Susan Giblett

MEK kinase activity is not necessary for Raf‐1 function

Raf‐1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf‐1 is achieved by Ras.GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a ‘knockout’ of the raf‐1 gene in mice as well as a rafFF mutant version of endogenous Raf‐1 with Y340FY341F mutations. Raf‐1−/− mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf‐1 from cells derived from raf‐1FF/FF mice has no detectable activity towards MEK in vitro, and yet raf‐1FF/FF mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf‐1−/− and raf‐1FF/FF mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf‐1 is not essential for normal mouse development and that Raf‐1 plays a key role in preventing apoptosis.

Prostanoids synthesized by cyclo‐oxygenase isoforms in rat spinal cord and their contribution to the development of neuronal hyperexcitability

1 The responses of wide dynamic range spinal dorsal horn neurones to noxious mechanical stimulation of the ankle or knee joint were tested before and after spinal administration of the non‐selective cyclo‐oxygenase (COX) inhibitors, indomethacin and meclofenamic acid. Neither of these drugs altered the responses of these neurones to noxious mechanical stimulation. 2 Wind‐up of a spinal nociceptive reflex evoked by electrical stimulation of the sural nerve at C‐fibre strength was dose‐dependently inhibited by intravenous administration of indomethacin, a non‐selective COX inhibitor, and SC58125, a selective COX‐2 inhibitor. Intrathecal administration of indomethacin also reduced the wind‐up of this nociceptive reflex. 3 Western blot analysis of proteins extracted from normal rat spinal cord revealed the presence of both cyclo‐oxygenase (COX)‐1 and COX‐2 proteins. 4 Immunocytochemistry of sections of normal rat spinal cord with specific COX‐1 antiserum revealed little specific COX‐1‐like immunoreactivity in the grey matter. With the same antiserum, intense COX‐1‐like immunoreactivity was observed in the cytoplasm, nuclear membrane and axonal processes of small to medium sized (<1000 μm2) dorsal root ganglion (DRG) cell bodies. 5 Immunocytochemistry of sections of normal rat spinal cord incubated with specific COX‐2 antiserum showed intense COX‐2‐like immunoreactivity (COX‐2‐li) in the superficial dorsal horn of the spinal cord (laminae I and II) and around the central canal (lamina X). COX‐2‐li was also observed in some neurones in deep dorsal horn and in individual motor neurones in ventral horn. COX‐2‐li was not observed in the cell bodies of DRG. 6 Superfusion of the lumbar spinal cord of normal rats with artificial CSF and subsequent radioimmunoassay revealed the presence of prostaglandin D2 (PGD2)

Disruption of the talin gene arrests mouse development at the gastrulation stage.

Studies on cultured cells show that the cytoskeletal protein talin plays a key role in cell spreading and the assembly of cell‐extracellular matrix junctions. To examine the role of talin in vivo, we have generated mice with a targeted disruption of the talin gene. Heterozygotes are normal, but no surviving homozygous mutant animals were obtained, proving that talin is required for embryogenesis. Mutant embryos develop normally to the blastocyst stage and implant, but there is a gross disorganization of the embryos at gastrulation (6.5‐7.5 days post coitum), and they die around 8.5‐9.5 days post coitum. The embryonic ectoderm is reduced in size, with fewer cells, and is incompletely organised compared with wild‐type embryos. The mutant embryos show disorganised extraembryonic tissues, and the ectoplacental and excocoelomic cavities are not formed. This seems to be because embryonic mesoderm accumulates as a mass on the posterior side of the embryos and fails to migrate to extraembryonic regions, although mesodermal cells are evident in the embryo proper. Spreading of trophoblast cells derived from cultured mutant blastocysts on fibronectin and laminin is also considerably reduced. Therefore, the fundamental deficit in these embryos seems to be a failure of cell migration at gastrulation.

Expression of Endogenous Oncogenic V600EB-raf Induces Proliferation and Developmental Defects in Mice and Transformation of Primary Fibroblasts

Mutations of the human B-RAF gene are detected in approximately 8% of cancer samples, primarily in cutaneous melanomas (70%). The most common mutation (90%) is a valine-to-glutamic acid mutation at residue 600 (V600E; formerly V599E according to previous nomenclature). Using a Cre/Lox approach, we have generated a conditional knock-in allele of (V600E)B-raf in mice. We show that widespread expression of (V600E)B-Raf cannot be tolerated in embryonic development, with embryos dying approximately 7.5 dpc. Directed expression of mutant (V600E)B-Raf to somatic tissues using the IFN-inducible Mx1-Cre mouse strain induces a proliferative disorder and bone marrow failure with evidence of nonlymphoid neoplasia of the histiocytic type leading to death within 4 weeks of age. However, expression of mutant B-Raf does not alter the proliferation profile of all somatic tissues. In primary mouse embryonic fibroblasts, expression of endogenous (V600E)B-Raf induces morphologic transformation, increased cell proliferation, and loss of contact inhibition. Thus, (V600E)B-Raf is able to induce several hallmarks of transformation in some primary mouse cells without evidence for the involvement of a cooperating oncogene or tumor suppressor gene.

V600EBraf induces gastrointestinal crypt senescence and promotes tumour progression through enhanced CpG methylation of p16INK4a

The majority of human colorectal cancers (CRCs) are initiated by mutations arising in the adenomatous polyposis coli (APC) tumour suppressor gene. However, a new class of non‐APC mutated CRCs has been defined that have a serrated histopathology and carry the V600EBRAF oncogene. Here we have investigated the pathogenesis of serrated CRCs by expressing V600EBraf in the proliferative cells of the mouse gastrointestinal tract. We show that the oncogene drives an initial burst of Mek‐dependent proliferation, leading to the formation of hyperplastic crypts. This is associated with β‐catenin nuclear localization by a mechanism involving Mapk/Erk kinase (Mek)‐dependent, Akt‐independent phosphorylation of Gsk3β. However, hyperplastic crypts remain dormant for prolonged periods due to the induction of crypt senescence accompanied by upregulation of senescence‐associated β‐galactosidase and p16Ink4a. We show that tumour progression is associated with down‐regulation of p16Ink4a through enhanced CpG methylation of exon 1 and knockout of Cdkn2a confirms this gene is a barrier to tumour progression. Our studies identify V600EBRAF as an early genetic driver mutation in serrated CRCs and indicate that, unlike APC‐mutated cancers, this subtype arises by the bypassing of a V600EBraf driven oncogene‐induced senescence programme.

V600EBraf induces gastrointestinal crypt senescence and promotes tumour progression through enhanced CpG methylation of p16INK4a.

The majority of human colorectal cancers (CRCs) are initiated by mutations arising in the adenomatous polyposis coli (APC) tumour suppressor gene. However, a new class of non‐APC mutated CRCs has been defined that have a serrated histopathology and carry the V600EBRAF oncogene. Here we have investigated the pathogenesis of serrated CRCs by expressing V600EBraf in the proliferative cells of the mouse gastrointestinal tract. We show that the oncogene drives an initial burst of Mek‐dependent proliferation, leading to the formation of hyperplastic crypts. This is associated with β‐catenin nuclear localization by a mechanism involving Mapk/Erk kinase (Mek)‐dependent, Akt‐independent phosphorylation of Gsk3β. However, hyperplastic crypts remain dormant for prolonged periods due to the induction of crypt senescence accompanied by upregulation of senescence‐associated β‐galactosidase and p16Ink4a. We show that tumour progression is associated with down‐regulation of p16Ink4a through enhanced CpG methylation of exon 1 and knockout of Cdkn2a confirms this gene is a barrier to tumour progression. Our studies identify V600EBRAF as an early genetic driver mutation in serrated CRCs and indicate that, unlike APC‐mutated cancers, this subtype arises by the bypassing of a V600EBraf driven oncogene‐induced senescence programme.

Cyclooxygenase‐1 is a marker for a subpopulation of putative nociceptive neurons in rat dorsal root ganglia

Immunocytochemical and morphometric techniques were used to quantify the distribution of cyclooxygenase (cox)‐containing neurons in rat L5 dorsal root ganglia (DRG). Cox‐1 immunolabelling was almost exclusively restricted to small diameter DRG neurons (< 1000 μm2), and was extensively colocalized with calcitonin gene‐related peptide (CGRP) and isolectin B4 (IB4). Cox‐1 was present in 65% and 70% of CGRP‐ and IB4‐labelled neurons, respectively. Cox‐1 labelling was also found in neurons expressing the sensory neuron‐specific (SNS) Na+ channel. Cox‐2 labelling was absent in DRG from normal rats. In the Freunds adjuvant model of monoarthritis, the proportion of cox‐1‐positive DRG neurons was unchanged and no neurons were found to be labelled for cox‐2. In primary tissue culture, cox‐1 immunolabelling persisted in vitro for up to 9 days and was present in morphologically identical neurons. The selective expression of cox‐1 in peripheral ganglia was confirmed by the small number of nodose ganglion neurons and superior cervical ganglion (SCG) neurons labelled for cox‐1. These data suggest that cox‐1 is a marker for a subpopulation of putative nociceptive neurons in vitro and in vivo, and suggests that the prostaglandins synthesized by these neurons may be important for nociceptor function. These data may have important implications for the mode and mechanism of action of non‐steroidal anti‐inflammatory drugs (NSAIDs).

CRAF autophosphorylation of serine 621 is required to prevent its proteasome-mediated degradation.

The CRAF protein kinase regulates proliferative, differentiation, and survival signals from activated RAS proteins to downstream effectors, most often by inducing MEK/ERK activation. A well-established model of CRAF regulation involves RAS-mediated translocation of CRAF to the plasma membrane, where it is activated by a series of events including phosphorylation. Here we have discovered a new mode of regulation that occurs prior to this step. By creating a kinase-defective version of CRAF in mice or by use of the RAF inhibitor sorafenib, we show that CRAF must first undergo autophosphorylation of serine 621 (S621). Autophosphorylation occurs in cis, does not involve MEK/ERK activation, and is essential to ensure the correct folding and stability of the protein. In the absence of S621 phosphorylation, CRAF is degraded by the proteasome by mechanisms that do not uniquely rely on the E3 ubiquitin ligase CHIP.

A study of regional gut endoderm potency by analysis of Cdx2 null mutant chimaeric mice

Inactivation of Cdx2 by homologous recombination results in the development of forestomach epithelium at ectopic sites in pericaecal areas of the midgut of heterozygote mice. Local factors subsequently result in the secondary induction of tissues exhibiting an orderly sequence of tissue types between the ectopic forestomach tissue and the surrounding colon. Clonal analysis of this secondarily generated tissue using Y chromosome painting in chimaeric mice indicates that once differentiated to express Cdx2, host colonic epithelium can only form small intestinal-type epithelium, while Cdx2 mutant cells give rise to a succession of gastric-type tissue but never to a small intestine morphology. Our results indicate a difference in potency between forestomach and midgut precursor endodermal cells.

Cyclooxygenase-1 is a marker for a subpopulation of putative nociceptive neurons in rat dorsal root ganglia

Immunocytochemical and morphometric techniques were used to quantify the distribution of cyclooxygenase (cox)‐containing neurons in rat L5 dorsal root ganglia (DRG). Cox‐1 immunolabelling was almost exclusively restricted to small diameter DRG neurons (< 1000 μm2), and was extensively colocalized with calcitonin gene‐related peptide (CGRP) and isolectin B4 (IB4). Cox‐1 was present in 65% and 70% of CGRP‐ and IB4‐labelled neurons, respectively. Cox‐1 labelling was also found in neurons expressing the sensory neuron‐specific (SNS) Na+ channel. Cox‐2 labelling was absent in DRG from normal rats. In the Freunds adjuvant model of monoarthritis, the proportion of cox‐1‐positive DRG neurons was unchanged and no neurons were found to be labelled for cox‐2. In primary tissue culture, cox‐1 immunolabelling persisted in vitro for up to 9 days and was present in morphologically identical neurons. The selective expression of cox‐1 in peripheral ganglia was confirmed by the small number of nodose ganglion neurons and superior cervical ganglion (SCG) neurons labelled for cox‐1. These data suggest that cox‐1 is a marker for a subpopulation of putative nociceptive neurons in vitro and in vivo, and suggests that the prostaglandins synthesized by these neurons may be important for nociceptor function. These data may have important implications for the mode and mechanism of action of non‐steroidal anti‐inflammatory drugs (NSAIDs).