H-2g, a glucose analog of blood group H antigen, mediates mononuclear cell recruitment via Src and phosphatidylinositol 3-kinase pathways

Objective Monocyte recruitment by proinflammatory cytokines is a hallmark of rheumatoid arthritis (RA). Lewis y-6 and H (Le y /H) are blood group antigens up-regulated on RA synovial endothelium. We have previously shown that both soluble Le y /H and a glucose analog of H, H-2g, are angiogenic and mediateleukocyte–endothelial adhesion via induction of intercellular adhesion molecule 1. We hypothesized that soluble Le y /H plays an important role in monocyte recruitment in RA. Methods We examined the role of H-2g in monocyte chemotaxis in vitro. We used an RA synovial tissue (ST)–SCID mouse chimera model to evaluate the role of H-2g in monocyte recruitment in vivo. We used Western blots to examine signaling molecules activated by H-2g in monocytes. Results H-2g induced human monocyte migration in vitro, which was mediated by Src and phosphatidylinositol 3-kinase (PI 3-kinase), since inhibitors and antisense oligodeoxynucleotides (ODNs) of Src and PI 3-kinase significantly decreased H-2g–induced monocyte migration ( P < 0.05). H-2g significantly increased mononuclear cell (MNC) homing in vivo into an RA ST–SCID mouse chimera ( P < 0.05). Transfection of MNCs with Src antisense ODNs blocked H-2g–induced MNC recruitment into the RA ST–SCID mouse chimera. Additionally, H-2g induced marked phosphorylation of protein kinase CΑI/ΒII (PKCΑI/ΒII), Src, IΚBΑ, and Akt in monocytes. Src, Akt, and NF-ΚB were shown to be downstream targets of PKCΑI/ΒII, since an inhibitor of PKCΑI/ΒII reduced H-2g–mediated phosphorylation of Src, Akt, and NF-ΚB in monocytes. Conclusion These data suggest that H-2g may be a novel mediator of monocyte recruitment in chronic inflammatory diseases like RA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58062/1/23296_ftp.pd

VEGF binding to NRP1 is essential for VEGF stimulation of endothelial cell migration, complex formation between NRP1 and VEGFR2, and signaling via FAK Tyr407 phosphorylation

In endothelial cells, neuropilin-1 (NRP1) binds vascular endothelial growth factor (VEGF)-A and is thought to act as a coreceptor for kinase insert domain-containing receptor (KDR) by associating with KDR and enhancing VEGF signaling. Here we report mutations in the NRP1 b1 domain (Y297A and D320A), which result in complete loss of VEGF binding. Overexpression of Y297A and D320A NRP1 in human umbilical vein endothelial cells reduced high-affinity VEGF binding and migration toward a VEGF gradient, and markedly inhibited VEGF-induced angiogenesis in a coculture cell model. The Y297A NRP1 mutant also disrupted complexation between NRP1 and KDR and decreased VEGF-dependent phosphorylation of focal adhesion kinase at Tyr407, but had little effect on other signaling pathways. Y297A NRP1, however, heterodimerized with wild-type NRP1 and NRP2 indicating that nonbinding NRP1 mutants can act in a dominant-negative manner through formation of NRP1 dimers with reduced binding affinity for VEGF. These findings indicate that VEGF binding to NRP1 has specific effects on endothelial cell signaling and is important for endothelial cell migration and angiogenesis mediated via complex formation between NRP1 and KDR and increased signaling to focal adhesions. Identification of key residues essential for VEGF binding and biological functions provides the basis for a rational design of antagonists of VEGF binding to NRP1

Mammalian Sperm Head Formation Involves Different Polarization of Two Novel LINC Complexes

Background: LINC complexes are nuclear envelope bridging protein structures formed by interaction of SUN and KASH proteins. They physically connect the nucleus with the peripheral cytoskeleton and are critically involved in a variety of dynamic processes, such as nuclear anchorage, movement and positioning and meiotic chromosome dynamics. Moreover, they are shown to be essential for maintaining nuclear shape. Findings: Based on detailed expression analysis and biochemical approaches, we show here that during mouse sperm development, a terminal cell differentiation process characterized by profound morphogenic restructuring, two novel distinctive LINC complexes are established. They consist either of spermiogenesis-specific Sun3 and Nesprin1 or Sun1g, a novel non-nuclear Sun1 isoform, and Nesprin3. We could find that these two LINC complexes specifically polarize to opposite spermatid poles likely linking to sperm-specific cytoskeletal structures. Although, as shown in co-transfection/ immunoprecipitation experiments, SUN proteins appear to arbitrarily interact with various KASH partners, our study demonstrates that they actually are able to confine their binding to form distinct LINC complexes. Conclusions: Formation of the mammalian sperm head involves assembly and different polarization of two novel spermiogenesis-specific LINC complexes. Together, our findings suggest that theses LINC complexes connect the differentiating spermatid nucleus to surrounding cytoskeletal structures to enable its well-directed shaping and elongation

Role of β-Catenin in Post-Meiotic Male Germ Cell Differentiation

Though roles of β-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that β-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis in mice. Spermatid-specific deletion of β-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of β-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids (apical ectoplasmic specialization; ES). These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that β-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since β-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell β-catenin complex to β-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation

Organization of multiprotein complexes at cell–cell junctions

The formation of stable cell–cell contacts is required for the generation of barrier-forming sheets of epithelial and endothelial cells. During various physiological processes like tissue development, wound healing or tumorigenesis, cellular junctions are reorganized to allow the release or the incorporation of individual cells. Cell–cell contact formation is regulated by multiprotein complexes which are localized at specific structures along the lateral cell junctions like the tight junctions and adherens junctions and which are targeted to these site through their association with cell adhesion molecules. Recent evidence indicates that several major protein complexes exist which have distinct functions during junction formation. However, this evidence also indicates that their composition is dynamic and subject to changes depending on the state of junction maturation. Thus, cell–cell contact formation and integrity is regulated by a complex network of protein complexes. Imbalancing this network by oncogenic proteins or pathogens results in barrier breakdown and eventually in cancer. Here, I will review the molecular organization of the major multiprotein complexes at junctions of epithelial cells and discuss their function in cell–cell contact formation and maintenance

Novel Role for p110β PI 3-Kinase in Male Fertility through Regulation of Androgen Receptor Activity in Sertoli Cells

We thank Anna-Lena Berg (AstraZeneca, Lund) and Cheryl Scudamore (MRC, Harwell, UK) for histological analysis, Julie Foster (Barts Cancer Institute, London) for CT scans, Johan Swinnen and Frank Claessens (Leuven University, Belgium) for discussion and AR-luciferase reporter plasmids, Florian Guillou (INRA, CNRS, Université de Tours, France) for the AMH-Cre mouse line and Laura Milne (MRC Centre for Reproductive Health, The University of Edinburgh) for technical support. We thank the members of the Cell Signalling group for critical input.International audienceThe organismal roles of the ubiquitously expressed class I PI3K isoform p110β remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110β, we document that full inactivation of p110β leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110β kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110β results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110β was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110β also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110β inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110β inactivation. In line with a crucial role for p110β in SCs, selective inactivation of p110β in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110β and AR have previously been reported to functionally interac

Signalling mechanisms mediating biological functions of vascular endothelial growth factor in endothelial cells

It was previously reported that VEGF stimulates prostacyclin (PGI2) production in human umbilical vein endothelial cells (HUVECs) via activation of the extracellular signal-regulated kinase (ERK) cascade. For this thesis, the contribution of other signalling pathways in VEGF-induced PGI2 production was investigated. Work carried out also intended to elucidate the signalling mechanisms that mediate VEGF-induced activation of ERK in HUVECs. VEGF- induced cytosolic phospholipase A2 (cPLA2) phosphorylation and PGI2 production were blocked by GF109203X and Calphostin C, two structurally unrelated, specific PKC inhibitors. These inhibitors also blocked VEGF-induced ERK and MEK (mitogen-activated protein kinase kinase) phosphorylation. VEGF increased PKC activity and immunoreactivity of PKCδ, α and ԑ in the particulate fraction of HUVECs while the [symbol] isoform was unaffected. Association of Raf 1 (the upstream activator of the MEK-ERK pathway) with PKCδ was increased by VEGF, as determined by co-immunoprecipitation of Raf 1 with PKCδ. A PKCδ-selective inhibitor, rottlerin, abrogated VEGF-induced ERK activation and PGI2 production while a PKCα-selective inhibitor, Go6976, had no effect on ERK activation but partially inhibited PGI2 production in response to VEGF. Antisense oligonucleotides against PKCδ, but not against PKCα, reduced VEGF-induced ERK activation. Inhibition of Ca2+ fluxes by BAPTA/AM inhibited PGI2 production but had no effect on VEGF-induced ERK activation. Neither inhibition nor activation of the NO/cGMP pathway had any effect on ERK activation and PGI2 synthesis. These findings indicate that activation of PKC plays a crucial role in VEGF signalling via the ERK cascade and implicate PKCδ as a key mediator of this pathway. The role of PKC in angiogenesis was also addressed. Inhibitors of PKC markedly decreased VEGF-induced angiogenesis, as determined in an in vitro model of tube formation. PKC inhibitors and PKC downregulation, induced by prolonged phorbol ester treatment, inhibited VEGF-induced phosphorylation of Akt, the PI3K effector. These findings suggest that PKC mediates VEGF signalling via the PI3K/Akt pathway. In conclusion, the results from this thesis indicate that PKC is a crucial mediator of VEGF signal transduction pathways and of two biological functions of VEGF, namely PGI2 production and angiogenesis. These results also suggest that distinct PKC isoforms are utilized by VEGF in eliciting distinct biological effects