c‐Jun promotes cell migration and drives expression of the motility factor ENPP2 in soft tissue sarcomas

Genomic amplification of the c‐Jun proto‐oncogene has been identified in ～30% of dedifferentiated liposarcomas (DDLPS), but the functional contribution of c‐Jun to the progression of DDLPS remains poorly understood. In previous work we showed that knock‐down of c‐Jun by RNA interference impaired the in vitro proliferation and in vivo growth of a DDLPS cell line (LP6) with genomic amplification of the c‐Jun locus. Here, we used gene expression analysis and functional studies in a broad panel of cell lines to further define the role of c‐Jun in DDLPS and other soft tissue sarcomas. We show that c‐Jun knock‐down impairs transition through the G₁ phase of the cell cycle in multiple DDLPS cell lines. We also found that high levels of c‐Jun expression are both necessary and sufficient to promote DDLPS cell migration and invasion in vitro. Our data suggest that high levels of c‐Jun enhance motility in part by driving the expression of ENPP2/Autotaxin. c‐Jun over‐expression has minimal effects on in vitro proliferation but substantially enhances the in vivo growth of weakly tumourigenic DDLPS cell lines. Finally, we provide evidence that c‐Jun genomic amplification and over‐expression may have similar functional consequences in other types of soft tissue sarcoma. Our data suggest a model in which relatively low levels of c‐Jun are sufficient for in vitro proliferation, but high levels of c‐Jun enhance invasiveness and capacity for in vivo tumour growth. These observations provide an explanation for the selective advantage provided by c‐Jun genomic amplification in vivo and suggest that sarcomas with elevated c‐Jun levels are likely to have a particularly high malignant potential. Data from exon array and RNA‐Seq experiments have been deposited in the GEO database (Accession No. GSE57531). Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

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.     

c‐Jun activating binding protein 1 binds to the IgA receptor and modulates protein levels of FcαRI and FcRγ‐chain

The receptor for IgA, FcαRI or CD89, is expressed on myeloid cells and can trigger phagocytosis, tumor cell lysis, and release of inflammatory mediators. These functions critically depend on the associated FcR γ‐chain; however, some biological functions, like receptor internalization, are solely mediated by FcαRI α‐chain. Little is known as to how FcαRI regulates these processes and the FcαRI intracellular domain does not contain recognized signalling motifs. We searched for associating proteins and identified c‐Jun activating binding protein 1 (JAB1) as a binding partner specifically for FcαRI. We found increased FcαRI surface expression after ectopic expression of JAB1 as well as diminished protein levels of total FcR γ‐chain levels after JAB1 knock‐down. These data functionally link JAB1 with controlling protein expression levels of FcαRI‐FcR γ‐chain protein complex.     

Induction of phosphorylated c‐Jun in neonatal spinal motoneurons after axonal injury is coincident with both motoneuron death and regeneration

c‐Jun activation has been implicated not only in neuronal degeneration, but also in survival and regeneration. Here, we investigated c‐Jun activation in injured motoneurons by using a nerve crush model in neonatal rats. We identified two distinct subpopulations of motoneurons: about 60% underwent degeneration following injury whereas the remaining 40% survived and induced a regeneration response at 3 weeks post injury. However, all motoneurons examined expressed phosphorylated‐c‐Jun‐immunoreactivity (p‐c‐Jun‐IR) at the early stage of 3 days following injury. These results suggest that active c‐Jun was induced in all neonatal motoneurons following nerve crush injury, regardless of whether they were destined to degenerate or undergo successful regeneration at a later stage. Our findings therefore support the hypothesis that active c‐Jun is involved in both neuronal degeneration and regeneration.     

c‐Rel is crucial for the induction of Foxp3+ regulatory CD4+ T cells but not TH17 cells

The NF‐κB/Rel family member c‐Rel was described to be required for the development of T_H1 responses. However, the role of c‐Rel in the differentiation of T_H17 and regulatory CD4⁺Foxp3⁺ T cells (Treg) remains obscure. Here, we show that in the absence of c‐Rel, in vitro differentiation of pro‐inflammatory T_H17 cells is normal. In contrast, generation of inducible Treg (iTreg) within c‐Rel‐deficient CD4⁺ T cells was severely hampered and correlated to reduced numbers of Foxp3⁺ T cells in vivo. Mechanistically, in vitro conversion of naive CD4⁺ T cells into iTreg was crucially dependent on c‐Rel‐mediated synthesis of endogenous IL‐2. The addition of exogenous IL‐2 was sufficient to rescue the development of c‐Rel‐deficient iTreg. Thus, c‐Rel is essential for the development of Foxp3⁺ Treg but not for T_H17 cells via regulating the production of IL‐2.     

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.     

Distinct roles for N‐Cadherin linked c‐Src and fyn kinases in lens development

Background: Src family tyrosine kinases (SFKs) are often coincidently expressed but few studies have dissected their individual functions in the same cell during development. Using the classical embryonic lens as our model, we investigated SFK signaling in the regulation of both differentiation initiation and morphogenesis, and the distinct functions of c‐Src and Fyn in these processes. Results: Blocking SFK activity with the highly specific inhibitor PP1 induced initiation of the lens differentiation program but blocked lens fiber cell elongation and organization into mini lens‐like structures called lentoids. These dichotomous roles for SFK signaling were discovered to reflect distinct functions of c‐Src and Fyn and their differentiation‐state‐specific recruitment to and action at N‐cadherin junctions. c‐Src was highly associated with the nascent N‐cadherin junctions of undifferentiated lens epithelial cells. Its siRNA knockdown promoted N‐cadherin junctional maturation, blocked proliferation, and induced lens cell differentiation. In contrast, Fyn was recruited to mature N‐cadherin junctions of differentiating lens cells and siRNA knockdown suppressed differentiation‐specific gene expression and blocked morphogenesis. Conclusions: Through inhibition of N‐cadherin junction maturation, c‐Src promotes lens epithelial cell proliferation and the maintenance of the lens epithelial cell undifferentiated state, while Fyn, signaling downstream of mature N‐cadherin junctions, promotes lens fiber cell morphogenesis. Developmental Dynamics 242:469–474, 2013. © 2013 Wiley Periodicals, Inc.