in its dimeric form, promotes functional recovery of spinal lesions by interfering with inhibitory signals triggered by Sema3A binding to NRP-1/PlexinA4 complex, supporting the use of this lectin for the treatment of SCI patients.

Inhibitory effect of semaphorin-3A, a known axon guidance molecule, in the human thymocyte migration induced by CXCL12.

Intrathymic T cell differentiation takes place within the thymic lobules and depends on interactions between developing thymocytes and cells of the thymic microenvironment. Along with differentiation, thymocytes migrate in an oriented progression, which is tightly regulated by a number of interactions, including one mediated by the chemokine CXCL12. It has been shown recently that SEMA-3A, a soluble member of the semaphorin family, is also involved in this human thymocyte migration and can have a chemorepulsive and de-adhesive role. Herein, we study the role of SEMA-3A on the CXCL12-driven migration of human thymocytes. We have shown that SEMA-3A is able to inhibit the chemotaxis triggered by CXCL12. Such an inhibition was seen in respect to immature and mature CD4/CD8-defined thymocyte subsets and can be reverted specifically by neutralizing anti-SEMA-3A mAb. We have also shown that SEMA-3A consistently down-regulates CXCR4 membrane expression in all CD4/CD8-defined thymocyte subsets, and this down-regulation is accompanied by a decrease in the phosphorylation of FAK and ZAP-70 protein kinases. Taken together, these results demonstrate the involvement of SEMA-3A in the regulation of CXCL12-driven human thymocyte migration, where it acts as a physiological antagonist.

Secreted human amyloid precursor protein binds semaphorin 3a and prevents semaphorin-induced growth cone collapse.

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-ß peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα(695) (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα(695) binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα(695) inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (K(d)≤8·10(-9) M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins.

Intrathymic T cell migration is a multivectorial process under a complex neuroendocrine control.

The thymus is responsible for normal T cell development, a process that includes cell proliferation, death, migration, and T cell receptor gene rearrangements. Moreover, it depends on interactions between developing thymocytes and thymic microenvironmental cells. Along with differentiation, thymocytes migrate and such oriented movement is regulated by several molecular interactions, comprising extracellular matrix (ECM) elements and chemokines. We postulated that intrathymic T cell migration is a multivectorial process; each individual vector being represented by a given molecular interaction. In vivo and in vitro experiments revealed that migration of developing thymocytes, including the export of mature T cells, is upregulated by hormones, such as growth hormone and triiodothyronine, through the modulation of ECM-mediated interactions, associated or not with the chemokine CXCL12. Recent data revealed that molecular interactions typically found in the nervous system also affect intrathymic T cell migration. Semaphorin-3A, a soluble member of the semaphorin family, is involved in the control of human thymocyte migration, bearing a chemorepulsive role. Such an effect is partially due to its downregulatory effect upon the interactions mediated by fibronectin and laminin, as well as CXCL12. These data unravel a complex neuroendocrine control intrathymic T cell migration, involving both endocrine and paracrine molecular interactions.

Chondroitin sulfate acts in concert with semaphorin 3A to guide tangential migration of cortical interneurons in the ventral telencephalon.

Chondroitin sulfate (CS) carrying proteoglycans (PGs) are widely expressed in the nervous system, and there is increasing evidence that they regulate developmental mechanisms like neurite outgrowth, axonal guidance and neuronal migration. Moreover, they can also act indirectly by organizing and/or modulating growth factors and guidance molecules. We found that chondroitin-4-sulfate is coexpressed with semaphorin 3A (Sema 3A) in the striatal mantle zone (SMZ), a nontarget region of neuropilin (Nrp)-1-expressing cortical interneurons flanking their migratory route in the subpallium. Using in vitro assays, we showed that CS PGs exert a repulsive effect on cortical interneurons, independently of Sema 3A, due to the CS side chains. We further showed that extracellular Sema 3A binds to CS. Disrupting Sema 3A-Nrp-1 signaling led migrating medial ganglionic eminence neurons to inappropriately invade the SMZ and even more so after removal of the CS side chains. Moreover, we found that soluble Sema 3A enhances the CS-induced repulsion in vitro. We concluded that CS acts as a repellent for cortical interneurons and that, in addition, CS restricts secreted Sema 3A within SMZ. Thus, both molecules act in concert to repel cortical interneurons from the SMZ during tangential migration toward the cerebral cortex.