Suppression of bone formation by osteoclastic expression of semaphorin 4D

Most of the currently available drugs for osteoporosis inhibit osteoclastic bone resorption; only a few drugs promote osteoblastic bone formation. It is thus becoming increasingly necessary to identify the factors that regulate bone formation. We found that osteoclasts express semaphorin 4D (Sema4D), previously shown to be an axon guidance molecule, which potently inhibits bone formation. The binding of Sema4D to its receptor Plexin-B1 on osteoblasts resulted in the activation of the small GTPase RhoA, which inhibits bone formation by suppressing insulin-like growth factor-1 (IGF-1) signaling and by modulating osteoblast motility. Sema4d-/- mice, Plxnb1-/- mice and mice expressing a dominant-negative RhoA specifically in osteoblasts showed an osteosclerotic phenotype due to augmented bone formation. Notably, Sema4D-specific antibody treatment markedly prevented bone loss in a model of postmenopausal osteoporosis. Thus, Sema4D has emerged as a new therapeutic target for the discovery and development of bone-increasing drugs.     

Estrogen-Dependent Proteolytic Cleavage of Semaphorin 4D and Plexin-B1 Enhances Semaphorin 4D-Induced Apoptosis during Postnatal Vaginal Remodeling in Pubescent Mice

Around the fifth week after birth, the vaginal cavity in female mouse pups opens to the overlaying skin. This postnatal tissue remodeling of the genital tract occurs during puberty, and it largely depends upon hormonally induced apoptosis that mainly occurs in the epithelium at the lower part of the mouse vaginal cavity. Previously, we showed that most BALB/c mice lacking the class IV Semaphorin (Sema4D) develop imperforate vagina and hydrometrocolpos; therefore, we reasoned that the absence of Sema4D-induced apoptosis in vaginal epithelial cells may cause the imperforate vagina. Sema4D signals via the Plexin-B1 receptor; nevertheless detailed mechanisms mediating this hormonally triggered apoptosis are not fully documented. To investigate the estrogen-dependent control of Sema4D signaling during the apoptosis responsible for mouse vaginal opening, we examined structural and functional modulation of Sema4D, Plexin-B1, and signaling molecules by analyzing both wild-type and Sema4D−/− mice with or without ovariectomy. Both the release of soluble Sema4D and the conversion of Plexin-B1 by proteolytic processing in vaginal tissue peaked 5 weeks after birth of wild-type BALB/c mice at the time of vaginal opening. Estrogen supplementation of ovariectomized wild-type mice revealed that both the release of soluble Sema4D and the conversion of Plexin-B1 into an active form were estrogen-dependent and concordant with apoptosis. Estrogen supplementation of ovariectomized Sema4D−/− mice did not induce massive vaginal apoptosis in 5-week-old mice; therefore, Sema4D may be an essential apoptosis-inducing ligand that acts downstream of estrogen action in vaginal epithelium during this postnatal tissue remodeling. Analysis of ovariectomized mice also indicated that Sema4D contributed to estrogen-dependent dephosphorylation of Akt and ERK at the time of vaginal opening. Based on our results, we propose that apoptosis in vaginal epithelium during postnatal vaginal opening is induced by enhanced Sema4D signaling that is caused by estrogen-dependent structural changes of Sema4D and Plexin-B1.     

Ephrin receptor A10 monoclonal antibodies and the derived chimeric antigen receptor T cells exert an antitumor response in mouse models of triple-negative breast cancer

Expression of the receptor tyrosine kinase ephrin receptor A10 (EphA10), which is undetectable in most normal tissues except for the male testis, has been shown to correlate with tumor progression and poor prognosis in several malignancies, including triple-negative breast cancer (TNBC). Therefore, EphA10 could be a potential therapeutic target, likely with minimal adverse effects. However, no effective clinical drugs against EphA10 are currently available. Here, we report high expression levels of EphA10 in tumor regions of breast, lung, and ovarian cancers as well as in immunosuppressive myeloid cells in the tumor microenvironment. Furthermore, we developed anti-EphA10 monoclonal antibodies (mAbs) that specifically recognize cell surface EphA10, but not other EphA family isoforms, and target tumor regions precisely in vivo with no apparent accumulation in other organs. In syngeneic TNBC mouse models, we found that anti-EphA10 mAb clone #4 enhanced tumor regression, therapeutic response rate, and T cell–mediated antitumor immunity. Notably, the chimeric antigen receptor T cells derived from clone #4 significantly inhibited TNBC cell viability in vitro and tumor growth in vivo. Together, our findings suggest that targeting EphA10 via EphA10 mAbs and EphA10-specific chimeric antigen receptor–T cell therapy may represent a promising strategy for patients with EphA10-positive tumors.     

Sema4D/Plexin-B1 promotes the progression of osteosarcoma cells by activating Pyk2-PI3K-AKT pathway

Objectives:Osteosarcoma (OS) is one of the two most common malignant bone tumors among children and teens but it is still a rare disorder. Semaphorin 4D (Sema4D) has been reported to play a specific role in human cancers. The aim of this study was to explore the function of Sema4D in the tumorigenesis and development of OS.Methods:10 pairs of OS tissues and paracancerous normal tissues from human OS samples and OS cell lines were used. Western blot assay was performed to detect the protein expression of Sema4D, Plexin-B1, and associated proteins of Pyk2-PI3K/AKT pathway. To explore the effect of Sema4D in the progression of OS, we reduced the expression of Sema4D. The effect of Sema4D knockdown on cell proliferation was explored by CCK-8 assay and clone formation assay. The effect of Sema4D knockdown on cell migration and invasion was assessed by Transwell assay.Results:Sema4D was overexpressed in OS tissues and cell lines. Sema4D knockdown notably suppressed cell proliferation in OS cells. Cell migration and invasion were reduced by Sema4D knockdown. Sema4D/Plexin-B1 facilitated OS, progression by promoting Pyk2-PI3K/AKT pathway.Conclusion:Sema4D/Plexin-B1 promoted the development of OS so Sema4D might be a potential target of treatment for patients with OS.     

Semaphorin 4D/Plexin-B1 Stimulates PTEN Activity through R-Ras GTPase-activating Protein Activity,Inducing Growth Cone Collapse in Hippocampal Neurons

Plexins are receptors for axonal guidance molecules semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin-B1, suppresses PI3K signaling through the R-Ras GTPase-activating protein (GAP) activity, inducing growth cone collapse. Phosphatidylinositol 3-phosphate level is critically regulated by PI3K and PTEN (phosphatase and tensin homologue deleted chromosome ten). Here we examined the involvement of PTEN in the Plexin-B1-induced repulsive response. Phosphorylation of PTEN at Ser-380 is known to suppress its phosphatase activity. Sema4D induced the dephosphorylation of PTEN at Ser-380 and stimulated PTEN phosphatase activity in hippocampal neurons. Knockdown of endogenous PTEN suppressed the Sema4D-induced growth cone collapse. Phosphorylation mimic PTEN mutant suppressed the Sema4D-induced growth cone collapse, whereas phosphorylation-resistant PTEN mutant by itself induced growth cone collapse. Plexin-B1-induced PTEN dephosphorylation through R-Ras GAP activity and R-Ras GAP activity was by itself sufficient for PTEN dephosphorylation and activation. We also suggested that the Sema4D-induced PTEN dephosphorylation and growth cone collapse were mediated by the inhibition of casein kinase 2 α activity. Thus, we propose that Sema4D/Plexin-B1 promotes the dephosphorylation and activation of PTEN through the R-Ras GAP activity, inducing growth cone collapse.     

Improvement in in?vitro fertilization outcome following in?vivo synchronization of oocyte maturation in mice

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.     

Semaphorin 4D/Plexin-B1-mediated R-Ras GAP activity inhibits cell migration by regulating beta(1) integrin activity

Plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. We recently reported that the semaphorin 4D (Sema4D) receptor Plexin-B1 functions as a GTPase-activating protein (GAP) for R-Ras, a member of Ras family GTPases implicated in regulation of integrin activity and cell migration. We characterized the role of R-Ras downstream of Sema4D/Plexin-B1 in cell migration. Activation of Plexin-B1 by Sema4D suppressed the ECM-dependent R-Ras activation, R-Ras-mediated phosphatydylinositol 3-kinase activation, and beta(1) integrin activation through its R-Ras GAP domain, leading to inhibition of cell migration. In addition, inactivation of R-Ras by overexpression of the R-Ras-specific GAP or knockdown of R-Ras by RNA interference was sufficient for suppressing beta(1) integrin activation and cell migration in response to the ECM stimulation. Thus, we conclude that R-Ras activity is critical for ECM-mediated beta(1) integrin activation and cell migration and that inactivation of R-Ras by Sema4D/Plexin-B1-mediated R-Ras GAP activity controls cell migration by modulating the activity of beta(1) integrins.     

Identification of thyroid hormone response elements in?vivo using mice expressing a tagged thyroid hormone receptor α1

TRα1 (thyroid hormone receptor α1) is well recognized for its importance in brain development. However, due to the difficulties in predicting TREs (thyroid hormone response elements) in silico and the lack of suitable antibodies against TRα1 for ChIP (chromatin immunoprecipitation), only a few direct TRα1 target genes have been identified in the brain. Here we demonstrate that mice expressing a TRα1–GFP (green fluorescent protein) fusion protein from the endogenous TRα locus provide a valuable animal model to identify TRα1 target genes. To this end, we analysed DNA–TRα1 interactions in vivo using ChIP with an anti-GFP antibody. We validated our system using established TREs from neurogranin and hairless, and by verifying additional TREs from known TRα1 target genes in brain and heart. Moreover, our model system enabled the identification of novel TRα1 target genes such as RNF166 (ring finger protein 166). Our results demonstrate that transgenic mice expressing a tagged nuclear receptor constitute a feasible approach to study receptor–DNA interactions in vivo, circumventing the need for specific antibodies. Models like the TRα1–GFP mice may thus pave the way for genome-wide mapping of nuclear receptor-binding sites, and advance the identification of novel target genes in vivo.     

A human IgE bispecific antibody shows potent cytotoxic capacity mediated by monocytes

The generation of bispecific antibodies (bsAbs) targeting two different antigens opens a new level of specificity and, compared to mAbs, improved clinical efficacy in cancer therapy. Currently, the different strategies for development of bsAbs primarily focus on IgG isotypes. Nevertheless, in comparison to IgG isotypes, IgE has been shown to offer superior tumor control in preclinical models. Therefore, in order to combine the promising potential of IgE molecules with increased target selectivity of bsAbs, we developed dual tumor-associated antigen-targeting bispecific human IgE antibodies. As proof of principle, we used two different pairing approaches - knobs-into-holes and leucine zipper–mediated pairing. Our data show that both strategies were highly efficient in driving bispecific IgE formation, with no undesired pairings observed. Bispecific IgE antibodies also showed a dose-dependent binding to their target antigens, and cell bridging experiments demonstrated simultaneous binding of two different antigens. As antibodies mediate a major part of their effector functions through interaction with Fc receptors (FcRs) expressed on immune cells, we confirmed FcεR binding by inducing in vitro mast cell degranulation and demonstrating in vitro and in vivo monocyte-mediated cytotoxicity against target antigen-expressing Chinese hamster ovary cells. Moreover, we demonstrated that the IgE bsAb construct was significantly more efficient in mediating antibody-dependent cell toxicity than its IgG1 counterpart. In conclusion, we describe the successful development of first bispecific IgE antibodies with superior antibody-dependent cell toxicity–mediated cell killing in comparison to IgG bispecific antibodies. These findings highlight the relevance of IgE-based bispecific antibodies for clinical application.     

Plexin-B1 activates NF-κB and IL-8 to promote a pro-angiogenic response in endothelial cells

Background

The semaphorins and their receptors, the plexins, are proteins related to c-Met and the scatter factors that have been implicated in an expanding signal transduction network involving co-receptors, RhoA and Ras activation and deactivation, and phosphorylation events. Our previous work has demonstrated that Semaphorin 4D (Sema4D) acts through its receptor, Plexin-B1, on endothelial cells to promote angiogenesis in a RhoA and Akt-dependent manner. Since NF-κB has been linked to promotion of angiogenesis and can be activated by Akt in some contexts, we wanted to examine NF-κB in Sema4D treated cells to determine if there was biological significance for the pro-angiogenic phenotype observed in endothelium.

Methods/Principal Findings

Using RNA interference techniques, gel shifts and NF-κB reporter assays, we demonstrated NF-κB translocation to the nucleus in Sema4D treated endothelial cells occurring downstream of Plexin-B1. This response was necessary for endothelial cell migration and capillary tube formation and protected endothelial cells against apoptosis as well, but had no effect on cell proliferation. We dissected Plexin-B1 signaling with chimeric receptor constructs and discovered that the ability to activate NF-κB was dependent upon Plexin-B1 acting through Rho and Akt, but did not involve its role as a Ras inhibitor. Indeed, inhibition of Rho by C3 toxin and Akt by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 blocked Sema4D-mediated endothelial cell migration and tubulogenesis. We also observed that Sema4D treatment of endothelial cells induced production of the NF-κB downstream target IL-8, a response necessary for angiogenesis. Finally, we could show through co-immunofluorescence for p65 and CD31 that Sema4D produced by tumor xenografts in nude mice activated NF-κB in vessels of the tumor stroma.

Conclusion/Significance

These findings provide evidence that Sema4D/Plexin-B1-mediated NF-κB activation and IL-8 production is critical in the generation a pro-angiogenic phenotype in endothelial cells and suggests a new therapeutic target for the anti-angiogenic treatment of some cancers.     

Hypoxia-inducible Factor-1-mediated Regulation of Semaphorin 4D Affects Tumor Growth and Vascularity

Tumor progression and metastasis depend on the ability of cancer cells to initiate angiogenesis to ensure delivery of oxygen, nutrients, and growth factors to tumor cells and provide access to the systemic circulation. Hypoxia-inducible factor-1 (HIF-1) can activate expression of a broad range of genes that mediate many of the adaptive responses to decreased oxygen concentration, such as enhanced glucose uptake and formation of new blood vessels. Acting through Plexin-B1 on endothelial cells, Semaphorin 4D (Sema4D) has been shown to promote angiogenesis and enhance invasive growth and proliferation in some tumors. Here we show that the gene for Sema4D, the product of which is elevated in head and neck squamous cell carcinoma (HNSCC) cells, contains upstream hypoxia response elements (HRE) and is strongly induced in hypoxia in a HIF-1-dependent manner. Knocking down Sema4D expression with short hairpin (sh) RNA reduces in vitro endothelial cell migration and growth and vascularity of HNSCC xenografts expressing a degradation resistant HIF-1α subunit. We also demonstrate a correlation between HIF-1 activity and Sema4D expression in HNSCC specimens. These findings indicate that Sema4D is induced by hypoxia in a HIF-1-dependent manner and influences endothelial cell migration and tumor vascularity. Expression of Sema4D may be a strategy by which carcinomas promote angiogenesis and therefore could represent a therapeutic target for these malignancies.     

The conserved C2 phospholipid‐binding domain in Delta contributes to robust Notch signalling

Accurate Notch signalling is critical for development and homeostasis. Fine‐tuning of Notch–ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N‐terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so‐called β1‐2 loop that is involved in phospholipid binding. Mutations in the β1‐2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the β1‐2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss‐of‐function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine‐tuning the balance of trans and cis ligand–receptor interactions.     

Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity

Functional delivery of mRNA has high clinical potential. Previous studies established that mRNAs can be delivered to cells in vitro and in vivo via RNA-loaded lipid nanoparticles (LNPs). Here we describe an alternative approach using exosomes, the only biologically normal nanovesicle. In contrast to LNPs, which elicited pronounced cellular toxicity, exosomes had no adverse effects in vitro or in vivo at any dose tested. Moreover, mRNA-loaded exosomes were characterized by efficient mRNA encapsulation (∼90%), high mRNA content, consistent size, and a polydispersity index under 0.2. Using an mRNA encoding the red light-emitting luciferase Antares2, we observed that mRNA-loaded exosomes were superior to mRNA-loaded LNPs at delivering functional mRNA into human cells in vitro. Injection of Antares2 mRNA-loaded exosomes also led to strong light emission following injection into the vitreous fluid of the eye or into the tissue of skeletal muscle in mice. Furthermore, we show that repeated injection of Antares2 mRNA-loaded exosomes drove sustained luciferase expression across six injections spanning at least 10 weeks, without evidence of signal attenuation or adverse injection site responses. Consistent with these findings, we observed that exosomes loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins induced long-lasting cellular and humoral responses to both. Taken together, these results demonstrate that exosomes can be used to deliver functional mRNA to and into cells in vivo.     

Direct interaction of Rnd1 with Plexin-B1 regulates PDZ-RhoGEF-mediated Rho activation by Plexin-B1 and induces cell contraction in COS-7 cells

Plexins are receptors for the axon guidance molecule semaphorins, and several lines of evidence suggest that Rho family small GTPases are implicated in the downstream signaling of Plexins. Recent studies have demonstrated that Plexin-B1 activates RhoA and induces growth cone collapse through Rho-specific guanine nucleotide exchange factor PDZ-RhoGEF. Here we show that Rnd1, a member of Rho family GTPases, directly interacted with the cytoplasmic domain of Plexin-B1. In COS-7 cells, coexpression of Rnd1 and Plexin-B1 induced cell contraction in response to semaphorin 4D (Sema4D), a ligand for Plexin-B1, whereas expression of Plexin-B1 alone or coexpression of Rnd1 and a Rnd1 interaction-defective mutant of Plexin-B1 did not. The Sema4D-induced contraction in Plexin-B1/Rnd1-expressing COS-7 cells was suppressed by dominant negative RhoA, a Rho-associated kinase inhibitor, a dominant negative form of PDZ-RhoGEF, or deletion of the carboxyl-terminal PDZ-RhoGEF-binding region of Plexin-B1, indicating that the PDZ-RhoGEF/RhoA/Rho-associated kinase pathway is involved in this morphological effect. We also found that Rnd1 promoted the interaction between Plexin-B1 and PDZ-RhoGEF and thereby dramatically potentiated the Plexin-B1-mediated RhoA activation. We propose that Rnd1 plays an important role in the regulation of Plexin-B1 signaling, leading to Rho activation during axon guidance and cell migration.     

Discovery of a small-molecule inhibitor of the TRIP8b–HCN interaction with efficacy in neurons

Major depressive disorder is a critical public health problem with a lifetime prevalence of nearly 17% in the United States. One potential therapeutic target is the interaction between hyperpolarization-activated cyclic nucleotide–gated (HCN) channels and an auxiliary subunit of the channel named tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b). HCN channels regulate neuronal excitability in the mammalian hippocampus, and recent work has established that antagonizing HCN function rescues cognitive impairment caused by chronic stress. Here, we utilize a high-throughput virtual screen to find small molecules capable of disrupting the TRIP8b–HCN interaction. We found that the hit compound NUCC-0200590 disrupts the TRIP8b–HCN interaction in vitro and in vivo. These results provide a compelling strategy for developing new small molecules capable of disrupting the TRIP8b–HCN interaction.     

Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication

Zinc pyrithione (1a), together with its analogues 1b–h and ruthenium pyrithione complex 2a, were synthesised and evaluated for the stability in biologically relevant media and anti-SARS-CoV-2 activity. Zinc pyrithione revealed potent in vitro inhibition of cathepsin L (IC₅₀=1.88 ± 0.49 µM) and PL^Pro (IC₅₀=0.50 ± 0.07 µM), enzymes involved in SARS-CoV-2 entry and replication, respectively, as well as antiviral entry and replication properties in an ex vivo system derived from primary human lung tissue. Zinc complexes 1b–h expressed comparable in vitro inhibition. On the contrary, ruthenium complex 2a and the ligand pyrithione a itself expressed poor inhibition in mentioned assays, indicating the importance of the selection of metal core and structure of metal complex for antiviral activity. Safe, effective, and preferably oral at-home therapeutics for COVID-19 are needed and as such zinc pyrithione, which is also commercially available, could be considered as a potential therapeutic agent against SARS-CoV-2.     

Bone morphogenetic protein 6–mediated crosstalk between endothelial cells and hepatocytes recapitulates the iron-sensing pathway in vitro

Liver sinusoidal endothelial cell–derived bone morphogenetic protein 6 (BMP6) and the BMP6–small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte–endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.