Author: Gianpiero Pescarmona
The semaphorins: versatile regulators of tumour progression and tumour angiogenesis 2008
Secreted class 3 semaphorins possess a conserved basic domain at their C-termini.
SEMA3A (semaphorin-3A), also known as SEMA1, SEMAD, SEMAL, coll-1, Hsema-I, SEMAIII or Hsema-III, is a 95 kDa secreted protein acting in a paracrine or autocrine manner. It belong to Class 3 semaphorins which are the only secreted vertebrate Semaphorins. SEMA3A was initially described as a guidance cue which affect the development of the central nervous system, both acting as a chemorepulsive agent, inhibiting axonal outgrowth, or as a chemoattractive agent, stimulating the growth of apical dendrites. However it was subsequently indicated that Sema3A can either promote or inhibit angiogenesis and cancer progression by multiple mechanisms. More recently a role for Sema3A has been found in osteoprotection, renal development , immunomodulation, and maturation of the lymphatic vascular network.
Sema3A cDNA predicts a 771 aa precursor protein with a putative 25 aa signal peptide.
A -> T Variant in position 131 has been found in a breast cancer sample.
P -> S Variant in position 396 has been found in a renal cell carcinoma sample.
SEMA3A belong to Class 3 semaphorins which are the only secreted vertebrate Semaphorins and are distinguished by the presence of a conserved Arg/Lys-rich (basic) domain at their C-termini (aa 727 – 769).
As all the other semaphorins, SEMA3A is characterized by the presence of an amino-terminal ~500 amino acid-long sema domain that is essential for signaling (aa 31 – 514). A 70 amino-acid stretch inside this domain was found to specify the biological activity and binding specificity of several class 3 semaphorins.
A conserved stretch of 54 amino-acid residues near the carboxy-terminal of the sema domain bears homology to the N-terminal region of β-integrins and is designated the PSI domain or MET-related sequence (MRS) domain. The X-ray structures of the sema domain reveal a conserved seven-blade β-propeller structure.
An Ig-like C2-type domain is present between the sema domain and the basic domain (aa 580 – 664).
Although Antipenko et al. saw a SEMA3A dimer in solution studies, SEMA3A formed a 1:1 complex in the presence of neuropilin. Also, the mutational studies they reported with the structure suggest that at least two of the loops involved in the formation of the SEMA3A dimer (4b–4c and a loop from the insertion between strands 5c–5d, shown in yellow and orange in the figure below) are involved in binding to neuropilin 1. This implies that not only is the dimer formed by the sema domain of SEMA3A weak, in agreement with earlier data indicating a requirement for a disulfide bond outside the sema domain for stability and biological activity of SEMA3A, but also that ligand binding to this semaphorin involves a decoupling of the sema domain– sema domain interaction. Although the overall conservation of mainchain structure in this region between SEMA3A and SEMA4D is striking (and the mode of dimerisation is essentially identical), there is lack of conservation between class 3 and class 4 semaphorin sequences for residues mediating SEMA4D dimerisation. A detailed examination of the structures reveals that the hydrophobic residues central to the dimerisation of SEMA4D (Phe223, Val224 and Phe225, loop 4b–4c) are not conserved in SEMA3A. The overall effect is a reduction in the hydrophobic nature of the dimer interface for SEMA3A compared to SEMA4D, which would be consistent with SEMA4D forming a more robust dimer (note that the class 4 semaphorins do not require neuropilin binding as part of the signalling complex).
Bioactive Sema3A is a disulfidelinked dimer. The bioactivity is increased after proteolytic processing by furinlike endoproteases near the carboxyterminus.
Molecular Weight of proSEMA3A: 125 kDa.
Molecular Weight of activated SEMA3A: 95 kDa.
Molecular Weight of SEMA3A proteolytic fragments: 65/45 kDa.
Predicted processing consensus sites are conserved in class III semaphorins. A schematic representation of SemD is shown, with the positions of the signal sequence (black), semaphorin domain (hatched), Ig homology (grey) and putative processing sites (arrowheads) indicated. The sequences of three clusters of basic residues found in SemD (SD, accession No. X85993) are aligned with the corresponding sequences of SemA (SA, X85990), SemE (SE, X85994), collapsin-1 (C1, G410078), collapsin-2 (C2, U28240) and semaphorin IV (S4, U33920). All consensus sequences derived from this alignment contain recognition sites for the endoprotease furin (K/RXRR). The predicted cleavage sites after the Processing Consensus Sequences (PCS) are indicated by arrowheads, and the theoretical molecular weights of cleavage products are shown in kDa. The sequence of PCS1 and 3 is conserved in all class III semaphorins. No evidence was found for cleavage at PCS2 (open arrowhead), a site unique to SemD and Coll-1.
Differential processing of proSemD generates several isoforms with distinct properties. SemD is synthesized as an inactive precursor, proSemD. Differential processing by furin or a related protease at three processing sites (PCS1, 3 or 4) during intracellular maturation results in the formation of several SemD isoforms [proSemD, SemD and SemD] which differ in their repulsive activity (indicated by a red bar on the left) over a range of three orders of magnitude. The individual PCSs have different functional properties. Processing at PCS3/4 coupled to a post-cleavage modification activates SemD. Secreted proSemD has the lowest, SemD (cleaved at PCS1/3/4) and SemD (cleaved at PCS3/4) the highest activity. After secretion, proSemD could be activated by cleavage at PCS3 or 4. Proteolysis of SemD at PCS1, in contrast, results in the reduction of its potency.
Furin is a cellular endoprotease ubiquitously expressed, which is localized in the Trans Golgi Network of the cell and recycle from plasma membrane. In optimal condition of pH it catalize the proteolytic activation of several substrate of the secretory pathway, using Calcium as co-factor. Since SEMA3A activation seems to be regulated during embryo development and given the abundance and ubiquitous expression of furin and related proteases, a specific mechanism is likely to exist that prevents complete processing of the 95 kDa Sema3A during its intracellular maturation.
These mechanisms could involve furin activation , transcriptional regulation , and cellular localization.
Intriguingly furin alterations are involved in many biological and clinical aspect similarly to SEMA3A such as cancer, angiogenesis, immunomodulation and neuronal development.
Its sema-related clinical role has been confirmed for other class 3 semaphorins such as SEMA3E.
However SEMA3A proteolytic consensus sequences are not exclusively recognized by furin and can be processed by other proteases; Although knock out experiments demonstrate that furin loss has an elevated impact and penetrance compared to other proteases, it can’t be excluded a redundancy of activation by other basic aa-specific PCs which share ≥50% sequence identity whit furin wide negatively charged catalytic pocket.
Moreover furin can activate numerous substrate (proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type-1 matrix metalloproteinase, beta subunit of pro-nerve growth factor and von Willebrand factor) and the biological effects of its alteration could be due to disregulation of these effectors alone, or in combination with Semaphorins.
SEMA3A regulates cellular adhesion, migration, proliferation, shape, survival and apoptosis by various mechanisms, such as influencing integrin function and cytoskeletal organization.
The effect of SEMA3A on focal contacts of endothelial cells is seen using an antibody directed against the focal contacts-associated protein vinculin. After 5 min all the focal contacts are disassembled.
Effects of SEMA3A on the actin cytoskeleton of endothelial cells.
SEMA3A play an essential function both in development and in adulthood physiology of CNS. Several knockout mice has been used to investigate SEMA3A role in CNS development
Sema3A induces the collapse of cultured DRG growth cones. (A) Morphology of an E15 DRG growth cone in vitro after addition of control medium. The actin cytoskeleton is stained using phalloidin-TRITC. (B) Collapsed growth cone. Addition of recombinant Sema3A-containing medium abolishes the spread morphology of the growth cone, and causes the growth cone to assume a collapsed appearance. Scale bar: 20 mm.
In adulthood a crush of facial and spinal motoneuron axons results in a downregulation of Sema3A expression, together with an upregulation of GAP-43. Upon target re-innervation, Sema3A and GAP-43 expression return to normal levels. When the regeneration of motor axons is prevented by ligation of the proximal nerve-stump, Sema3A expression continues to be downregulated, indicating that Sema3A expression in motoneurons is controlled by retrogradely transported signals derived from the target area. Expression levels of Sema3A receptor components are not affected by nerve crush, suggesting that regenerating motoneurons retain their sensitivity to Sema3A. Interestingly, Sema3A expression is induced in terminal Schwann cells of target muscle endplates following nerve crush, which could function to stabilize restored synaptic connections.
In vitro studies showed that there is an upregulation of Sema3A and CRMP expression in cultured chick sympathetic neurons that undergo dopamineinduced apoptosis. Antibodies directed against Sema3A as well as antibodies against NP-1 could attenuate cell death. Furthermore, exposure of sympathetic neurons to Sema3A protein, or a small peptide derived from Sema3A, resulted in the induction of neuronal apoptosis.
Several class 3 semaphorins, including SEMA3A and SEMA3F, are expressed by endothelial cells, suggesting that they may function in an autocrine fashion. SEMA3A can inhibit the binding of VEGF165 to NP1 and is able to inhibit in vitro angiogenesis. However, SEMA3A repulses endothelial cells and functions as an inducer of apoptosis even in the absence of VEGF, suggesting that inhibition of VEGF-induced endothelial cell proliferation and migration by SEMA3A is not caused solely by competition with VEGF but is also the result of inhibitory SEMA3A-induced signalling mediated by NP1.
SEMA3A does not seem to have a role in the regulation of developmental angiogenesis. However, exogenous SEMA3A inhibited in vivo VEGF induced angiogenesis in the chick chorioallantoic angiogenesis assay and functioned in addition as a vascular permeability factor.
SEMA3A inhibits the migration and spreading of MDA-MB-231 breast cancer cells and inhibits the invasiveness of prostate cancer cells in in vitro assays. However, there have also been contradictory reports suggesting, for example, that SEMA3A contributes to the progression of pancreatic cancer and colon cancer. These contradictory results might be due to interplay between VEGF and SEMA3A. In normal mesothelial cells VEGF induces a p38 (MAPK14)-mediated expression of SEMA3A that causes inhibition of cell proliferation. This suggests that the abrogation of regulated SEMA3A expression is responsible for VEGF-driven growth of tumour cells. SEMA3A was recently found to induce the permeabilization of blood vessels, a process that can contribute to tumour development.
SEMA3A is expressed in several types of tumour cells and inhibits the proliferation of primary human T cells and cytokine production by these cells. Inhibition of SEMA3A synthesis in SEMA3A-producing tumour cells augmented the activation of T cells, suggesting that SEMA3A synthesis may contribute to T-cell dysfunction in the tumour microenvironment and indirectly contribute to the escape of tumour cells from immune surveillance.
The functional receptor complex for Sema3 is composed of two distinct transmembrane proteins: Neuropilin1 (Npn1) and PlexinA. Npn1 binds directly to Sema3A with highaffinity and confers specificity. PlexinA interacts with Npn1 to increase the affinity of the complex for Sema3A and serves as the signaling subunit in the receptor complex.
The neuropilin extracellular domain contains two CUB motifs (domain A: A1, A2), followed by two domains with similarity to coagulation factor V/VIII (domain B: B1, B2) and one MAM domain (domain C). Both domains A and B are essential for binding the sema domain of SEMA3A, while only B1 is required for the interaction with VEGF165 or the basic carboxy terminus of SEMA3A. While the C terminus does not contribute to the biological specificity of semaphorins, it is a major determinant of their affinity for neuropilins.
SEMA3A signaling has numerous different and complex biological effect, which sometimes result to be opposite and contraddictory depending from the SEMA3A concentration, from the target cell microenviroment and from the set of surface receptor expression.
This happen because of the existence of alternative receptors for semaphorins (such as glycosaminoglycans for SEMA3A), and for the eventual presence of alternative ligand for Neuropilin1 resulting in competition whit SEMA3A for receptor binding.
Moreover NP1 and PLEXA1 can play different crosstalks with other cell surface receptors, resulting in different cell signaling cascade and biological effect.
The main signal transduction pathways by which SEMA3A activate plexin A1 is derived mainly from the study of neuronal cells.
Prior to the binding of SEMA3A, the sema domain of PLEXA1 auto-inhibits the spontaneous activation of PLEXA1 signalling. The binding of SEMA3A to NP1 induces a conformational change in the plexin that relieves this auto-inhibition. The four-point-one, ezrin, radixin, moesin (FERM) domain-containing GEF FARP2 is associated with PLEXA1 in the presence of NP1. Following SEMA3A binding to NP1, FARP2 dissociates and promotes the binding of GTP to the small GTPase RAC1. Activated RAC1 is recruited to PLEXA1 and promotes in turn the recruitment of the small constitutively active GTPase RND1. This induces the activation of the PLEXA1 GAP domain, resulting in the recruitment of the small extracellular matrix activated GTPase RRAS. The PLEXA1 GAP domain activates the intrinsic GTPase function of RRAS resulting in RRAS inactivation. RRAS is a regulator of integrin function and its inactivation leads to the inactivation of integrin and promotes the detachment of target cells from the extracellular matrix. The interaction of RND1 with PLEXA1 can be inhibited by RHOD, another small GTPase that binds to the same binding site on PLEXA1. In addition, free FARP2 associates with a phosphatidylinositol- 4-phosphate 5 kinase (PIPKI, also know as PIP5K1C) and inhibits its interaction with the focal adhesion protein talin, thereby leading to further suppression of integrin function and to the disassembly of focal adhesions.
Activation of PLEXA1 by SEMA3A also leads to the collapse of the actin cytoskeleton. However, it is not yet completely clear how the collapse of the cytoskeleton is orchestrated. SEMA3A induces the dephosphorylation and activation of the actin-degrading enzyme cofilin. The enzymatic activity of cofilin is inhibited following phosphorylation by activated LIM domain kinase 1 (LIMK1). LIMK1 is activated by p21-activated kinase 1 (PAK1), which is in turn activated by RAC1. The activation of PLEXA1 by SEMA3A is followed by activation of RAC1, which might account for a transient SEMA3A-induced phosphorylation of cofilin that is observed in axonal growth cones. However, active RAC1 is subsequently sequestered by PLEXA1, resulting in the dephosphorylation of PAK, activation of cofilin and finally actin depolymerization. It is also possible, although there is no experimental proof yet, that SEMA3A is able to activate cofilin phosphatases belonging to the slingshot family, which might also contribute to the activation of cofilin. SEMA3A also activates additional intracellular signalling pathways. In neuronal cells the intracellular tyrosine kinases FES (also known as FPS), FYN and FER are activated by SEMA3A. Activation of FYN, a kinase constitutively associated with PLEXA1, leads to the recruitment and activation of the serine/threonine kinase CDK5 and phosphorylates tyrosine residues within the intracellular domain of PLEXA1. In neuronal cells collapsin response mediator proteins (CRMPs) such as CRMP2 (also known as DPYSL2) are recruited to the activated PLEXA1 and are phosphorylated by FES, CDK5 and activated glycogen synthase kinase 3(GSK3). Phosphorylated CRMP2 promotes tubulin depolymerization, and thus regulates microtubule organization . These responses are also modulated by activated Rho-associated coiled coil-containing kinase (ROCK), which is also able to phosphorylate CRMP2. Additional proteins that have essential roles in SEMA3A signalling are the members of the MICAL (molecule interacting with CasL) family. These are flavoprotein monooxygenases that associate with type A plexins. Indeed, the effects of SEMA3A on the cytoskeleton are inhibited by the flavoprotein monooxygenase inhibitor epigallocatechin gallate. Interestingly, in neuronal cells SEMA3A promotes the formation of complexes between MICAL and CRMP and both bind PLEXA1. This association then activates the enzymatic function of MICALs. Another signal transducer that has an important role in SEMA3A signal transduction is RANBPM (also know as RANBP9) but not much is known about its mode of action. Semaphorin-induced localized changes in cytoskeletal organization represent the major mechanism by which semaphorins repulse axonal growth cones. Class 3 semaphorins such as SEMA3A and SEMA3F repel endothelial cells and the effects that they have on the cytoskeleton of endothelial cells and responsive mural cells might enable them to repel blood vessels, a novel mechanism that might contribute to the anti-angiogenic properties of these semaphorins. Lastly, it was found that prolonged stimulation with class 3 semaphorins such as SEMA3A and SEMA3F can induce apoptosis of neuronal cells and endothelial cells concomitant with inhibition of extracellular signalregulated kinases 1 and 2 phosphorylation and induction of caspases such as caspase 3.
Soluble guanylate cyclase is asymmetrically localized to the developing apical dendrite, and is required for the chemoattractive effect of SEMA3A. Thus, the asymmetric localization of soluble guanylate cyclase confers distinct SEMA3A responses to axons and dendrites.
p38 MAPK signal transduction pathways mediates VEGF-induced Sema-3A in mesothelial cells.