Polysialyltransferase expression is linked to neuronal migration in the developing and adult zebrafish.

Modulation of cell-cell adhesion is crucial for regulating neuronal migration and maintenance of structural plasticity in the embryonic and mature brain. Such modulation can be obtained by the enzymatic attachment of polysialic acid (PSA) to the neural cell adhesion molecule (NCAM) by means of the polysialyltransferases STX and PST. Thus, differential expression of STX and PST is likely to be responsible for varying functions of PSA-NCAM during neuronal differentiation, maintenance, plasticity, and regeneration. We have isolated the zebrafish homologues of STX (St8sia2) and PST (St8sia4) and demonstrate that their expression in the embryonic and adult nervous system is often confined to regions of neuronal migration. Moreover, in the adult cerebellum, the complementary expression pattern of both polysialyltransferases suggests a function in regulating cerebellar neuronal plasticity. Enzymatic removal of PSA in the embryonic cerebellum results in impaired neuronal migration, suggesting that PSA-NCAM is a key regulator of motility for cerebellar neuronal progenitors.     

Sphingosine‐1‐phosphate activates chemokine‐promoted myeloma cell adhesion and migration involving α4β1 integrin function

Myeloma cell adhesion dependent on α4β1 integrin is crucial for the progression of multiple myeloma (MM). The α4β1‐dependent myeloma cell adhesion is up‐regulated by the chemokine CXCL12, and pharmacological blockade of the CXCL12 receptor CXCR4 leads to defective myeloma cell homing to bone marrow (BM). Sphingosine‐1‐phosphate (S1P) regulates immune cell trafficking upon binding to G‐protein‐coupled receptors. Here we show that myeloma cells express S1P1, a receptor for S1P. We found that S1P up‐regulated the α4β1‐mediated myeloma cell adhesion and transendothelial migration stimulated by CXCL12. S1P promoted generation of high‐affinity α4β1 that efficiently bound the α4β1 ligand VCAM‐1, a finding that was associated with S1P‐triggered increase in talin‐β1 integrin association. Furthermore, S1P cooperated with CXCL12 for enhancement of α4β1‐dependent adhesion strengthening and spreading. CXCL12 and S1P activated the DOCK2‐Rac1 pathway, which was required for stimulation of myeloma cell adhesion involving α4β1. Moreover, in vivo analyses indicated that S1P contributes to optimizing the interactions of MM cells with the BM microvasculture and for their lodging inside the bone marrow. The regulation of α4β1‐dependent adhesion and migration of myeloma cells by CXCL12‐S1P combined activities might have important consequences for myeloma disease progression. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Prognostic significance of urokinase plasminogen activator receptor and its cleaved forms in blood from patients with non‐small cell lung cancer

Urokinase plasminogen activator (uPA) cleaves its three‐domain cell surface receptor, uPAR, liberating domain I [uPAR(I)] and leaving the cleaved uPAR(II‐III) on the cell surface. Both intact and cleaved uPAR can be shed from the cell surface. uPAR(I) was previously shown to be a prognostic factor in lung tumour extracts. Here we analyse uPAR forms in blood from patients with non‐small cell lung cancer (NSCLC). Preoperatively sampled plasma/serum from 32 patients with NSCLC was analysed. Three time‐resolved fluoroimmunoassays (TR‐FIAs) measuring intact uPAR(I‐III) (TR‐FIA 1), uPAR(I‐III) + uPAR(II‐III) (TR‐FIA 2) and uPAR(I) (TR‐FIA 3) were applied. The Spearman rank correlations between plasma and serum levels of uPAR(I‐III), uPAR(I‐III) + uPAR(II‐III), and uPAR(I) were 0.89, 0.94 and 0.68 respectively. Survival analysis demonstrated that high levels of all uPAR forms were associated with shorter survival. Adjusted for histological subtype high plasma uPAR(I‐III) and uPAR(I) levels as well as serum uPAR(I) levels were significantly associated with shorter OS (hazards ratios = 4.3, 2.8 and 3.8 respectively). High blood levels of intact uPAR and its cleaved forms are associated with poor prognosis in NSCLC.     

Selected signalling proteins recruited to the T‐cell receptor–CD3 complex

The T‐cell receptor (TCR)–CD3 complex, expressed on T cells, determines the outcome of a T‐cell response. It consists of the TCR‐αβ heterodimer and the non‐covalently associated signalling dimers of CD3εγ, CD3εδ and CD3ζζ. TCR‐αβ binds specifically to a cognate peptide antigen bound to an MHC molecule, whereas the CD3 subunits transmit the signal into the cytosol to activate signalling events. Recruitment of proteins to specialized localizations is one mechanism to regulate activation and termination of signalling. In the last 25 years a large number of signalling molecules recruited to the TCR–CD3 complex upon antigen binding to TCR‐αβ have been described. Here, we review knowledge about five of those interaction partners: Lck, ZAP‐70, Nck, WASP and Numb. Some of these proteins have been targeted in the development of immunomodulatory drugs aiming to treat patients with autoimmune diseases and organ transplants.     

Endoplasmic reticulum chaperone gp96 in macrophages is essential for protective immunity during Gram‐negative pneumonia

Klebsiella pneumoniae is among the most common Gram‐negative bacteria that cause pneumonia. Gp96 is an endoplasmic reticulum chaperone that is essential for the trafficking and function of Toll‐like receptors (TLRs) and integrins. To determine the role of gp96 in myeloid cells in host defence during Klebsiella pneumonia, mice homozygous for the conditional Hsp90b1 allele encoding gp96 were crossed with mice expressing Cre‐recombinase under control of the LysM promoter to generate LysMcre‐Hsp90b1‐flox mice. LysMcre‐Hsp90b1‐flox mice showed absence of gp96 protein in macrophages and partial depletion in monocytes and granulocytes. This was accompanied by almost complete absence of TLR2 and TLR4 on macrophages. Likewise, integrin subunits CD11b and CD18 were not detectable on macrophages, while being only slightly reduced on monocytes and granulocytes. Gp96‐deficient macrophages did not release pro‐inflammatory cytokines in response to Klebsiella and displayed reduced phagocytic capacity independent of CD18. LysMcre‐Hsp90b1‐flox mice were highly vulnerable to lower airway infection induced by K. pneumoniae, as reflected by enhanced bacterial growth and a higher mortality rate. The early inflammatory response in Hsp90b1‐flox mice was characterized by strongly impaired recruitment of granulocytes into the lungs, accompanied by attenuated production of pro‐inflammatory cytokines, while the inflammatory response during late‐stage pneumonia was not dependent on the presence of gp96. Blocking CD18 did not reproduce the impaired host defence of LysMcre‐Hsp90b1‐flox mice during Klebsiella pneumonia. These data indicate that macrophage gp96 is essential for protective immunity during Gram‐negative pneumonia by regulating TLR expression. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

IgSF molecule MDGA1 is involved in radial migration and positioning of a subset of cortical upper‐layer neurons

Mdga1, encoding a GPI‐anchored immunoglobulin superfamily molecule containing an MAM domain, is expressed by a specific subset of neurons, including layer II/III projection neurons, in the mouse neocortex. To investigate the function of Mdga1 in corticogenesis, we generated Mdga1‐deficient mice and backcrossed them to obtain a congenic background. Gross anatomy of the Mdga1‐deficient brain at postnatal day (P) 14 showed no obvious phenotype. However, the migration of Mdga1‐mutant neurons to the superficial cortical plate was clearly delayed. Most Mdga1‐mutant neurons reached the lower portion of the upper cortical layer by embryonic day 18.5 and stayed there through P0. By P7, the location of the mutant cells was the same as wild‐type. The location of Cux2‐expressing upper‐layer neurons in the cortical plate was largely unaffected. These observations indicated that Mdga1 is involved in the migration and positioning of a subset of cortical neurons and suggested that the radial migration of upper‐layer neurons might be differentially regulated. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

Plasmacytoid dendritic cell‐induced migration and activation of NK cells in vivo

NK cells are cytotoxic cells of the innate immune system. They have been found to be critical in the defense against infections and also against some tumors. Recent studies have shown that NK cells require signals from accessory cells to induce their recruitment and activation at the site of infection or tumor growth. In this study, we examined whether plasmacytoid DC (pDC) could recruit and activate NK cells in vivo. When CpG‐stimulated pDC were injected i.p. to C57BL/6 mice, they efficiently recruited NK cells, a process that was dependent on NK cell CXCR3 and CD62L and in part on CCR5. NK cells isolated from the peritoneum of mice inoculated with TLR7/8 or TLR9‐stimulated pDC exhibited greater cytotoxicity against YAC‐1 tumor cells than NK cells recovered from mice inoculated with control pDC. The present results are discussed in relation to pDC‐induced NK cell migration and activation in vivo.     

Macrophage migration inhibitory factor of Sciaenops ocellatus regulates immune cell trafficking and is involved in pathogen-induced immune response

Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine involved in immunoregulation and inflammation. In this study, we examined the expression and biological function of a MIF, SoMIF, from red drum Sciaenops ocellatus. SoMIF is composed of 115 residues and shares 85–99% overall sequence identities with the MIF of a number of teleost. SoMIF expression was detected in a wide range of tissues and upregulated by bacterial and viral infection in a time-dependent manner. In head kidney (HK) leukocytes, pathogen infection induced SoMIF expression, and the expressed SoMIF was secreted into the extracellular milieu. Recombinant SoMIF (rSoMIF) purified from Escherichia coli inhibited the migration of both HK monocytes and lymphocytes, and this inhibitory effect was abolished by the presence of anti-rSoMIF antibodies. When rSoMIF was administered into red drum, it stimulated the production of reactive oxygen species in HK monocytes both in the presence and absence of pathogen infection. In vivo infection study showed that compared to untreated fish, fish pre-treated with rSoMIF before bacterial infection exhibited significantly lower bacterial loads in blood, kidney, spleen, and liver. Taken together, these results indicate that SoMIF is a secreted protein that regulates immune cell trafficking and is involved in pathogen-induced immune response.     

Nasal embryonic LHRH factor plays a role in the developmental migration and projection of gonadotropin‐releasing hormone 3 neurons in zebrafish

The initiation of puberty and the functioning of the reproductive system depend on proper development of the hypophysiotropic gonadotropin‐releasing hormone (GnRH) system. One critical step in this process is the embryonic migration of GnRH neurons from the olfactory area to the hypothalamus. Using a transgenic zebrafish model, Tg(gnrh3:EGFP), in which GnRH3 neurons and axons are fluorescently labeled, we investigated whether zebrafish NELF is essential for the development of GnRH3 neurons. The zebrafish nelf cDNA was cloned and characterized. During embryonic development, nelf is expressed in GnRH3 neurons and in target sites of GnRH3 projections and perikarya, before the initiation of their migration. Nelf knockdown resulted in a disruption of the GnRH3 system which included absence or misguiding of GnRH3 axonal outgrowth and incorrect or arrested migration of GnRH3 perikarya. These results suggest that Nelf is an important factor in the developmental migration and projection of GnRH3 neurons in zebrafish. Developmental Dynamics 238:66–75, 2009. © 2008 Wiley‐Liss, Inc.     

Myosin‐X is required for cranial neural crest cell migration in Xenopus laevis

Myosin‐X (MyoX) belongs to a large family of unconventional, nonmuscle, actin‐dependent motor proteins. We show that MyoX is predominantly expressed in cranial neural crest (CNC) cells in embryos of Xenopus laevis and is required for head and jaw cartilage development. Knockdown of MyoX expression using antisense morpholino oligonucleotides resulted in retarded migration of CNC cells into the pharyngeal arches, leading to subsequent hypoplasia of cartilage and inhibited outgrowth of the CNC‐derived trigeminal nerve. In vitro migration assays on fibronectin using explanted CNC cells showed significant inhibition of filopodia formation, cell attachment, spreading and migration, accompanied by disruption of the actin cytoskeleton. These data support the conclusion that MyoX has an essential function in CNC migration in the vertebrate embryo. Developmental Dynamics 238:2522–2529, 2009. © Published 2009 Wiley‐Liss, Inc.^?     

A novel role of CXCR4 and SDF‐1 during migration of cloacal muscle precursors

The cloaca acts as a common chamber into which gastrointestinal and urogenital tracts converge in lower vertebrates. The distal end of the cloaca is guarded by a ring of cloacal muscles or sphincters, the equivalent of perineal muscles in mammals. It has recently been shown that the development of the cloacal musculature depends on hindlimb muscle formation. The signaling molecules responsible for the outward migration of hindlimb myogenic precursors are not known. Based on the expression studies for CXCR4 and SDF‐1, we hypothesized a role of this signaling pair during cloacal muscle precursor migration. The aim of our study was to investigate the role of SDF‐1/CXCR4 during cloacal muscle precursor migration in the chicken embryos. We show that SDF‐1 is expressed in the cloacal region, and by experimentally manipulating the SDF‐1/CXCR4 signaling, we can show that SDF‐1 guides the migration of CXCR4‐expressing cloacal muscle precursors. Developmental Dynamics 239:1622–1631, 2010. © 2010 Wiley‐Liss, Inc.     

Tetraspanins CD9 and CD151 at the immune synapse support T‐cell integrin signaling

Understanding how the immune response is activated and amplified requires detailed knowledge of the stages in the formation of the immunological synapse (IS) between T lymphocytes and antigen‐presenting cells (APCs). We show that tetraspanins CD9 and CD151 congregate at the T‐cell side of the IS. Silencing of CD9 or CD151 blunts the IL‐2 secretion and expression of the activation marker CD69 by APC‐conjugated T lymphocytes, but does not affect the accumulation of CD3 or actin to the IS, or the translocation of the microtubule‐organizing center toward the T‐B contact area. CD9 or CD151 silencing diminishes the relocalization of α4β1 integrin to the IS and reduces the accumulation of high‐affinity β1 integrins at the cell–cell contact. These changes are accompanied by diminished phosphorylation of the integrin downstream targets FAK and ERK1/2. Our results suggest that CD9 and CD151 support integrin‐mediated signaling at the IS.     

Comparison of indirect peroxidase and avidin‐biotin‐peroxidase complex (ABC) immunohistochemical staining procedures for c‐fos in rat brain

c‐Fos is the product of a gene expressed within neurons in the brain that serves as an anatomical marker of cellular activation. Immunohistochemical staining for c‐fos allows a characterization of the effects of many different types of experimental manipulations on neuronal activity, making it a powerful technique for understanding brain, drug and behavior relationships. This study compared visualization of an anti‐c‐fos primary antibody in 40‐μm‐thick cryostat sections of formaldehyde‐fixed rat brainstem using either a peroxidase enzyme‐conjugated secondary antibody (indirect peroxidase) or the peroxidase‐conjugated avidin‐biotin complex (ABC) method. All sections were treated with H₂O₂ to quench endogenous peroxidase enzyme and sodium borohydride to enhance permeability of the tissue and improve staining quality. Every other section was used to examine either the indirect peroxidase or the ABC method. Sections for the indirect peroxidase method were treated with Triton X‐100 detergent to increase tissue permeability, goat serum to reduce non‐specific binding of the secondary antibody and, in some cases, bovine serum albumin (BSA) to reduce non‐specific binding of the primary antibody. Sections for the ABC method were treated with dilute normal serum, and avidin and biotin solutions and, in some cases BSA. Alternate sections were incubated for 72 h in either rabbit anti‐c‐fos primary antibody (1 : 20 000) or its vehicle (negative control). For the indirect peroxidase protocol, tissues were treated with peroxidase‐conjugated goat anti‐rabbit secondary antibody. For the ABC protocol, tissues were treated with biotinylated goat anti‐rabbit secondary antibody and ABC peroxidase complex. All sections were reacted with 3,3′‐diaminobenzadine (DAB) and H₂O₂, mounted and coverslipped. Both methods produced specific staining of c‐fos‐containing neurons, relative to the negative control sections. The indirect peroxidase protocol produced clear staining of c‐fos‐containing neurons, with very little background in the negative control sections. Staining for c‐fos was enhanced using the ABC method in that c‐fos stained neurons were darker and more clearly visible after shorter treatment with DAB. However, negative control sections showed a greater amount of non‐specific staining with the ABC method. Thus, the ABC method was more sensitive but showed reduced specificity, with BSA treatment slightly reducing the level of non‐specific staining. Overall, the ABC method produced better visualization and contrast of c‐fos‐containing neurons against the background color of the tissue.     

Delta‐like 4‐mediated Notch signaling is required for early T‐cell development in a three‐dimensional thymic structure

Delta‐like 4 (Dll4)‐mediated Notch signaling is critical for specifying T‐cell fate, but how Dll4‐mediated Notch signaling actually contributes to T‐cell development in the thymus remains unclear. To explore this mechanism in the thymic three‐dimensional structure, we performed fetal thymus organ culture using Dll4‐deficient mice. DN1a/b+DN2mt cells, which had not yet committed to either the αβ T or γδ T/NK cell lineage, did not differentiate into the αβ T‐cell lineage in Dll4‐deficient thymus despite the lack of cell fate conversion into other lineages. However, DN3 cells efficiently differentiated into a later developmental stage of αβ T cells, the double‐positive (DP) stage, although the proliferation was significantly impaired during the differentiation process. These findings suggest that the requirement for Notch signaling differs between the earliest and pre‐TCR‐bearing precursors and that continued Notch signaling is required for proper differentiation with active proliferation of αβ T lineage cells. Furthermore, we showed that Notch signaling increased the c‐Myc expression in DN3 cells in the thymus and that its overexpression rescued the proliferation and differentiation of DN3 cells in the Dll4‐null thymus. Therefore, c‐Myc plays a central role in the transition from stage DN3 to DP as a downstream target of Notch signaling.     

Defective antiviral CD8 T‐cell response and viral clearance in the absence of c‐Jun N‐terminal kinases

The c‐Jun N‐terminal kinase (JNK) signalling pathway appears to act as a critical intermediate in the regulation of lymphocyte activation and proliferation. The majority of studies on the importance of JNK are focused on its role in T helper responses, with very few reports addressing the mechanisms of JNK in governing CD8 T‐cell‐mediated immunity. By using a well‐defined mousepox model, we demonstrate that JNK is involved in CD8⁺ T‐cell‐mediated antiviral responses. Deficiency of either JNK1 or JNK2 impaired viral clearance, subsequently resulting in an increased susceptibility to ectromelia virus in resistant mice. The impairment of CD8 responses in JNK‐deficient mice was not directly due to an inhibition of effector T‐cell expansion, as both JNK1 and JNK2 had limited effect on the activation‐induced cell death of CD8⁺ T cells, and only JNK2‐deficient mice exhibited a significant change in CD8⁺ T‐cell proliferation after acute ectromelia virus infection. However, optimal activation of CD8⁺ T cells and their effector functions require signals from both JNK1 and JNK2. Our results suggest that the JNK pathway acts as a critical intermediate in antiviral immunity through regulation of the activation and effector function of CD8⁺ T cells rather than by altering their expansion.     

MiR‐26b is down‐regulated in carcinoma‐associated fibroblasts from ER‐positive breast cancers leading to enhanced cell migration and invasion

Carcinoma‐associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor‐positive cancers, and explore the influences of one of these, miR‐26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR‐26b was the most highly deregulated microRNA. Using qPCR, miR‐26b was confirmed as down‐regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR‐26b expression changed breast fibroblast behaviour. Reduced miR‐26b expression caused fibroblast migration and invasion to increase by up to three‐fold in scratch‐closure and trans‐well assays. Furthermore, in co‐culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR‐26b expression enhanced both MCF7 migration in trans‐well assays and MCF7 invasion from three‐dimensional spheroids by up to five‐fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR‐26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR‐26b. In addition, three novel miR‐26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR‐26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor‐positive cancers, and we have identified key genes and molecular pathways that act downstream of miR‐26b in CAFs. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

CD5 expression is regulated during human T‐cell activation by alternative polyadenylation,PTBP1, and miR‐204

T lymphocytes stimulated through their antigen receptor (TCR) preferentially express mRNA isoforms with shorter 3´ untranslated regions (3´‐UTRs) derived from alternative pre‐mRNA cleavage and polyadenylation (APA). However, the physiological relevance of APA programs remains poorly understood. CD5 is a T‐cell surface glycoprotein that negatively regulates TCR signaling from the onset of T‐cell activation. CD5 plays a pivotal role in mediating outcomes of cell survival or apoptosis, and may prevent both autoimmunity and cancer. In human primary T lymphocytes and Jurkat cells we found three distinct mRNA isoforms encoding CD5, each derived from distinct poly(A) signals (PASs). Upon T‐cell activation, there is an overall increase in CD5 mRNAs with a specific increase in the relative expression of the shorter isoforms. 3´‐UTRs derived from these shorter isoforms confer higher reporter expression in activated T cells relative to the longer isoform. We further show that polypyrimidine tract binding protein (PTB/PTBP1) directly binds to the proximal PAS and PTB siRNA depletion causes a decrease in mRNA derived from this PAS, suggesting an effect on stability or poly(A) site selection to circumvent targeting of the longer CD5 mRNA isoform by miR‐204. These mechanisms fine‐tune CD5 expression levels and thus ultimately T‐cell responses.