Down-regulated by mos (Drm)/Gremlin is a highly conserved protein whose properties and expression pattern suggest a role in early development, tissue-specific differentiation, and cell transformation. We have investigated the biosynthesis and processing of Drm expressed endogenously in rat fibroblasts or overexpressed following transient or stable transfection. Analysis of metabolically labeled cells revealed that Drm exists in secreted and cell-associated forms that exhibit similar mobilities in SDS-polyacrylamide gel electrophoresis. Protein analysis indicated that Drm is present in two major species: a slow migrating glycosylated form and a nonglycosylated form. Both forms of Drm are able to undergo phosphorylation. Drm is released into the media within 30 min of synthesis and is detectable for up to 4 -5 h, whereas the cell-associated form has a half-life of about 1 h. Confocal immunofluorescent microscopy indicates that Drm is present both on the external surface of expressing cells, as well as within the endoplasmic reticulum and the Golgi. Both glycosylated and nonglycosylated forms of Drm exhibit identical distributions and are able to antagonize bone morphogenetic protein signaling. Like the soluble form, the cell-associated forms are capable of binding 125

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an autosomal dominant small-vessel disease of the brain caused by mutations in the NOTCH3 receptor. The highly stereotyped nature of the mutations, which alter the number of cysteine residues within the epidermal growth factor-like repeats (EGFR), predicts that all mutations share common mechanisms. Prior in vitro assays and genetic studies in the mouse support the hypothesis that common mutations do not compromise canonical Notch3 function but instead convey a non-physiological and deleterious activity to the receptor through the unpaired cysteine residue. Intriguingly, in vitro studies predict that mutations located in the Delta/Serrate/LAG-2 ligand binding domain-(EGFR10-11) may result in a loss of Notch3 receptor function. However, the in vivo relevance and functional significance of this with respect to the pathogenic mechanisms and clinical expression of the disease remain largely unexplored. To ascertain, in vivo, the functional significance of EGFR10-11 mutations, we generated transgenic mice with one representative mutation (C428S) in EGFR10 of Notch3. These mice, like those with a common R90C mutation, developed characteristic arterial accumulation of Notch3 protein and granular osmiophilic material upon aging. By introducing the mutant C428S transgene into a Notch3 null background, we found that, unlike the R90C mutant protein, the C428S mutant protein has lost wild-type Notch3 activity and exhibited mild dominant-negative activity in three different biological settings. From a large prospectively recruited cohort of 176 CADASIL patients, we identified 10 patients, from five distinct pedigrees carrying a mutation in EGFR10 or 11. These mutations were associated with significantly higher Mini-Mental State Examination and Mattis Dementia Rating Scale scores (P < 0.05), when compared with common mutations. Additionally, we found a strong effect of this genotype on the burden of white matter hyperintensities (P < 0.01). Collectively, these results highlight distinctive functional and phenotypic features of EGFR10-11 mutations relative to the common CADASIL mutations. Our findings are compatible with the hypothesis that EGFR10-11 mutations cause the disease through the same gain of novel function as the common mutations, and lead us to propose that reduced Notch3 signalling acts as a modifier of the CADASIL phenotype.

Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53Δ31, a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis, hallmarks of syndromes caused by short telomeres. Indeed, p53Δ31/Δ31 mice had short telomeres and other phenotypic traits associated with the telomere disease dyskeratosis congenita and its severe variant the Hoyeraal-Hreidarsson syndrome. Heterozygous p53+/Δ31 mice were only mildly affected, but decreased levels of Mdm4, a negative regulator of p53, led to a dramatic aggravation of their symptoms. Importantly, several genes involved in telomere metabolism were downregulated in p53Δ31/Δ31 cells, including Dyskerin, Rtel1, and Tinf2, which are mutated in dyskeratosis congenita, and Terf1, which is implicated in aplastic anemia. Together, these data reveal that a truncating mutation can activate p53 and that p53 plays a major role in the regulation of telomere metabolism.

Notch signaling is repeatedly used during animal development to specify cell fates. Using atomic force microscopy on live cells, chemical inhibitors, and conventional analyses, we show that the rate of Notch signaling is linked to the adhesion force between cells expressing Notch receptors and Delta ligand. Both the Notch extracellular and intracellular domains are required for the high adhesion force with Delta. This high adhesion force is lost within minutes, primarily due to the action of Presenilin on Notch. Reduced turnover or Delta pulling accelerate this loss. These data suggest that strong adhesion between Notch and Delta might serve as a booster for initiating Notch signaling at a high rate.

Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53Δ31, a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53Δ31/Δ31 fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53Δ31/Δ31 fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop.

No abstract

Mdm4, a protein related to the ubiquitin-ligase Mdm2, is an essential inhibitor of tumor suppressor protein p53. In both human and mouse cells, the Mdm4 gene encodes two major transcripts: one encodes the full-length oncoprotein (designated below as Mdm4-FL), whereas the other, resulting from a variant splicing that skips exon 6, encodes the shorter isoform Mdm4-S. Importantly, increased Mdm4-S mRNA levels were observed in several human cancers, and correlated with poor survival. However, the role of Mdm4-S in cancer progression remains controversial, because the Mdm4-S protein appeared to be a potent p53 inhibitor when overexpressed, but the splice variant also leads to a decrease in Mdm4-FL expression. To unambiguously determine the physiological impact of the Mdm4-S splice variant, we generated a mouse model with a targeted deletion of the Mdm4 exon 6, thereby creating an obligatory exon skipping. The mutant allele (Mdm4(ΔE6)) prevented the expression of Mdm4-FL, but also led to increased Mdm4-S mRNA levels. Mice homozygous for this allele died during embryonic development, but were rescued by a concomitant p53 deficiency. Furthermore in a hypomorphic p53(ΔP/ΔP) context, the Mdm4(ΔE6) allele led to p53 activation and delayed the growth of oncogene-induced tumors. We next determined the effect of Mdm4(+/ΔE6) heterozygosity in a hypermorphic p53(+/Δ31) genetic background, recently shown to be extremely sensitive to Mdm4 activity. Mdm4(+/ΔE6) p53(+/Δ31) pups were born, but suffered from aplastic anemia and died before weaning, again indicating an increased p53 activity. Our results demonstrate that the main effect of a skipping of Mdm4 exon 6 is not the synthesis of the Mdm4-S protein, but rather a decrease in Mdm4-FL expression. These and other data suggest that increased Mdm4-S mRNA levels might correlate with more aggressive cancers without encoding significant amounts of a potential oncoprotein. Hypotheses that may account for this apparent paradox are discussed.

The drm gene encodes a cystine knot-containing secreted and cell membrane-associated glycoprotein shown to be an antagonist of BMPs. Drm was recently reported to play a crucial role in limb bud development, by its capacity to bind BMPs. Here, we have studied the expression pattern of drm transcripts during chicken development, by using whole-mount in situ hybridization. We show that, from stage 22HH to stage 26HH, in addition to limb buds, drm is expressed in cephalic neural crest-derived branchial arches I, II and III, in the medio-dorsal lip of the myotome and in the superficial dermatome