Sema3E Expression Research Articles

Abstract 5753: An Axon-guiding Molecule Semaphorin3E is Critically Involved in Impaired Angiogenesis of Diabetes

The axon-guiding molecules known as semaphorins and their specific receptors (plexins) regulate the vascular pattern and play an important role in development of the vascular network during embryogenesis. However, it remains unclear whether these molecules are involved in postnatal angiogenesis. Here we report that semaphorin3E (Sema3E), one of the class 3 semaphorins, and its specific receptor plexinD1 inhibit angiogenesis in adults. Sema3E inhibited cell growth and tube formation by suppressing the vascular endothelial growth factor (VEGF) signaling pathway. Expression of Sema3E and plexinD1 was markedly up-regulated in ischemic limbs of mice, and inhibition of this pathway by introduction of the plexinD1-Fc gene or disruption of Sema3E led to significant improvement of revascularization. We also found that Sema3E-deficient mice showed better blood recovery and a larger vessel area in their ischemic limbs than wild-type mice, suggesting that Sema3E/plexinD1 negatively regulate postnatal angiogenesis. Next, we identified a putative binding element of p53, the tumor suppressor protein, within the promotor region of the Sema3E gene, and found that hypoxia up-regulated Sema3E expression by activating p53 in endothelial cells. Consistent with our in vitro data, up-regulation of Sema3E expression was abolished in the ischemic limbs of p53-deficient mice, suggesting that an increase of p53 promotes Sema3E expression in ischemic tissue and thus inhibits blood flow recovery. It has been reported that angiogenic response to ischemia is attenuated in diabetes. We found that expression of p53 was increased in diabetic mice and this increase was further enhanced by ischemia. Likewise, expression levels of Sema3E were significantly higher in diabetic mice than in control mice. Consequently, blood flow recovery after VEGF treatment was impaired in diabetic mice compared with VEGF-treated control mice. These changes were effectively reversed by additional introduction of the plexinD1-Fc gene. These results indicate that Sema3E negatively regulates postnatal angiogenesis and suggest that inhibition of Sema3E would be a novel strategy for therapeutic angiogenesis, especially when VEGF treatment is ineffective, such as in the diabetic state.

Specificity of sensory–motor connections encoded by Sema3e–Plxnd1 recognition

Spinal reflexes are mediated by synaptic connections between sensory afferents and motor neurons. The organization of these circuits shows several levels of specificity. Only certain classes of proprioceptive sensory neurons make direct, monosynaptic connections with motor neurons. Those that do are bound by rules of motor pool specificity: they form strong connections with motor neurons supplying the same muscle, but avoid motor pools supplying antagonistic muscles. This pattern of connectivity is initially accurate and is maintained in the absence of activity, implying that wiring specificity relies on the matching of recognition molecules on the surface of sensory and motor neurons. However, determinants of fine synaptic specificity here, as in most regions of the central nervous system, have yet to be defined. To address the origins of synaptic specificity in these reflex circuits we have used molecular genetic methods to manipulate recognition proteins expressed by subsets of sensory and motor neurons. We show here that a recognition system involving expression of the class 3 semaphorin Sema3e by selected motor neuron pools, and its high-affinity receptor plexin D1 (Plxnd1) by proprioceptive sensory neurons, is a critical determinant of synaptic specificity in sensory-motor circuits in mice. Changing the profile of Sema3e-Plxnd1 signalling in sensory or motor neurons results in functional and anatomical rewiring of monosynaptic connections, but does not alter motor pool specificity. Our findings indicate that patterns of monosynaptic connectivity in this prototypic central nervous system circuit are constructed through a recognition program based on repellent signalling.

Semaphorin 3E Expression Correlates Inversely with Plexin D1 During Tumor Progression

Plexin D1 (PLXND1) is broadly expressed on tumor vessels and tumor cells in a number of different human tumor types. Little is known, however, about the potential functional contribution of PLXND1 expression to tumor development. Expression of semaphorin 3E (Sema3E), one of the ligands for PLXND1, has previously been correlated with invasive behavior and metastasis, suggesting that the PLXND1-Sema3E interaction may play a role in tumor progression. Here we investigated PLXND1 and Sema3E expression during tumor progression in cases of melanoma. PLXND1 was not expressed by melanocytic cells in either naevi or melanomas in situ, whereas expression increased with invasion level, according to Clark's criteria. Furthermore, 89% of the metastatic melanomas examined showed membranous PLXND1-staining of tumor cells. Surprisingly, expression of Sema3E was inversely correlated with tumor progression, with no detectable staining in melanoma metastasis. To functionally assess the effects of Sema3E expression on tumor development, we overexpressed Sema3E in a xenograft model of metastatic melanoma. Sema3E expression dramatically decreased metastatic potential. These results show that PLXND1 expression during tumor development is strongly correlated with both invasive behavior and metastasis, but exclude Sema3E as an activating ligand.

Abstract 3654: Semaphorin3E is a Novel Angiogenic Regulator

Semaphorin3E (sema3E) and its specific receptor plexinD1 are known to regulate the patterning of vessels during embryogenesis. However, it remains unclear whether these molecules are involved in postnatal angiogenesis. To elucidate the role of sema3E/plexinD1, we performed in vitro assay using human umbilical vein endothelial cells (HUVECs). Treatment with vascular endothelial growth factor (VEGF) increased proliferation and tube formation and this increase was significantly inhibited by sema3E. Moreover, treatment with the plexinD1-Fc fusion protein antagonized this anti-angiogenic activity of sema3E. Western blot analyses revealed that sema3E suppressed VEGF-induced phosphorylation of VEGFR2, suggesting that sema3E negatively regulates angiogenesis by inhibiting the VEGF signaling pathway. Expression of sema3E and plexinD1 was markedly upregulated in ischemic limbs. Immunohistochemistry showed that sema3E was expressed by the arterioles, myocytes, and capillary endothelial cells in ischemic tissue. Introduction of the plexinD1-Fc gene into ischemic limbs led to significant improvement of blood flow recovery and an increase in the number of CD31-positive cells. It has been reported that other members of the sema3 family are transcriptionally regulated by p53, a tumor suppressor protein that inhibits neovascularization in tumors. Consistent with these reports, forced expression of p53 was found to upregulate sema3E expression in HUVECs. We also found that the expression of p53 was markedly increased in ischemic limbs and that this increase was further enhanced in ischemic tissues of diabetic mice. Consequently, expression of sema3E was significantly higher in ischemic limbs of diabetic mice than in control mice, and the blood flow recovery after ischemia was strongly impaired in these mice even though treated with VEGF. In contrast, treatment with both VEGF and PlexinD1-Fc markedly improved blood flow recovery in diabetic mice. These results indicate that sema3E/plexinD1 negatively regulates postnatal angiogenesis under the regulation of p53 and suggest that inhibition of sema3E would be a novel strategy for therapeutic angiogenesis, especially when VEGF treatment is ineffective.

Differential expression of Class 3 and 4 semaphorins and netrin in the lamprey spinal cord during regeneration

To explore the role of axon guidance molecules during regeneration in the lamprey spinal cord, we examined the expression of mRNAs for semaphorin 3 (Sema3), semaphorin 4 (Sema4), and netrin during regeneration by in situ hybridization. Control lampreys contained netrin-expressing neurons along the length of the spinal cord. After spinal transection, netrin expression was downregulated in neurons close (500 mum to 10 mm) to the transection at 2 and 4 weeks. A high level of Sema4 expression was found in the neurons of the gray matter and occasionally in the dorsal and the edge cells. Fourteen days after spinal cord transection Sema4 mRNA expression was absent from dorsal and edge cells but was still present in neurons of the gray matter. At 30 days the expression had declined to some extent in neurons and was absent in dorsal and edge cells. In control animals, Sema3 was expressed in neurons of the gray matter and in dorsal and edge cells. Two weeks after transection, Sema3 expression was upregulated near the lesion, but absent in dorsal cells. By 4 weeks a few neurons expressed Sema3 at 20 mm caudal to the transection but no expression was detected 1 mm from the transection. Isolectin I-B(4) labeling for microglia/macrophages showed that the number of Sema3-expressing microglia/macrophages increased dramatically at the injury site over time. The downregulation of netrin and upregulation of Sema3 near the transection suggests a possible role of netrin and semaphorins in restricting axonal regeneration in the injured spinal cord.

Differential impact of semaphorin 3E and 3A on CNS axons

During the development of the central nervous system (CNS), the correct wiring of outgrowing neurites is mediated by antagonistic mechanisms. Aberrant growth is prevented by repulsive factors such as semaphorins. Expression of the ligands Sema3A and -3E and the receptors neuropilin Npn-1, -2a and -2b in the chick visual system were analyzed by RT-PCR. Whereas Sema3A and its major receptor Npn-1 were abundant, Sema3E and Npn-2 isoform expression was highly restricted and developmentally regulated. Peak expression occurred during retinal axon innervation of the tectum. Functional in vitro assays with recombinant proteins revealed a topography-specific growth cone collapsing activity of Sema3A for tectal axons. Interestingly, whereas tectal axons collapsed in a topographic-specific manner only in the presence of Sema3A, retinal axons responded only to Sema3E. The collapsing activity was intracellularly mediated by cGMP. For a detailed analysis of neuronal responses to sempahorins, time lapse video recording was performed. When tectal and retinal axons were pre-exposed to brain-derived neurotrophic factor (BDNF), a protective effect was evident only in the case of retinal axons. Our results suggest a molecular mechanism whereby ingrowth of retinal axons into the tectum can be regulated by Sema3E/BDNF modulation without disturbing tectal axon growth out of the tectum mediated by Sema3A.

KDR and Sema3 genes expression in bone marrow stromal cells and hematopoietic cells from leukemia patients and normal individuals

Kinase domain receptor (KDR) and Semaphorin3 (Sema3) have a functional relationship and are expressed in human bone marrow (BM). We cultured in vitro bone marrow stromal cells (BMSCs) and collected nonadherent cells from patients with acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL) and normal individuals. Reverse transcriptase polymerase chain reaction enzyme-linked immunosorbent assay (RT-PCR-ELISA) was performed to examine KDR and Sema3 genes expression, using β2 microglobulin as an internal reference. KDR expression ratio in normal control BMSCs (97.0%, 32/33) was higher than in its corresponding nonadherent cells (70.8%, 17/24). KDR expression levels in ALL BMSCs and AML nonadherent cells were significantly higher than in normal controls. Sema3 expression ratios in nonadherent cells from AML (78.6%, 11/14) and CML (71.4%, 10/14) were both significantly lower than in normal control (100%, 27/27), its expression levels were also significantly lower than in normal control. Sema3 expression level in normal BMSCs was significantly lower than in nonadherent cells. Sema3 and KDR genes expression levels displayed a significantly positive correlation in normal control and ALL nonadherent cells (r = 0.703, P = 0.002; r = 0.999, P = 0.001). These results suggests that KDR may play a critical role in sustaining the hematopoietic microenvironment due to its high expression, and that KDR may be involved in pathogenesis of AML and ALL. Sema3 could also sustain the survival of hematopoietic cells with its high expression, while Sema3 gene expression may be inhibited in AML and CML.

Proteolytic Processing Converts the Repelling Signal Sema3E into an Inducer of Invasive Growth and Lung Metastasis

We have previously shown that the expression of a semaphorin, known as a repelling cue in axon guidance, Sema3E, correlates with the ability to form lung metastasis in murine adenocarcinoma cell models. Now, besides providing evidence for the relevance of SEMA3E to human disease by showing that SEMA3E is frequently expressed in human cancer cell lines and solid tumors from breast cancer patients, we show biological activities of Sema3E, which support the implication of Sema3E in tumor progression and metastasis. In vivo, expression of Sema3E in mammary adenocarcinoma cells induces the ability to form experimental lung metastasis, and in vitro, the Sema3E protein exhibits both migration and growth promoting activity on endothelial cells and pheochromocytoma cells. This represents the first evidence of a metastasis-promoting function of a class 3 semaphorin, as this class of genes has hitherto been implicated in tumor biology only as tumor suppressors and negative regulators of growth. Moreover, we show that the full-size Sema3E protein is converted into a p61-Sema3E isoform due to furin-dependent processing, and by analyzing processing-deficient and truncated forms, we show that the generation of p61-Sema3E is required and sufficient for the function of Sema3E in lung metastasis, cell migration, invasive growth, and extracellular signal-regulated kinase 1/2 activation of endothelial cells. These findings suggest that certain breast cancer cells may increase their lung-colonizing ability by converting the growth repellent, Sema3E, into a growth attractant and point to a type of semaphorin signaling different from the conventional signaling induced by full-size dimeric class 3 semaphorins.