Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines

Embryonic stem cells are established directly from the pluripotent epiblast of the preimplantation mouse embryo. Their derivation and propagation are dependent upon cytokine-stimulated activation of gp130 signal transduction. Embryonic stem cells maintain a close resemblance to epiblast in developmental potency and gene expression profile. The presumption of equivalence between embryonic stem cells and epiblast is challenged, however, by the finding that early embryogenesis can proceed in the absence of gp130. To explore this issue further, we have examined the capacity of gp130 mutant embryos to accommodate perturbation of normal developmental progression. Mouse embryos arrest at the late blastocyst stage when implantation is prevented. This process of diapause occurs naturally in lactating females or can be induced experimentally by removal of the ovaries. We report that gp130(-/-) embryos survive unimplanted in the uterus after ovariectomy but, in contrast to wild-type or heterozygous embryos, are subsequently unable to resume development. Inner cell masses explanted from gp130(-/-) delayed blastocysts produce only parietal endoderm, a derivative of the hypoblast. Intact mutant embryos show an absence of epiblast cells, and Hoechst staining and TUNEL analysis reveal a preceding increased incidence of cell death. These findings establish that gp130 signalling is essential for the prolonged maintenance of epiblast in vivo, which is commonly required of mouse embryos in the wild. We propose that the responsiveness of embryonic stem cells to gp130 signalling has its origin in this adaptive physiological function.     

gp130-Dependent signalling pathway is not enhanced in gp130 transgenic heart after LIF stimulation

Activation of gp130 transduces a hypertrophic signal in the heart, but it is not clear whether signalling through gp130 is enhanced when gp130 is overexpressed in vivo. We generated gp130 transgenic mice (TG) and examined the activation of signalling pathways downstream of gp130 in the hearts. The tyrosine phosphorylation of gp130 was enhanced, the phosphorylation of STAT3 and ERK (extracellular signal regulated kinase) 1/2 was increased and induction of the beta-myosin heavy chain (MHC) gene was observed in TG hearts without significant phenotypic changes. Intravenous administration of leukaemia inhibitory factor (LIF) induced tyrosine phosphorylation of STAT3 and ERK 1/2 and expression of c-fos and beta-MHC mRNAs in wild-type littermates' (WT) hearts. However, enhancement of STAT3 and ERK 1/2 phosphorylation or augmented mRNA expressions was not observed in TG hearts after LIF stimulation. Next, STAT-induced STAT inhibitor (SSI) mRNA expression was examined. The expression of SSI-1, SSI-2, and SSI-3 mRNAs was significantly augmented in TG hearts after LIF stimulation. These results indicate that overexpressed gp130 does not always enhance downstream signals in the hearts and suggest that the SSI family plays a role in the regulation of the gp130-dependent signalling pathway in the hearts.     

Receptor recognition by gp130 cytokines

Cytokines of the gp130 family exert their diverse biological effects by formation of stable high affinity transmembrane receptor complexes that are characterized by the presence of the shared transmembrane signalling receptor gp130. Different gp130 ligands form signalling complexes that vary in both composition and stoichiometry. Analysis of the three-dimensional structure of selected ligands and receptor elements indicates that ligands display three topologically conserved receptor recognition epitopes that interact with complementary ligand recognition elements. The composition of the signalling complex and downstream biological responses is defined by the relative affinity of different receptor components for these epitopes. The detailed structure of receptor recognition epitopes indicates that the generation of small molecule cytokine mimetics may be a feasible objective.     

The role of the glycoprotein gp130 in serotonin mediator system in mouse brain

Glycoprotein gp130 is involved in signaling out of significant cytokine receptors as interleukin-6 (IL-6), leukemia inhibitory factor and ciliary neurotrophic factor, which play critical role in immunity, inflammation and neurogenesis. IL-6 and brain neurotransmitter serotonin are involved in the mechanism of depression. The aim of this work was to investigat the role of protein gp130 in the regulation of expression of genes, coding the key enzyme of serotonin synthesis--tryptophan hydroxylase 2 (TPH2), 5-HT-transporter, 5-HT(1A)- and 5-HT(2A)-receptors of serotonin. The study was carried out on adult mouse males of AKR and congenic AKR.CBA-D13Mit76 strains, created by transfer of the fragment of chromosome 13 containing the gene coding gp130 protein from CBA/Lac strain to the genome of AKR/J strain. Decreased expression of 5-HT(1A) - 5-HT(2A)-receptor genes in hippocampus midbrain and TPH2 gene in midbrain in AKR.CBA-D13Mit76 mice compared with AKR mice were shown. Activation of nonspecific immunity by bacterial endotoxin lipopolysaccharide (LPS) administration did not affect the genes expression in AKR mice, but increased 5-HT(2A)-receptor expression in midbrain and decreased 5-HT(1A)-receptor expression in cortex in AKR.CBA-D13Mit76 mice. The results indicate: 1) the participation of gp130 in the regulation of TPH2, 5-HT(1A)- and 5-HT(2A)-receptor genes and 2) association of this protein in the genetically determined sensitivity to LPS.     

N-Glycans modulate the activation of gp130 in mouse embryonic neural precursor cells

gp130 is a ubiquitously expressed glycoprotein and signal transducer of interleukin 6 family of cytokines. It has been reported that gp130 has 11 potential N-glycosylation sites in the extracellular domain, and nine of them are actually N-glycosylated. However, the structure and functional role of the carbohydrate chains carried by gp130 are totally unknown. In this study, we examined the functional role of N-glycans of gp130 in mouse neuroepithelial cells. In neuroepithelial cells treated with tunicamycin, an N-glycosylation inhibitor, unglycosylated form of gp130 was detected. The unglycosylated gp130 was not phosphorylated in response to leukemia inhibitory factor stimulation. Although the unglycosylated gp130 was found to be expressed on the cell surface, it could not form a heterodimer with leukemia inhibitory factor receptor. These results suggest that N-glycans are required for the activation, but not for the localization, of gp130 in neuroepithelial cells.     

Transactivation of gp130 in myeloma cells

Receptor transactivation, i.e., interaction between unrelated receptor systems, is a growing theme in cytokine and growth factor signaling. In this study we reveal for the first time the ability of IFN-alpha to transactivate gp130 in myeloma cells. An epidermal growth factor receptor/gp130 chimeric receptor previously shown by us to transactivate endogenous gp130, provided a complementary tool to study the underlying mechanisms of receptor cross-talk. Further analysis revealed that transactivation of gp130 by IFN-alpha did not require the extracellular or trans-membrane domain of gp130. Moreover, transactivation of gp130 was critically dependent upon Janus kinase activation by the initiating receptor and correlated with rapid and sustained Janus kinase 1 and tyrosine kinase (Tyk) 2 tyrosine phosphorylation. Finally, transactivation of gp130 may be a common theme in myeloma cells, perhaps providing a mechanism for enhanced or qualitatively distinct cellular responses to specific stimuli.     

gp130 Cytokine family and bone cells

Bone tissue is continually being remodelled according to physiological circumstances. Two main cell populations (osteoblasts and osteoclasts) are involved in this process, and cellular activities (including cell differentiation) are modulated by hormones, cytokines and growth factors. Within the last 20 years, many factors involved in bone tissue metabolism have been found to be closely related to the inflammatory process. More recently, a cytokine family sharing a common signal transducer (gp130) had been identified, which appears to be a key factor in bone remodelling. This family includes interleukin 6, interleukin 11, oncostatin M, leukaemia inhibitory factor, ciliary neurotrophic factor and cardiotrophin-1. This paper provides an exhaustive review of recent knowledge on the involvement of gp130 cytokine family in bone cell (osteoblast, osteoclast, etc.) differentiation/activation and in osteoarticular pathologies.     

Trace element binding in the copper deficient mottled mutants in the mouse.

In contrast to the situation for copper, the levels of cadmium, nickel and zinc in various tissues from mice hemizygous for the neonatallethal mottled allele $\text{Mo}{}^{\mathrm{br}}$ are entirely normal. A developmental study of copper levels in the semi-viable $\text{Mo}{}^{\mathrm{vbr}}$ has revealed that the accumulation of copper in the kidney and the deficiency in the brain are maintained at about the same level throughout the first 4 weeks of life. In contrast, the accumulation in the gut mucosa disappears in older animals, and the deficiency in the liver is much reduced by 21–30 days. A reduced gut accumulation and liver deficiency is also seen in adult $\text{Mo}{}^{\mathrm{br}}$/+ heterozygotes. The deficiency in the liver and the accumulation in the kidney of $\text{Mo}{}^{\mathrm{br}}$ male mice is associated with a protein fraction of about 14000 daltons. This fraction isolated from liver tissue has been further divided by ion-exchange chromatography into 4 sub-fractions, with each sub-fraction from mutant tissue showing an approximately proportionate reduction in copper content.     

The role of the glycoprotein gp130 in the serotonin mediator system in the mouse brain

Glycoprotein gp130 is involved in signal transduction from the receptors of such important cytokines as interleukin-6 (IL-6), leukemia inhibitory factor, and ciliary neurotrophic factor, which play a critical role in immunity, inflammation, and neurogenesis. Both IL-6 and the brain neurotransmitter serotonin are involved in the mechanism of depression. The aim of this work was to investigate the role of gp130 in regulating the gene expression of the tryptophan hydroxylase 2 (TPH2), the key enzyme of the serotonin synthesis, as well as of the 5-HT transporter and the 5-HT_1A and 5-HT_2A receptors. The study was carried out on adult male mice of the congenic strains AKR and AKR.CBA-D13Mit76; the latter was created by transferring a gp130-containing fragment of chromosome 13 from the CBA/Lac strain into the AKR/J genome. The expression of 5-HT_1A and 5-HT_2A receptor genes in the hippocampus and midbrain and of the TPH2 gene in the midbrain was decreased in AKR.CBA-D13Mit76 mice in comparison to AKR mice. Activation of nonspecific immunity by administration of a bacterial endotoxin lipopolysaccharide did not affect the gene expression in AKR mice but increased the 5-HT_2A receptor expression in the midbrain and decreased the 5-HT_1A receptor expression in the cortex in AKR.CBA-D13Mit76 mice. These results suggest that gp130 is involved in the regulation of TPH2, 5-HT_1A and 5-HT_2A receptor genes and is associated with the genetically determined sensitivity to lipopolysaccharides.     

Conditional lethality of the recA441 and recA730 mutants of Escherichia coli deficient in DNA polymerase I

E. coli strains bearing the recA441 mutation and various mutations in the polA gene resulting in enzymatically well-defined deficiencies of DNA polymerase I have been constructed. It was found that the recA441 strains bearing either the polA1 or polA12 mutation causing deficiency of the polymerase activity of pol I are unable to grow at 42 degrees C on minimal medium supplemented with adenine, i.e., when the SOS response is continuously induced in strains bearing the recA441 mutation. Under these conditions the inhibition of DNA synthesis is followed in recA441 polA12 by DNA degradation and loss of cell viability. A similar lethal effect is observed with the recA730 polA12 mutant. The recA441 strain bearing the polA107 mutation resulting in the deficiency of the 5'-3' exonuclease activity of pol I shows normal growth under conditions of continuous SOS response. We postulate that constitutive expression of the SOS response leads to an altered requirement for the polymerase activity of pol I.     

Exon-intron organization of the human gp130 gene

The exon-intron organization and sequences of the exon-intron boundaries of the human gp130 transmembrane receptor gene have been determined using genomic DNAs as samples. The gp130 gene comprises 17 exons and 16 introns. The positions of the exon-intron boundaries show good correlation to the functional/homology regions of gp130. Exons 3-17 code for the gp130 protein, and each subdomain of the receptor is encoded by a set of exons. The coding potential of exons and the intron phasing of the human gp130 gene conform to the patterns observed previously for other cytokine receptor genes. This supports the notions that the gp130 gene evolved from the same ancestral gene that gave rise to other members of the cytokine receptor family.     

c-Cbl-dependent monoubiquitination and lysosomal degradation of gp130

Interleukin 6 (IL-6), a pleiotropic cytokine, functions in cells through its interaction with its receptor complex, which consists of two ligand-binding α subunits and two signal-transducing subunits known as gp130. There is a wealth of studies on signals mediated by gp130, but its downregulation is less well understood. Here we found that IL-6 stimulation induced lysosome-dependent degradation of gp130, which correlated with an increase in the K63-linked polyubiquitination of gp130. The stimulation-dependent ubiquitination of gp130 was mediated by c-Cbl, an E3 ligase, which was recruited to gp130 in a tyrosine-phosphorylated SHP2-dependent manner. We also found that IL-6 induced a rapid translocation of gp130 from the cell surface to endosomal compartments. Furthermore, the vesicular sorting molecule Hrs contributed to the lysosomal degradation of gp130 by directly recognizing its ubiquitinated form. Deficiency of either Hrs or c-Cbl suppressed gp130 degradation, which leads to a prolonged and amplified IL-6 signal. Thus, our present report provides the first evidence for involvement of a c-Cbl/SHP2 complex in ubiquitination and lysosomal degradation of gp130 upon IL-6 stimulation. The lysosomal degradation of gp130 is critical for cessation of IL-6-mediated signaling.     

Respiratory deficient mutants of Rhodopseudomonas capsulata

The facultative photosynthetic bacterium Rhodopseudomonas capsulata was mutagenized by transfer of the plasmid pSUP201::Tn5 from Escherichia coli to R. capsulata. Mutants defective in cytochrome oxidase and other respiratory functions were selected by replica plating, NADI-reaction and immunological methods. Among 20,000 mutants no clone was detected, which lacks the 65,000-protein of the cytochrome oxidase, but many mutants have been isolated which were cytochrome oxidase deficient (or inactive). Other mutants excrete heme and cytochrome c into the medium and lack cytochrome c ₂.Abbreviations Ap ampicillin - CIE crossed immunoelectrophoresis - cyt cytochrome - Cm chloramphenicol - Km kanamycin - SDS sodium dodecylsulfate - Tc tetracycline     

Soluble glycoprotein 130 (gp130) attenuates OSM- and LIF-induced cartilage proteoglycan catabolism

Oncostatin M (OSM) and leukaemia inhibitory factor (LIF) exhibit pleiotropic biological activities and share many structural and genetic features. The two cytokines bind with high affinity to the same receptor (LIF/OSM receptor), which consists of the LIF receptor alpha chain (LIFRalpha) and the signal transduction unit gp130. A soluble form of the beta chain of the receptor complex called soluble gp130 (sgp130) has been cloned. In this study, we sought to determine whether recombinant sgp130 or anti-gp130 Ab could attenuate the resorption of proteoglycans induced by OSM and LIF in articular cartilage explants. The results show that at high concentrations sgp130 is capable of attenuating both LIF and OSM mediated resorption. In contrast, anti-gp130 Ab selectively inhibited the stimulation of proteoglycan (PG) release by OSM, albeit minimally. The failure of anti-gp130 to attenuate LIF stimulated PG resorption may be due to the normal interaction of LIF with LIFRalpha and unfettered heterodimerization of LIFRalpha with gp130 in the presence of the antibody. The results indicate that sgp130 and anti-gp130 can modulate cartilage PG metabolism in vitro. Whether sgp130 may have therapeutic activity in models of arthritis or indeed in arthritic diseases remains to be determined.     

Acquiring signalling specificity from the cytokine receptor gp130

Repeated use of a relatively small number of intracellular signalling molecules specifies tissue- and cell-type-specific responses to pleiotropic-acting growth factors and cytokines. Currently, gaining a better understanding of these mechanisms is a major challenge. The IL-6 family of cytokines shares a common receptor subunit called gp130. Phenotypic comparisons of mice with amino acid knock-in substitutions that disable individual signalling modules in gp130, with knockout mice lacking ligand-specific gp130 activation or transgenic mice with constitutive gp130 activation, has led to the identification of two molecular mechanisms. One mechanism is based on differential target-gene responsiveness to signalling threshold levels transduced by either the STAT1/3 or the SHP2/ERK cascade, which are under reciprocal negative regulation and together account for the majority of intracellular gp130 signalling. The second mechanism is based on the capacity of certain cell types to integrate the often-conflicting information transduced by these two pathways, and to prevent pathological responses.