Caracterización del receptor de hormonas tiroideas en células nerviosas en cultivo regulación del receptor y efectos biológicos de la T3 en células de neuroblastoma

Tesis doctoral inédita leida en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 11 de Octubre de 1989

Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization

The intestinotrophic and cytoprotective actions of glucagon-like peptide-2 (GLP-2) are mediated by the GLP-2 receptor (GLP-2R), a member of the class II glucagon-secretin G protein-coupled receptor superfamily. Although native GLP-2 exhibits a short circulating half-life, long-acting degradation-resistant GLP-2 analogues are being evaluated for therapeutic use in human subjects. Accordingly, we examined the mechanisms regulating signaling, internalization, and trafficking of the GLP-2R to identify determinants of receptor activation and desensitization. Heterologous cells expressing the transfected rat or human GLP-2R exhibited a rapid, dose-dependent, and prolonged desensitization of the GLP-2–stimulated cAMP response and a sustained GLP-2–induced decrease in levels of cell surface receptor. Surprisingly, inhibitors of clathrin-dependent endocytosis failed to significantly decrease GLP-2R internalization, whereas cholesterol sequestration inhibited ligand-induced receptor internalization and potentiated homologous desensitization. The hGLP-2R localized to both Triton X-100–soluble and –insoluble (lipid raft) cellular fractions and colocalized transiently with the lipid raft marker caveolin-1. Although GLP-2R endocytosis was dependent on lipid raft integrity, the receptor transiently associated with green fluorescent protein tagged-early endosome antigen 1–positive vesicles and inhibitors of endosomal acidification attenuated the reappearance of the GLP-2R on the cell surface. Our data demonstrate that GLP-2R desensitization and raft-dependent trafficking represent distinct and independent cellular mechanisms and provide new evidence implicating the importance of a clathrin- and dynamin-independent, lipid raft-dependent pathway for homologous G protein-coupled receptor internalization

Proliferation and differentiation are not directly related to H1 0 accumulation in cultured glial cells

A basic nuclear chromatin protein with electrophoretic mobility of H1 0 histone is present in C6 rat glial cells and in primary cultures of rat brain astroglial cells. That this protein is identical to H1 0 is further demonstrated by the finding that it accumulates in C6 cells in a time- and dose-dependent manner in response to butyrate, an agent which is known to induce this protein in other cell types. Other short-chain fatty acids were found to influence H1 0 levels similarly although to a lesser extent than butyrate. There was a very close correlation between the induction of H1 0 and the inhibition of growth induced by different concentrations of short-chain fatty acids which supports the idea that the concentration of this protein is higher in non-proliferating cells. However, when cell growth was inhibited by dexamethasone or agents that increase intracellular cyclic adenosine monophosphate levels, H1 0 levels were not affected, even though these compounds also blocked DNA synthesis and induced morphologic changes in C6 cells. These observations suggest that, at least in glial cells, the accumulation of H1 0 is specifically caused by short-chain fatty acids and that suppression of cell division or commitment to differentiation are not sufficient 'per se' for the induction of this protein

Mechanism of L-triiodothyronine (T3) uptake by glial C6 cells: Regulation by butyrate

The mode of entry of triiodothyronine (T3) and its regulation by butyrate was studied in cultured glial C6 cells. Uptake of [125I]T3 increases for at least 60 min in C6 cells. The amount of cell-associated radioactivity is 2- to 4-fold higher during the entire time-course in cells previously exposed to 2 mM butyrate for 48 h. Uptake was non-saturable since uptake velocity was linearly related to the extracellular hormone concentration between 0.2 and 800 nM T3 in control and butyrate-treated cells. Uptake velocity increased by more than 3-fold in the cells incubated with the fatty acid. T3 uptake was temperature dependent and the effect of butyrate was observed at the different temperatures examined. Preincubation with metabolic inhibitors did not block [125I]T3 uptake in either group, and monodansylcadaverine was also ineffective. Present results suggest that in C6 cells T3 uptake proceeds by a passive, energy-independent, non-saturable process, that is markedly affected by short-chain fatty acids. Additonally, this is the first study documenting that a natural compound directly influences the entry of thyroid hormones into cells

Comparison of the effects of forskolin and dibutyryl cyclic AMP in neuroblastoma cells: Evidence that some of the actions of dibutyryl cyclic AMP are mediated by butyrate

We have compared the effects of forskolin, N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP, Bt2-cAMP), and butyrate on several aspects of neuroblastoma cell physiology. The morphology of Neuro 2A cells was similar after incubation with forskolin and Bt2-cAMP, which caused extensive neurite outgrowth, whereas in the presence of butyrate some rudimentary neurites were formed but they were not nearly as extensive. All compounds produced a dose-dependent inhibition of cell proliferation, but the effect of Bt2-cAMP was more marked than that caused by forskolin, thus showing that the effect of Bt2-cAMP is due partially to the butyrate released. Acetylcholinesterase activity was lower in the cells incubated with butyrate or Bt2-cAMP than in untreated cells or in forskolin-treated cells. This suggests that cyclic AMP does not play a role in the regulation of this enzyme. Bt2-cAMP produced histone acetylation, a well-known effect of butyrate in cultured cells, whereas forskolin did not affect this modification. Consequently, the levels of thyroid hormone receptor, a nuclear protein whose concentration is regulated by butyrate through changes in acetylation of chromatin proteins, were decreased in cells incubated with Bt2-cAMP or butyrate, but were unaffected by forskolin. Butyrate elevated the concentration of histone H10, a protein that increases in neuroblastoma cells as a result of different treatments that block cell division. The concentration of H10 in the cells treated with Bt2-cAMP was at a level intermediate between that found after treatment with butyrate and with forskolin. The present results clearly indicate that some of the effects of Bt2-cAMP on neuroblastoma cells can be attributed to the butyryl moiety of this compound rather than to the cylic nucleotide itself.Peer Reviewe

Organization of the thyroid hormone receptor in the chromatin of C6 glial cells: Evidence that changes in receptor levels are not associated with changes in receptor distribution

The association of [125I]T3-receptor complexes with C6 cell chromatin was analyzed after a limited digestion with micrococcal nuclease (MN) or DNase I. Both nucleases solubilized up to 60-70% of receptor and 0.4 M KCl extracted 70% of the non-digested receptor, thus showing that only a residual fraction of receptor is associated with the nuclear matrix. With DNase I the receptor was released 2-3-fold faster than the bulk of chromatin, whereas a preferential release of receptor over total chromatin was not observed with MN. The digestion of receptor with DNase I and MN occurred 14- and 6-fold faster, respectively, than the appearance of PCA-soluble chromatin. Preincubation for 48 h with 4 nM T3 of 2 mM butyrate significantly altered receptor levels but did not change sensitivity to the nucleases. These results suggest that the thyroid hormone receptor is associated with chromatin highly sensitive to nuclease digestion, and that changes in receptor number are not associated with changes in its distribution in chromatin.Supported by grants from CAYCIT and CICYT (PM88-0007). The technical assistance of Ms A. Villa is gratefully acknowledged

Triiodothyronine (T3) induces neurite formation and increases synthesis of a protein related to MAP 1B in cultured cells of neuronal origin

Neuroblastoma (N2A) cells were found to develop axon-like neurite extensions when grown in the presence of triiodothyronine (T3), while C6 cells (of glial origin) did not. Analysis of radiolabelled protein synthesis showed that, in N2A only, T3 increased the synthesis of a polypeptide corresponding in electrophoretic mobility to the microtubule-associated protein MAP 1B. Immunoblotting of total cell proteins with a monoclonal antibody confirmed that this polypeptide was immunologically related to MAP 1B. Further studies using indirect immunofluorescence with monoclonal antibodies against both tubulin and MAP 1B showed that both antigens were present in neurites. Taken together, these results suggest that T3 may control maturation of neural tissue via effects on the microtubule-associated proteins in cells of neuronal origin.This work has been supported by grants from ‘Comision Asesora de Investigation Cientifica y Tecnica’, ‘Fondo de Investigaciones Sanitarias de la Seguridad Social’ and partially by the U.S. - Spain committee for Scientific and Technological Cooperation.Peer reviewe