epidermal-growth-factor and Abnormalities--Multiple

Protein O-glucosylation is a conserved post-translational modification that occurs on epidermal growth factor-like (EGF) repeats harboring the C(1)-X-S-X-P-C(2) consensus sequence. The Drosophila protein O-glucosyltransferase (Poglut) Rumi regulates Notch signaling, but the contribution of protein O-glucosylation to mammalian Notch signaling and embryonic development is not known. Here, we show that mouse Rumi encodes a Poglut, and that Rumi(-/-) mouse embryos die before embryonic day 9.5 with posterior axis truncation and severe defects in neural tube development, somitogenesis, cardiogenesis and vascular remodeling. Rumi knockdown in mouse cell lines results in cellular and molecular phenotypes of loss of Notch signaling without affecting Notch ligand binding. Biochemical, cell culture and cross-species transgenic experiments indicate that a decrease in Rumi levels results in reduced O-glucosylation of Notch EGF repeats, and that the enzymatic activity of Rumi is key to its regulatory role in the Notch pathway. Genetic interaction studies show that removing one copy of Rumi in a Jag1(+/-) (jagged 1) background results in severe bile duct morphogenesis defects. Altogether, our data indicate that addition of O-glucose to EGF repeats is essential for mouse embryonic development and Notch signaling, and that Jag1-induced signaling is sensitive to the gene dosage of the protein O-glucosyltransferase Rumi. Given that Rumi(-/-) embryos show more severe phenotypes compared to those displayed by other global regulators of canonical Notch signaling, Rumi is likely to have additional important targets during mammalian development.

Topics: Abnormalities, Multiple; Animals; Bile Ducts, Intrahepatic; Calcium-Binding Proteins; Cardiovascular Abnormalities; Cell Line; Drosophila Proteins; Embryonic Development; Epidermal Growth Factor; Female; Gene Dosage; Glucosyltransferases; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Liver; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Mice, Transgenic; Phenotype; Pregnancy; Receptors, Notch; RNA, Messenger; Serrate-Jagged Proteins; Signal Transduction

Costello syndrome (CS) is a rare congenital disorder characterized by failure to thrive, craniofacial dysmorphisms, cardiac and skin abnormalities, mental retardation, and predisposition to malignancies. CS is caused by heterozygous gain-of-function mutations in HRAS that also occur as somatic alterations in human tumors. HRAS is one of the three classical RAS proteins and cycles between an active, GTP- and an inactive, GDP-bound conformation. We used primary human skin fibroblasts from patients with CS as a model system to study the functional consequences of HRAS mutations on endogenous signaling pathways. The GTP-bound form of HRAS was significantly enriched in CS compared with normal fibroblasts. Active HRAS is known to stimulate both the RAF-MEK-ERK and the PI3K-AKT signaling cascade. Phosphorylation of MEK and ERK was normal in CS fibroblasts under basal conditions and slightly prolonged after epidermal growth factor (EGF) stimulation. Interestingly, basal phosphorylation of AKT was increased yet more in CS fibroblasts. Moreover, AKT phosphorylation was diminished in the early and enhanced in the late phase of EGF stimulation. Taken together, these results document that CS-associated HRAS mutations result in prolonged signal flux in a ligand-dependent manner. Our data suggest that altered cellular response to growth factors rather than constitutive activation of HRAS downstream signaling molecules may contribute to some of the clinical features in patients with CS.

Topics: Abnormalities, Multiple; Cells, Cultured; Chromones; Craniofacial Abnormalities; Enzyme Inhibitors; Epidermal Growth Factor; Failure to Thrive; Fibroblasts; Guanosine Triphosphate; Humans; Immunoblotting; Intellectual Disability; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Morpholines; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Signal Transduction; Skin; Skin Abnormalities; Syndrome

Leprechaunism features a clinical constellation characterized by extreme insulin resistance, growth retardation, and several distinct developmental abnormalities. One puzzling observation about leprechaunism is that mutations in the insulin receptor gene frequently associated with this syndrome cannot account for the aberrant responses of cultured cells to other growth factors. Here we report that the generation of reactive oxygen species (ROS) is impaired in cells from leprechaunism patients, thus shedding new light on this issue. Stimulation of patients' skin fibroblast cells with platelet-derived growth factor (PDGF) resulted in a lower-level tyrosine phosphorylation of cytosolic proteins compared with that seen in normal cells. In addition, consistent with the hypothesis that ROS mediate the level of tyrosine phosphorylation of cytosolic proteins through inactivation of protein tyrosine phosphatases (PTPases), patient fibroblast cells showed a significantly higher phosphatase activity than normal cells. We further showed that the lower-level tyrosine phosphorylation in response to growth factors results from the downregulation of an NADPH oxidase, Nox4, which in turn results in the reduction of ROS generation. Ectopic expression of Nox4 in the patient fibroblast cells consistently restored PDGF-induced ROS production and regulation of PTPase activities. Taken together, these data provide insight into the mechanisms through which growth retardation is associated with leprechaunism syndrome.

Topics: Abnormalities, Multiple; Cells, Cultured; Down-Regulation; Epidermal Growth Factor; Fibroblasts; Gene Expression Regulation, Enzymologic; Humans; Insulin Resistance; NADPH Oxidase 4; NADPH Oxidases; Phenotype; Phosphorylation; Platelet-Derived Growth Factor; Reactive Oxygen Species; Tyrosine

We have investigated and compared GH and epidermal growth factor (EGF) signaling in primary human skin fibroblasts from normal subjects and subjects with GH-binding protein-positive Laron syndrome (LS). In normal human fibroblasts, GH and EGF activate the tyrosine phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT5b; in LS fibroblasts, EGF does, but GH does not. GH also activates the tyrosine phosphorylation of Janus kinase (JAK)2 in normal, but not LS, fibroblasts. Similarly, both GH and EGF activate MAPK in normal fibroblasts, but only EGF does in the LS fibroblasts. As in the 3T3-F442A mouse preadipocyte cell line, GH signaling to mitogen-activated protein kinase is partially inhibited by wortmannin treatment, indicating a role for phosphatidylinositol 3-kinase (PI3K) in this signaling pathway. The exogenous expression of the GH receptor in one family of LS fibroblasts (H1) but not the other (M) restores signaling to a STAT5 reporter element. Together, these results indicate that the mechanism of defective GH signaling in two families of LS fibroblasts are different but that both occur at a level close to, and specific for, the GH receptor.

Topics: Abnormalities, Multiple; Androstadienes; Blotting, Western; Child; Epidermal Growth Factor; Female; Fibroblasts; Growth Hormone; Humans; Indicators and Reagents; Janus Kinase 2; Luciferases; Mitogen-Activated Protein Kinases; Phosphorylation; Precipitin Tests; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Somatotropin; Signal Transduction; Syndrome; Transfection; Tyrosine; Wortmannin

During the immediate-early response of mammalian cells to mitogens, histone H3 is rapidly and transiently phosphorylated by one or more unidentified kinases. Rsk-2, a member of the pp90rsk family of kinases implicated in growth control, was required for epidermal growth factor (EGF)-stimulated phosphorylation of H3. RSK-2 mutations in humans are linked to Coffin-Lowry syndrome (CLS). Fibroblasts derived from a CLS patient failed to exhibit EGF-stimulated phosphorylation of H3, although H3 was phosphorylated during mitosis. Introduction of the wild-type RSK-2 gene restored EGF-stimulated phosphorylation of H3 in CLS cells. In addition, disruption of the RSK-2 gene by homologous recombination in murine embryonic stem cells abolished EGF-stimulated phosphorylation of H3. H3 appears to be a direct or indirect target of Rsk-2, suggesting that chromatin remodeling might contribute to mitogen-activated protein kinase-regulated gene expression.

Topics: 3T3 Cells; Abnormalities, Multiple; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Transformed; Cell Nucleus; Cells, Cultured; Epidermal Growth Factor; Gene Expression Regulation; Gene Targeting; Histones; Humans; Mice; Mitosis; Mutation; Phosphorylation; Ribosomal Protein S6 Kinases; Signal Transduction; Stem Cells; Syndrome

The anatomical substrate of Marfan's syndrome is a degeneration of elastic fibres and disorganization of the collagen. It is now known that these lesions are due to mutation of genes localised on chromosome 15. The first of them (FBN1) codes for the main constitutive protein of the elastic tissue: fibrillin 1, present mainly in structures which must resist load and stress (aortic adventitia, the suspending ligament of the lens, skin); the second (FBN2) codes for fibrillin 2: responsible for the orientation of the elastin and mainly present in cartilage, the aortic media, the bronchi, and all tissues rich in elastin. Mutations of FBN1 are very common and are associated not only with Marfan's syndrome but also fibrillinopathies: incomplete forms, neonatal forms, ectopic lens, isolated aneurysms of the thoracic aorta. The widespread distribution of fibrillin explains the pleiotropic nature of Marfan's syndrome and its clinical presentation. The variability of interfamilial expression is due to genetic heterogeneity (at least two genes) and alletic differences (different mutations of FBN1 from one family to another), also explaining mild forms due to quantitative reduction in normal fibrillin and severe forms by "negative dominance" where the fibrillin is structurally abnormal because of alteration of the polymerisation mechanism. The biologic diagnosis of fibrillopathy can be made by a protein test analysing fibrillin on a culture of the patient's fibroblast obtained by skin biopsy. At present, molecular diagnosis of the mutation within the FBN1 gene is not feasible as a routine procedure.

Topics: Abnormalities, Multiple; Amino Acid Sequence; Child; Child, Preschool; Chromosomes, Human, Pair 15; Epidermal Growth Factor; Fibrillin-1; Fibrillin-2; Fibrillins; Humans; Infant; Infant, Newborn; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Mutation; Pedigree

We have used rats with epidermal growth factor (EGF) autoantibodies to study the role of EGF deficiency during perinatal development. The study was focused on organs known to contain EGF or its receptor. Compared with controls, the offspring of autoimmune rats had a higher perinatal mortality and a lower birth weight. The weight of the lungs was particularly low in the offspring of EGF-immunized rats, and morphologically the lungs from the surviving pups seemed atelectatic and had alveolar duct dilatation, which indicates mild respiratory distress syndrome. Judged from immunohistochemical studies, the amount of surfactant protein-A was decreased, suggesting a delayed lung maturation. The offspring of EGF-immunized rats had dry and wrinkled skin. The skin was thin and the hair follicles were immature. This suggests a role for EGF in the growth and development of the skin. The liver/body weight ratio was lower in pups from EGF-immunized rats. This difference was, however, not significant (p = 0.07), but flow cytometric analyses showed a significantly lower proportion of the liver cells from newborn EGF-deficient pups to be in S-phase and indicated that these cells were larger than liver cells from controls. To study possible alterations in EGF binding, 125I-EGF was injected i.v. in newborn rats. 125I-EGF bound in all the organs investigated. The binding is listed in decreasing order: liver, gut, skin, kidney, and lungs. In the pups from EGF-immunized rats, the lungs and the skin bound a significantly higher amount than the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Abnormalities, Multiple; Animals; Autoantibodies; Birth Weight; Digestive System; Embryonic and Fetal Development; Epidermal Growth Factor; Female; Immunization; Liver; Lung; Pregnancy; Pregnancy Complications; Pulmonary Atelectasis; Rats; Rats, Wistar; Skin

Leprechaunism is an inherited disorder characterized by growth restriction and severe insulin resistance and caused by mutations in the insulin receptor gene. Cells from these patients have defective insulin binding. Fibroblasts from some patients have concomitant defects in other tyrosine kinase receptors, such as those for Insulin-like Growth Factor I (IGF-I) and Epidermal Growth Factor (EGF). In this report, binding of insulin, IGF-I, EGF, and Platelet derived growth factor (PDGF) AA and PDGF-BB is compared among fibroblasts of patients with defined mutations in their insulin receptor gene. Fibroblasts from patient Atl-1, homozygous for a R86P substitution in the insulin receptor, had reduced binding of Platelet-Derived Growth Factor AA (PDGF-AA), a specific ligand for type alpha PDGF receptors. The reduction in PDGF binding impaired the ability of this growth factor to stimulate DNA synthesis.

Topics: Abnormalities, Multiple; Becaplermin; DNA; Epidermal Growth Factor; Fibroblasts; Humans; Insulin; Insulin-Like Growth Factor I; Kinetics; Mutation; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Receptor, Insulin; Receptors, Platelet-Derived Growth Factor; Recombinant Proteins; Reference Values; Skin; Thymidine

Rieger syndrome is an autosomal dominant disorder of morphogenesis in which previous cytogenetic arrangements have suggested chromosome 4 as a candidate chromosome. Using a group of highly polymorphic short tandem repeat polymorphisms (STRP), including a new tetranucleotide repeat for epidermal growth factor (EGF), significant linkage of Rieger syndrome to 4q markers has been identified. Tight linkage to EGF supports its role as a candidate gene, although a recombinant in an unaffected individual has been identified. This study demonstrates the utility of using polymorphic STRP markers when only a limited number of small families are available for study.

Topics: Abnormalities, Multiple; Anterior Eye Segment; Base Sequence; Chromosomes, Human, Pair 4; DNA; Epidermal Growth Factor; Female; Genes, Dominant; Genetic Linkage; Genetic Markers; Humans; Male; Molecular Sequence Data; Pedigree; Repetitive Sequences, Nucleic Acid; Tooth Abnormalities

Topics: Abnormalities, Multiple; Child; Child, Preschool; Endocrine System Diseases; Epidermal Growth Factor; Female; Follow-Up Studies; Growth Disorders; Hirsutism; Humans; Phenotype; Syndrome