Abdolrahman S. Nateri

The AP-1 transcription factor c-Jun is required for efficient axonal regeneration

Nerve injury triggers numerous changes in the injured neurons and surrounding nonneuronal cells that ultimately result in successful target reinnervation or cell death. c-Jun is a component of the heterodimeric AP-1 transcription factor, and c-Jun is highly expressed in response to neuronal trauma. Here we have investigated the role of c-jun during axonal regeneration using mice lacking c-jun in the central nervous system. After transection of the facial nerve, the absence of c-Jun caused severe defects in several aspects of the axonal response, including perineuronal sprouting, lymphocyte recruitment, and microglial activation. c-Jun-deficient motorneurons were atrophic, resistant to axotomy-induced cell death, and showed reduced target muscle reinnervation. Expression of CD44, galanin, and alpha7beta1 integrin, molecules known to be involved in regeneration, was greatly impaired, suggesting a mechanism for c-Jun-mediated axonal growth. Taken together, our results identify c-Jun as an important regulator of axonal regeneration in the injured central nervous system.

Interaction of phosphorylated c-Jun with TCF4 regulates intestinal cancer development

The proto-oncoprotein c-Jun is a component of the AP-1 transcription factor, the activity of which is augmented in many tumour types. An important mechanism in the stimulation of AP-1 function is amino-terminal phosphorylation of c-Jun by the c-Jun N-terminal kinases (JNKs). Phosphorylated c-Jun is biologically more active, partially because it acquires the ability to interact with binding partners. Here we show that phosphorylated c-Jun interacts with the HMG-box transcription factor TCF4 to form a ternary complex containing c-Jun, TCF4 and β-catenin. Chromatin immunoprecipitation assays revealed JNK-dependent c-Jun–TCF4 interaction on the c-jun promoter, and c-Jun and TCF4 cooperatively activated the c-jun promoter in reporter assays in a β-catenin-dependent manner. In the ApcMin mouse model of intestinal cancer, genetic abrogation of c-Jun N-terminal phosphorylation or gut-specific conditional c-jun inactivation reduced tumour number and size and prolonged lifespan. Therefore, the phosphorylation-dependent interaction between c-Jun and TCF4 regulates intestinal tumorigenesis by integrating JNK and APC/β-catenin, two distinct pathways activated by WNT signalling.

JNK signalling modulates intestinal homeostasis and tumourigenesis in mice

Wnt signalling is a crucial signalling pathway controlling intestinal homeostasis and cancer. We show here that the JNK MAP kinase pathway and one of its most important substrates, the AP‐1 transcription factor c‐Jun, modulates Wnt signalling strength in the intestine. Transgenic gut‐specific augmentation of JNK signalling stimulated progenitor cell proliferation and migration, resulting in increased villus length. In the crypt, c‐Jun protein was highly expressed in progenitor cells and the absence of c‐Jun resulted in decreased proliferation and villus length. In addition to several known c‐Jun/AP‐1 target genes, expression of Wnt target genes Axin2 and Lgr5 were stimulated by JNK activation, suggesting a cross talk of JNK to Wnt signalling. Expression of the Wnt pathway component TCF4 was controlled by JNK activity, and chromatin immunoprecipitation and reporter assays identified tcf4 as a direct c‐Jun target gene. Consequently, increased JNK activity accelerated tumourigenesis in a model of colorectal carcinogenesis. As c‐jun is a direct target of the TCF4/β‐catenin complex, the control of tcf4 expression by JNK/c‐Jun leads to a positive feedback loop that connects JNK and Wnt signalling. This mechanism regulates the physiological function of progenitor cells and oncogenic transformation.

FBXW7 influences murine intestinal homeostasis and cancer, targeting Notch, Jun, and DEK for degradation

The E3 ubiquitin ligase component FBXW7 modulates homeostasis and inhibits tumorigenesis in the murine intestine.

ERK activation causes epilepsy by stimulating NMDA receptor activity

The ERK MAPK signalling pathway is a highly conserved kinase cascade linking transmembrane receptors to downstream effector mechanisms. To investigate the function of ERK in neurons, a constitutively active form of MEK1 (caMEK1) was conditionally expressed in the murine brain, which resulted in ERK activation and caused spontaneous epileptic seizures. ERK activation stimulated phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and augmented NMDA receptor 2B (NR2B) protein levels. Pharmacological inhibition of NR2B function impaired synaptic facilitation in area cornus ammonicus region 3 (CA3) in acute hippocampal slices derived from caMEK1‐expressing mice and abrogated epilepsy in vivo. In addition, expression of caMEK1 caused phosphorylation of the transcription factor, cAMP response element‐binding protein (CREB) and increased transcription of ephrinB2. EphrinB2 overexpression resulted in increased NR2B tyrosine phosphorylation, which was essential for caMEK1‐induced epilepsy in vivo, since conditional inactivation of ephrinB2 greatly reduced seizure frequency in caMEK1 transgenic mice. Therefore, our study identifies a mechanism of epileptogenesis that links MAP kinase to Eph/Ephrin and NMDA receptor signalling.

Embryonic NANOG Activity Defines Colorectal Cancer Stem Cells and Modulates through AP1‐ and TCF‐dependent Mechanisms

Embryonic NANOG (NANOG1) is considered as an important regulator of pluripotency while NANOGP8 (NANOG‐pseudogene) plays a role in tumorigenesis. Herein, we show NANOG is expressed from both NANOG1 and NANOGP8 in human colorectal cancers (CRC). Enforced NANOG1‐expression increases clonogenic potential and tumor formation in xenograft models, although it is expressed only in a small subpopulation of tumor cells and is colocalized with endogenous nuclear β‐cateninHigh. Moreover, single NANOG1‐CRCs form spherical aggregates, similar to the embryoid body of embryonic stem cells (ESCs), and express higher levels of stem‐like Wnt‐associated target genes. Furthermore, we show that NANOG1‐expression is positively regulated by c‐JUN and β‐catenin/TCF4. Ectopic expression of c‐Jun in murine ApcMin/+‐ESCs results in the development of larger xenograft tumors with higher cell density compared to controls. Chromatin immunoprecipitation assays demonstrate that c‐JUN binds to the NANOG1‐promoter via the octamer M1 DNA element. Collectively, our data suggest that β‐Catenin/TCF4 and c‐JUN together drive a subpopulation of CRC tumor cells that adopt a stem‐like phenotype via the NANOG1‐promoter. STEM Cells2012;30:2076–2087

The Links between Transcription, β-catenin/JNK Signaling, and Carcinogenesis

Interactions between transcription and signaling are fundamentally important for understanding both the structure and function of genetic pathways and their role in diseases such as cancer. The finding that β-catenin/TCF4 and JNK/c-Jun cooperate has important implications in carcinogenesis. Previously, we found that binding of c-Jun and β-catenin/TCF4 to the c-jun promoter is dependent upon JNK activity, thus one role for this complex is to contribute to the repression and/or activation of genes that may mediate cell maintenance, proliferation, differentiation, and death, whereas deregulation of these signals may contribute to carcinogenesis. Here we address the functional links reported between activated β-catenin/JNK signaling pathways, their component genes, and their common targets, and discuss how alterations in the properties of these genes lead to the development of cancer. (Mol Cancer Res 2009;7(8):1189–96)

In Vivo and In Vitro Identification of Structural and Sequence Elements of the Human Parechovirus 5′ Untranslated Region Required for Internal Initiation

ABSTRACT Sequence analysis of the picornavirus echovirus 22 led to its classification as the first member of a new genus,Parechovirus, and renaming as human parechovirus type 1 (HPeV1). Although distinct from other genera in most of the genome, the 5′ untranslated region (5′UTR) shows similarities to that of cardio/aphthoviruses in some of its structural domains (A to L). The 5′UTR plays an important role in picornavirus translation initiation and in RNA synthesis. To investigate translation in HPeV1, we engineered an extensive range of mutations (including precise deletions and point mutations) into the 5′UTR. Their effects were studied both by in vitro transcription-translation using a bicistronic construct and by in vivo studies using an infectious, full-length HPeV1 cDNA. These approaches allowed the HPeV1 internal ribosome entry site (IRES) to be mapped. Deletions within the first 298 nucleotides had little impact in the in vitro system, while deletions of nucleotides 298 to 538 had a significant effect. Precise removal of domains H and L (nucleotides 287 to 316 and 664 to 682, respectively) did not significantly reduce translation efficiency in vitro, while domains I, J, and K (nucleotides 327 to 545, 551 to 661, and 614 to 645, respectively) appeared to have much more important roles. Mutation of a phylogenetically conserved GNRA motif (positions 421 to 424) within domain I severely reduced translation. We also confirmed the identity of the AUG (positions 710 to 712) which initiates the open reading frame, the positive identification of which has not been possible previously, as the N terminus of the polyprotein is blocked and not amenable to sequence analysis. This is therefore important in understanding parechovirus genome organization. Mutation of the AUG or an upstream polypyrimidine tract leads to aberrant translation, suggesting they both form part of the parechovirus Yn-Xm-AUG motif. In vivo experiments confirmed the importance of domains I, J, and K, the conserved GNRA motif, polypyrimidine sequences, and AUG, as mutations here were lethal. These features are also important in the IRES elements of cardio/aphthoviruses, but other features reported to be part of the IRES of some members of these genera, notably domains H and L, do not appear to be critical in HPeV1. This adds weight to the idea that there may be functional differences between the IRES elements of different picornaviruses, even when they share significant structural similarity.

C‐terminal Tensin‐like (CTEN) is an oncogene which alters cell motility possibly through repression of E‐cadherin in colorectal cancer

The Tensin gene family encodes proteins thought to modulate integrin function. C‐terminal Tensin‐like (CTEN) is a member of the Tensin gene family which lacks the N‐terminus actin‐binding domain. Cten is reported to have both oncogenic and tumour‐suppressor functions. We investigated the role that Cten may play in colorectal cancer (CRC). By quantitative RT–PCR CTEN is up‐regulated (i.e. > two‐fold increase) in 62% of cell lines and 69% of tumours compared with normal mucosa, consistent with CTEN being a possible oncogene. Stable transfection of HCT116 and SW480 (CRC cell lines with low endogenous Cten expression) with a Cten expression vector gave identical results in both cell lines. Forced Cten expression did not cause change in cell numbers, although it did confer resistance to staurosporine‐induced apoptosis (p < 0.005). Cten also induced epithelial–mesenchymal transition (EMT) in tumour cells accompanied by a significant increase in both cell migration (transwell migration and cell wounding assays, p < 0.001 and p < 0.05, respectively) and cell invasion (invasion through Matrigel, p < 0.001). Given the observed EMT, we investigated the levels of E‐cadherin. Cten induction was associated with a reduction in E‐cadherin protein expression but not levels of E‐cadherin mRNA. These data suggest that CTEN is an oncogene in CRC which stimulates EMT, cell migration and invasion and may therefore have a role in tumour invasion/spread. Furthermore, Cten induction is associated with post‐transcriptional repression of E‐cadherin. Copyright

Terminal RNA Replication Elements in Human Parechovirus 1

ABSTRACT To define structural elements critical for RNA replication in human parechovirus 1 (HPeV1), a replicon with chloramphenicol acetyltransferase as a reporter gene and an infectious virus cDNA clone have been used. It was observed that there are cis-acting signals required for HPeV1 replication located within the 5′-terminal 112 nucleotides of the genome and that these include two terminal stem-loops, SL-A and SL-B, together with a pseudoknot element. Significant disruption of any of these structures impaired both RNA replication and virus growth. In view of the similarity in terminal structures to several picornaviruses, such as cardioviruses and hepatoviruses, the insights generated in this work are of wider significance for understanding picornavirus replication.