Plexin-A2 enables the proliferation and the development of tumors from glioblastoma derived cells.

The semaphorin guidance factors receptor plexin-A2 transduces sema6A and sema6B signals and may mediate, along with plexin-A4, the anti-angiogenic effects of sema6A. When associated with neuropilins plexin-A2 also transduces the anti-angiogenic signals of sema3B. Here we show that inhibition of plexin-A2 expression in glioblastoma derived cells that express wild type p53 such as U87MG and A172 cells, or in primary human endothelial cells, strongly inhibits cell proliferation. Inhibition of plexin-A2 expression in U87MG cells also results in strong inhibition of their tumor forming ability. Knock-out of the plexin-A2 gene in U87MG cells using CRISPR/Cas9 inhibits cell proliferation which is rescued following plexin-A2 re-expression, or expression of a truncated plexin-A2 lacking its extracellular domain. Inhibition of plexin-A2 expression results in cell cycle arrest at the G2/M stage, and is accompanied by changes in cytoskeletal organization, cell flattening, and enhanced expression of senescence associated ß-galactosidase. It is also associated with reduced AKT phosphorylation and enhanced phosphorylation of p38MAPK. We find that the pro-proliferative effects of plexin-A2 are mediated by FARP2 and FYN and by the GTPase activating (GAP) domain located in the intracellular domain of plexin-A2. Point mutations in these locations inhibit the rescue of cell proliferation upon re-expression of the mutated intracellular domain in the knock-out cells. In contrast re-expression of a plexin-A2 cDNA containing a point mutation in the semaphorin binding domain failed to inhibit the rescue. Our results suggest that plexin-A2 may represent a novel target for the development of anti-tumorigenic therapeutics.

CD72-semaphorin3A axis: A new regulatory pathway in systemic lupus erythematosus.

CD72 is a regulatory co-receptor on B cells, with a role in the pathogenesis of systemic lupus erythematosus (SLE) in both human and animal models. Semaphorin3A (sema3A) is a secreted member of the semaphorin family that can reconstruct B cells' regulatory functions by upregulating IL-10 expression and inhibiting the pro-inflammatory activity of B and T cells in autoimmune diseases. The aim of our present study was to identify a new ligand for CD72, namely sema3A, and exploring the signal transduction pathways following its ligation in B cells. We established that CD72 functions as sema3A binding and signal-transducing receptor. These functions of CD72 are independent of neuropilin-1 (NRP-1) (the known sema3A receptor). We discovered that sema3A induces the phosphorylation of CD72 on tyrosine residues and the association of CD72 with SHP-1 and SHP-2. In addition, the binding of sema3A to CD72 on B cells inhibits the phosphorylation of STAT-4 and HDAC-1 and induces the phosphorylation of p38-MAPK and PKC-theta in B-cells derived B-lymphoblastoid (BLCL) cells, and in primary B-cells isolated from either healthy donors or SLE patients. We concluded that sema3A is a functional regulatory ligand for CD72 on B cells. The sema3A-CD72 axis is a crucial regulatory pathway in the pathogenesis of autoimmune and inflammatory diseases namely SLE, and modulation of this pathway may have a potential therapeutic value for autoimmune diseases.

Favorable outcome of empagliflozin treatment in two pediatric glycogen storage disease type 1b patients.

Background: Glycogen storage disease type 1b (GSD1b) is an ultra-rare autosomal recessive disorder, caused by mutations in SLC37A4 gene. Affected patients present with episodes of fasting hypoglycemia and lactic acidosis, hepatomegaly, growth retardation, hyperlipidemia and renal impairment. In addition, patients present neutropenia, neutrophil dysfunction and oral, and skin infections as well as a significant predisposition to develop inflammatory bowel disease (IBD). Low neutrophil counts and function is related to the toxic accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P). Recently, several reports have shown that off-label treatment with empagliflozin (EMPA), an inhibitor of the renal glucose transporter SGLT2, decreased blood 1,5-anhydroglucitol (1,5-AG), and neutrophil 1,5-AG6P, thus resulting in a new therapeutic option for neutropenia and neutrophil dysfunction in patients. Methods: Off-label treatment with EMPA was established in two GSD1b patients after signed informed consent. The patients were followed clinically. We monitored neutrophil counts and function, 1,5-AG levels in plasma and its renal clearance before and during EMPA treatment. Results: A 17 year-old girl who had long standing oral ulcers and developed IBD, requiring systemic steroid and regular granulocyte colony-stimulating factor (GCSF) therapy and an 8 year-old boy who had steady non healing oral lesions were treated with empagliflozin during 18-24 months. Treatment led to increase of neutrophil counts and function with substantial clinical improvement. This included remission of IBD in the first patient which allowed to discontinue both GCSF and steroid therapy and resolution of oral lesions in both patients. The concentration of 1,5-AG in blood was greatly decreased within two weeks of treatment and remained stable thereafter. Conclusions: Repurposing of empagliflozin to treat neutropenia in two GSD1b patients was safe and resulted in the urinary excretion of 1,5-AG, the normalization of neutrophil function, and a remarkable improvement of neutropenia-related clinical traits. We showed for the first time that empagliflozin increases concomitantly the renal clearance of both 1,5-anhydroglucitol and glucose in GSD1b patients.

Semaphorin3A: A Potential Therapeutic Tool for Lupus Nephritis.

Background: The immune regulatory properties of semaphorin3A (sema3A) (both innate and adaptive) are well established in many in vitro studies. The injection of sema3A into a mice model of rheumatoid arthritis was proven to be highly beneficial, both in attenuating clinical symptoms and in decreasing inflammatory mechanisms. Objectives: This study was designed in order to assess the possible therapeutic benefits of sema3A following its injection into female NZB/W mice. Methods: Forty-eight NZB/W mice were recruited for this study. Thirty mice were treated as a "prevention group" and 18 were used as a "treatment group." Eight-week-old mice were acclimated and then divided into the two abovementioned groups. Results: The injection of sema3A into young mice (at week 12) before the onset of disease (the prevention group) delayed the appearance of proteinuria. Here, the median time to severe proteinuria was 110 days, 95% CI: 88-131. However, in mice in which the empty vector was injected, the median time to severe proteinuria was 63 days, 95% CI: 0-139. sema3A treatment, significantly reduced renal damage, namely, it prevented the deposition of immune complexes in the glomeruli. When sema3A was injected at the onset of proteinuria (the treatment group), aiming to treat rather than to prevent disease in these mice, survival was increased and the deterioration of proteinuria was delayed. Conclusion: Semaphorin3A is highly beneficial in reducing lupus nephritis in NZB/W mice. It delays the appearance and deterioration of proteinuria, and increases the survival rates in these mice. The regulatory mechanisms of sema3A involve both innate and adaptive immune responses. Further studies will establish the idea of applying sema3A in the treatment of lupus nephritis.

The role of the plexin-A2 receptor in Sema3A and Sema3B signal transduction.

Class 3 semaphorins are anti-angiogenic and anti-tumorigenic guidance factors that bind to neuropilins, which, in turn, associate with class A plexins to transduce semaphorin signals. To study the role of the plexin-A2 receptor in semaphorin signaling, we silenced its expression in endothelial cells and in glioblastoma cells. The silencing did not affect Sema3A signaling, which depended on neuropilin-1, plexin-A1 and plexin-A4, but completely abolished Sema3B signaling, which also required plexin-A4 and one of the two neuropilins. Interestingly, overexpression of plexin-A2 in plexin-A1- or plexin-A4-silenced cells restored responses to both semaphorins, although it nullified their ability to differentiate between them, suggesting that, when overexpressed, plexin-A2 can functionally replace other class A plexins. By contrast, although plexin-A4 overexpression restored Sema3A signaling in plexin-A1-silenced cells, it failed to restore Sema3B signaling in plexin-A2-silenced cells. It follows that the identity of plexins in functional semaphorin receptors can be flexible depending on their expression level. Our results suggest that changes in the expression of plexins induced by microenvironmental cues can trigger differential responses of different populations of migrating cells to encountered gradients of semaphorins.

Semaphorin-3D and semaphorin-3E inhibit the development of tumors from glioblastoma cells implanted in the cortex of the brain.

Class-3 semaphorins are secreted axon guidance factors. Some of these semaphorins have recently been characterized as suppressors of tumor progression. To determine if class-3 semaphorins can be used to inhibit the development of glioblastoma-multiforme tumors, we expressed recombinant sema-3A, 3B, 3D, 3E, 3F or 3G in U87MG glioblastoma cells. Sema3A and sema3B expressing cells contracted and changed shape persistently while cells expressing other semaphorins did not. Sema3A and sema3F differed from other semaphorins including sema3B as they also inhibited the proliferation of the cells and the formation of soft agar colonies. With the exception of sema3G and sema3B, expression of these semaphorins in U87MG cells inhibited significantly tumor development from subcutaneously implanted cells. Strong inhibition of tumor development was also observed following implantation of U87MG cells expressing each of the class-3 semaphorins in the cortex of mouse brains. Sema3D and sema3E displayed the strongest inhibitory effects and their expression in U373MG or in U87MG glioblastoma cells implanted in the brains of mice prolonged the survival of the mice by more then two folds. Furthermore, most of the mice that died prior to the end of the experiment did not develop detectable tumors and many of the mice survived to the end of the experiment. Most of the semaphorins that we have used here with the exception of sema3D were characterized previously as inhibitors of angiogenesis. Our results indicate that sema3D also functions as an inhibitor of angiogenesis and suggest that the anti-tumorigenic effects are due primarily to inhibition of tumor angiogenesis. These results indicate that class-3 semaphorins such as sema3D and sema3E could perhaps be used to treat glioblastoma patients.

Semaphorins in angiogenesis and tumor progression.

The semaphorins were initially described as axon guidance factors, but have recently been implicated in a variety of physiological and developmental functions, including regulation of immune response, angiogenesis, and migration of neural crest cells. The semaphorin family contains more than 30 genes divided into seven subfamilies, all of which are characterized by the presence of a sema domain. The semaphorins transduce their signals by binding to one of the nine receptors belonging to the plexin family, or, in the case of the class 3 semaphorins, by binding to one of the two neuropilin receptors. Additional receptors, which form complexes with these primary semaphorin receptors, are also frequently involved in semaphorin signaling. Recent evidence suggests that some semaphorins can act as antiangiogenic and/or antitumorigenic agents whereas other semaphorins promote tumor progression and/or angiogenesis. Furthermore, loss of endogenous inhibitory semaphorin expression or function on one hand, and overexpression of protumorigenic semaphorins on the other hand, is associated with the progression of some tumor types.

The structural context of disease-causing mutations in gap junctions.

Gap junctions form intercellular channels that mediate metabolic and electrical signaling between neighboring cells in a tissue. Lack of an atomic resolution structure of the gap junction has made it difficult to identify interactions that stabilize its transmembrane domain. Using a recently computed model of this domain, which specifies the locations of each amino acid, we postulated the existence of several interactions and tested them experimentally. We introduced mutations within the transmembrane domain of the gap junction-forming protein connexin that were previously implicated in genetic diseases and that apparently destabilized the gap junction, as evidenced here by the absence of the protein from the sites of cell-cell apposition. The model structure helped identify positions on adjacent helices where second-site mutations restored membrane localization, revealing possible interactions between residue pairs. We thus identified two putative salt bridges and one pair involved in packing interactions in which one disease-causing mutation suppressed the effects of another. These results seem to reveal some of the physical forces that underlie the structural stability of the gap junction transmembrane domain and suggest that abrogation of such interactions bring about some of the effects of disease-causing mutations.

Connexins in hearing loss: a comprehensive overview.

Connexins are a family of transmembrane proteins that form gap junctions between adjacent cells and allow intercellular communication. Connexin proteins are involved in pathological conditions in humans, mainly in hearing loss, neurodegenerative disorders and skin diseases. The association between connexin proteins and the inner ear is well established. The abundant expression of connexins in the auditory system of the inner ear demonstrates their importance in inner ear development and the hearing process. Most compelling, there are over 100 mutations in genes encoding connexins that are associated with deafness. Most prominent is the remarkable involvement of connexin 26 in hearing loss. Mutations in the gene GJB2, encoding connexin 26, are responsible for around 50% of genetic cases of severe to profound non-syndromic hearing loss in some parts of the world. Learning more about the connexin family in general and about connexin 26 in particular can shed light on the pathogenesis of the inner ear and bring us closer to finding clinical solutions for the hearing impaired.