RESUMEN
OBJECTIVES: In the hypothalamus, the molecular actions of receptors for growth hormone secretagogue (ghrelin) receptor-GHSR, leptin receptor-b (LEPRb), Melanocortin-4 receptor (MC4R) and Cannabinoid-1 receptor (CB1R) regulate energy homeostasis and body weight. We hypothesized that the acute loss of stomach tissue upon sleeve gastrectomy (SG), performed to treat obesity, imposes modulations on the expression of these receptors in the brain to sustain weight loss. METHODS: Rats, induced to obesity with high-fat diet were randomized to SG- or sham-operation groups and killed at 30 or 90 days post surgery, when the expression of Ghrl, Mboat4 and Cnr1 in the stomach, and Ghsr, Leprb, Mc4r and Cnr1 in distinct brain areas was assessed by reverse transcription-PCR and western blotting. RESULTS: SG acutely reduced body weight and fat mass and suppressed the remnant stomach mRNA levels of preproghrelin and ghrelin O-acyltransferase, which correlated well with long-term decreases in CB1R mRNA. In the hypothalamus, increases in GHSR and decreases in CB1R and LEPRb by 30 days were followed by further downregulation of CB1R and an increase in MC4R by 90 days. CONCLUSIONS: Post SG, acyl-ghrelin initiates a temporal hierarchy of molecular events in the gut-brain axis that may both explain the sustained lower body weight and suggest intervention into the cannabinoid pathways for additional therapeutic benefits.
RESUMEN
Children with neurofibromatosis (NF1) typically develop central nervous system (CNS) abnormalities, including aberrant proliferation of astrocytes and formation of benign astrocytomas. The NF1 gene encodes neurofibromin, a Ras-GAP, highly expressed in developing neural cells; the mechanism of regulation of neurofibromin as a Ras-GAP, remains however unknown. We now show that, in response to EGF, neurofibromin is in vivo phosphorylated on serine residues by PKC-alpha, in human, rat, and avian CNS cells and cell lines. EGF-induced PKC phosphorylation was prominent in the cysteine/serine-rich domain (CSRD) of neurofibromin, which lies in the N-terminus and upstream of the Ras-GAP domain (GRD), and this modification significantly increased the association of neurofibromin with actin in co-immunoprecipitations. In addition, we show that Ras activation in response to EGF was significantly lowered when C62B cells overexpressed a construct encoding both CSRD + GRD. Moreover, when PKC-alpha was downregulated, the Ras-GAP activity of CSRD + GRD was significantly diminished, whereas overexpressed GRD alone acted as a weaker GAP and in a PKC-independent manner. Most importantly, functional Ras inhibition and EGF signaling shifts were established at the single cell level in C6-derived cell lines stably overexpressing CSRD + GRD, when transient co-overexpression of Ras and PKC-depletion prior to stimulation with EGF-induced mitosis. Taken together, these data provide the first evidence of a functional, allosteric regulation of GRD by CSRD, which requires neurofibromin phosphorylation by PKC and association with the actin cytoskeleton. Our data may suggest a novel mechanism for regulating biological responses to EGF and provide a new aspect for the understanding of the aberrant proliferation seen in the CNS of children with NF1.
Asunto(s)
Receptores ErbB/metabolismo , Neurofibromina 1/metabolismo , Neuronas/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Humanos , Inmunohistoquímica , Neuronas/citología , Fosforilación , Ratas , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
The protein product of the neurofibromatosis 1 gene, neurofibromin, is abundantly expressed in the cerebral cortex during development, but its physiological role remains unknown. To gain insights into the functions of neurofibromin in neurons, we examined patterns of expression and subcellular localization of neurofibromin during neuronal differentiation. Western blot analysis of telencephali homogenates throughout chick embryogenesis revealed that neurofibromin expression increased during embryonic development. Further analysis showed that telencephalic neurons were also enriched in neurofibromin in culture and that a biphasic gain in expression correlated well with both phases of differentiation in culture, first with a massive outgrowth of processes and gains in neurotransmitter phenotype differentiation, and then with synapse formation. Compared to proteins associated with distinct cytoskeleton systems, the pattern of neurofibromin expression correlated closely with that of the cortical cytoskeleton protein paxillin. Moreover, analysis of immunofluorescence staining of neurofibromin showed that in the presence of a protein crosslinker which preserves both soluble and filamentous cytoskeleton proteins after extraction with Triton X-100, neurofibromin colocalized with F-actin only during the first differentiation phase. This colocalization persisted when the actin cytoskeleton was collapsed with cytochalasin D treatment. In contrast, during the second phase of differentiation neurofibromin colocalized with microtubules, but not F-actin, and the staining pattern was disrupted with nocodazole, but not cytochalasin. A constant finding under all conditions was the presence of neurofibromin in the nucleus, which supports the idea that the bipartite nuclear targeting sequence between residues 2555 and 2572 of neurofibromin may be functional. In summary, we have shown that telencephalic neurons and astroblasts are enriched in neurofibromin and that the subcellular targeting of neurofibromin toward the actin or the microtubule cytoskeleton is developmentally regulated.
Asunto(s)
Actinas/análisis , Microtúbulos/química , Neurofibromina 1/análisis , Neuronas/citología , Telencéfalo/embriología , Citoesqueleto de Actina/química , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Antineoplásicos/farmacología , Astrocitos/química , Astrocitos/citología , Astrocitos/metabolismo , Western Blotting , Diferenciación Celular/fisiología , Células Cultivadas , Embrión de Pollo , Citocalasina D/farmacología , Inmunohistoquímica , Microscopía Confocal , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Neuronas/química , Neuronas/metabolismo , Nocodazol/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Polímeros/metabolismo , Telencéfalo/citologíaRESUMEN
There is increasing evidence that proteoglycans, particularly chondroitin sulfate proteoglycans (CSPGs), are integral components in the assembly of the extracellular matrix during early stages of histogenesis. The differential expression of several CSPGs in the developing CNS has raised questions on their origin, phenotype (chemical and structural characteristics), regulation of expression and function. The S103L monoclonal antibody has been an invaluable specific reagent to identify and study a large and abundant CSPG in embryonic chick brain. In the present study we demonstrate that during embryogenesis of the chick CNS, the S103L CSPG (B-aggrecan) is synthesized by neurons of all major neuronal cell types but not by astrocytes, is developmentally regulated, and is associated predominantly with neuronal somata, suggesting that neuronal-specific regulatory mechanisms control the expression of the S103L CSPG in culture. Neurons also exhibit differential expression of glycosaminoglycan type (i.e., KS) and sulfation patterns on different CSPGs when compared to astrocytes, meningial cells or chondrocytes, implying the existence of additional, cell type-specific modes of regulation of the final CSPG phenotype (chemical and structural posttranslational characteristics). A specific temporal pattern of expression of the S103L-CSPG was observed which may contribute to conditions that induce or stabilize specific cell phenotypes during CNS development. In contrast, the other major CSPG in the CNS recognized by the HNK-1 antibody, is synthesized by all cell types of different cell lineages over the entire embryonic period, suggesting a more global cell maintenance function for this CSPG.
Asunto(s)
Sistema Nervioso Central/enzimología , Sistema Nervioso Central/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Animales , Anticuerpos Monoclonales/biosíntesis , Astrocitos/metabolismo , Western Blotting , Células Cultivadas , Sistema Nervioso Central/citología , Cesio/química , Embrión de Pollo , Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Matriz Extracelular/metabolismo , Técnicas para Inmunoenzimas , Inmunohistoquímica , Marcaje Isotópico , Meninges/citología , Meninges/metabolismo , Sulfotransferasas/biosíntesis , Radioisótopos de AzufreRESUMEN
In normal development, embryonic astrocytes progress through their cell lineage by acquiring differentiation, by apoptosis, and by proliferation. In this study, we show that embryonic astrocytes may maintain and make gains in differentiation as they simultaneously progress through one cell cycle when induced by prolactin (PRL). Prolactin induced the majority of astrocytes to incorporate bromodeoxyuridine (BrdU) with a four-fold increase over controls after 18 h of exposure. Investigating possible mitogenic signaling pathways we show for the first time that prolactin is coupled to a sustained phospholipase D (PLD) activation, with an efficacy similar to the phorbol ester and astrocytic mitogen 12-tetradecanoylphorbol-13-acetate (TPA). Both cyclosporine and suramin abolished this activation. Staurosporine and calphostin C also inhibited the PRL effect by 50%, consistent with involvement of protein kinase C-(PKC)-alpha, the major PKC isoform in astrocytes. Genistein and PP1 blocked the activation indicating additional regulation by cytosolic tyrosine kinases. This profile of PLD activation was suggestive of a PLD I isoform and a mitogenic response. Upon completion of the cell cycle, analysis of glia fibrillary acidic protein (GFAP) and vimentin abundance, and glutamine synthetase (GS) activity showed that astrocytes had gained in expression of differentiation markers. Moreover, the intensity of GFAP immunofluorescence was greater per cell, as was the length of the cell processes. In exploring the signaling for prolactin-induced differentiation we found that prolactin activated the tyrosine kinase Janus kinase (JAK) 2 and significantly stimulated tyrosine, phosphorylation of the prolactin receptor. Stat 1 and 3 were also activated presumably downstream to JAK2 activation. A rapid translocation of the cytosolic Stats over the nucleus was seen in nearly every astrocyte corresponding well with the gains in GFAP per cell. The Stats translocation did not depend on MEK-ERK inhibition by PD98059, inhibition of p38 by 1 microm SB203580, or Src kinase family inhibition by PP1. Our results demonstrate the ability of PRL to concurrently induce activation of PLD, a mitogenic signaling pathway in astrocytes, and prolonged stimulation of Stat1, compatible with the increased GFAP upregulation and cell differentiation. Considered together this data may provide an explanation on the fast gain in both numbers and differentiation in the astrocytic population during development (HD 09402, CRF).
Asunto(s)
Astrocitos/fisiología , Fosfolipasa D/fisiología , Prolactina/fisiología , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal/fisiología , Familia-src Quinasas/fisiología , Animales , Antimetabolitos , Western Blotting , Bromodesoxiuridina , Diferenciación Celular/fisiología , División Celular/fisiología , Células Cultivadas , Glutamato-Amoníaco Ligasa/metabolismo , Inmunohistoquímica , Janus Quinasa 3 , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Pruebas de Precipitina , Proteína Quinasa C/fisiologíaRESUMEN
Excitatory and inhibitory neuronal cell fates require specific expression of both neurotransmitter and morphological phenotypes. The role of the F-actin cytoskeleton in morphological phenotypes has been well documented, but its role in neurotransmitter phenotype expression remains unknown. Here we present evidence that the F-actin binding protein cortactin participates in determining both aspects of cell fate in large telencephalic neurons. We show that the expression of cortactin was upregulated early in development just prior to appearance of GABAergic neurons in the chick telencephalon at embryonic day 6. This program was faithfully maintained in primary neuronal cultures derived from E6 telencephalon, where immature neurons differentiate either to large pyramidal and large stellate excitatory neurons or to small inhibitory GABAergic neurons. Immunostaining revealed that cortactin was enriched in areas of membrane budding, growth cones, and in the cell cortex of immature neurons. With differentiation, intense punctate staining was also observed in an extraction-resistant cytosolic compartment of the soma and processes. More importantly, suppression of cortactin by inhibition of cortactin mRNA translation with antisense oligonucleotides caused permanent phenotypic changes. Specifically, a transient suppression of cortactin was achieved in immature neurons with a single exposure to antisense oligonucleotides. This inhibition first induced both the expression of mRNA and the enzymatic activity of GAD significantly earlier than in control neurons. Second, cortactin-suppressed large projectional neurons exhibited significantly shorter processes and growth cones with protrusive filopodia and an enlarged lamellipodia veil. Most importantly, this remodeling of neuritic outgrowth in projectional somata was accompanied by the ectopic induction of GABA (*-aminobutyric acid) expression. Considering this data altogether, it appears that cortactin may function to suppress concurrently several parameters of the GABAergic program in large developing neurons.
Asunto(s)
Proteínas de Microfilamentos/metabolismo , Neuronas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Diferenciación Celular , División Celular , Células Cultivadas , Embrión de Pollo , Cortactina , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/genética , Neuronas/citología , Oligonucleótidos Antisentido , Fenotipo , Telencéfalo/citologíaRESUMEN
The effects of pituitary and extrapituitary prolactin include cellular proliferation and differentiation. PC12 cells was used as a model to delineate respective signaling of prolactin. Prolactin acted as a mitogen for undifferentiated PC12 cells, as measured by significant increases in bromodeoxyuridine incorporation and in cell numbers, with an efficacy equal to epidermal growth factor. Both the long and short form of the prolactin receptor was expressed, yet only the long isoform was tyrosine-phosphorylated upon agonist binding. Functional prolactin receptor signaling was further demonstrated in the activation of JAK2 and phosphorylation activation of the transcription factors Stat1, -3, and -5a. Surprisingly, prolactin stimulated a sustained activation of Raf-B, without activation of the MAP kinases ERK1 or -2. Instead, in solid phase kinase assays using a glutathione S-transferase-c-Jun fusion protein (amino acids 1-79) as the substrate, a significant activation of the mitogen-activated protein Janus kinase (c-Jun N-terminal kinase; JNK) was observed. The prolactin-induced activation of JNK was prolonged and accompanied by a significant increase in c-Jun mRNA abundance and c-Jun protein synthesis. Moreover, analysis of bromodeoxyuridine incorporation at the single cell level revealed that epidermal growth factor-dependent incorporation was inhibited by PD98059 and independent of SB203580, whereas prolactin-induced incorporation was ERK and mitogen-activated protein kinase p38 independent but was abolished with JNK inhibition by 30 microm SB203580. Our studies suggest that prolactin may have a role in the growth of PC12 cells, where it stimulates concurrent mitogenic and differentiation-promoting signaling pathways.
Asunto(s)
División Celular/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Prolactina/fisiología , Animales , Secuencia de Bases , Cartilla de ADN , Activación Enzimática , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Células PC12 , Fosforilación , Ratas , Receptores de Prolactina/metabolismo , Tirosina/metabolismoRESUMEN
Embryonic astrocytes respond readily to serine/threonine kinase regulation in terms of cytoskeleton assembly, mitotic activity, and cell fate. We now present evidence that these responses include apoptosis. Staurosporine induced apoptosis in astrocyte cultures derived from chick embryo cerebral hemispheres, as assayed both by immunocytochemical detection of new 3-hydroxy DNA ends and production of 200-bp DNA fragment laddering. Staurosporine treatment also resulted in the prolonged (>24 h) activation of a 60-kDa serine/threonine protein kinase (PK60), increased ceramide formation (fourfold after 24 h), increased glutamine synthetase activity, and significant apoptosis (40%) after 24 h. PK60 was shown to be cytoskeleton associated and its activity, as measured by phosphorylation of myelin basic protein, was rapid, increased for up to 3 h, and was stable for at least 24 h. Other protein kinase C inhibitors, H8, sphingosine, calphostin C, or the protein kinase A inhibitor KT5720 did not induce either PK60 activation or apoptosis. The dose-dependent increase in [3H]palmitate labeling of ceramide and a specific decrease in labeling of its precursor sphingomyelin were not blocked by the biosynthetic inhibitor fumonisin beta1 but were increased (in a dose-dependent manner) by the coaddition of the ceramidase inhibitor oleoylethanolamine. Exogenous addition of C2-ceramide induced apoptosis but did not activate PK60. These results suggest that apoptosis in embryonic astrocytes involves pathways similar to those described in other cell types and that the activation of PK60 and formation of ceramide are early events in the pathway.
Asunto(s)
Apoptosis/fisiología , Astrocitos/fisiología , Ceramidas/biosíntesis , Corteza Cerebral/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Astrocitos/efectos de los fármacos , Corteza Cerebral/citología , Corteza Cerebral/embriología , Embrión de Pollo , Citoesqueleto/enzimología , Relación Dosis-Respuesta a Droga , Endocannabinoides , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Etanolaminas/farmacología , Ácidos Oléicos , Fosfotransferasas/metabolismo , Estaurosporina/farmacología , Factores de TiempoRESUMEN
This review explores the role of individual opioid receptor types and signal transduction pathways on cell growth and differentiation. The findings reviewed herein provide suggestive evidence that while no single opioid receptor or peptide type exclusively regulates growth, depending on the cell type, the activation of all three (mu, delta, or kappa) opioid receptor types can affect maturation in a cell type-dependent manner. Specific developmental responses are determined primarily by how a particular opioid receptor type is coupled to intracellular signaling effectors. Moreover, the coupling of opioid receptors appears to be developmentally regulated, and these protein-protein interactions change during ontogeny. The diversity of opioid receptor types and intracellular effectors may be a mechanism by which individual cells discriminate among different opioid signals, and may permit diverse opioid signals to be translated into a unique developmental logic in distinct neuronal and glial subpopulations.
Asunto(s)
Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Neuronas/citología , Péptidos Opioides/fisiología , Receptores Opioides/fisiología , Transducción de Señal , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Diferenciación Celular , División Celular , Humanos , Neuronas/fisiologíaRESUMEN
We have investigated the signal transduction pathway of the G-protein mu-opioid receptor upstream of phospholipase D (PLD) and protein kinase C-epsilon (PKC-epsilon) activation in postmitotic E6CH chick embryo cortical neurons. The mu-opioid receptor and PLD-PKC-epsilon functional coupling depends on upstream tyrosine kinase activation. We now report that the mu-opioid agonists specifically stimulated tyrosine phosphorylation and activation of the focal adhesion kinase (FAK) in a time-dependent manner. We also demonstrate that met-enkephalin, a mu-opioid agonist in E6CH cultures, significantly increases tyrosine phosphorylation of another Src kinase substrate, the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin led to drastic changes in subcellular localization, an estimated 2-fold enrichment in the cytosol. Similarly, opioids stimulated a sustained tyrosine phosphorylation of vinculin, a protein enriched in focal adhesion sites. These data provide novel evidence that opioid receptor intracellular signaling engages the specific activation of tyrosine kinase FAK and regulates the neuronal cytoskeleton during central nervous system morphogenesis.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Encefalina Metionina/farmacología , Proteínas de Microfilamentos/metabolismo , Neuronas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores Opioides mu/fisiología , Vinculina/metabolismo , Animales , Moléculas de Adhesión Celular/biosíntesis , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/embriología , Embrión de Pollo , Cortactina , Proteínas del Citoesqueleto/biosíntesis , Activación Enzimática , Proteína-Tirosina Quinasas de Adhesión Focal , Cinética , Proteínas de Microfilamentos/biosíntesis , Neuronas/citología , Neuronas/efectos de los fármacos , Fosforilación , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/biosíntesis , Receptores Opioides mu/efectos de los fármacos , Vinculina/biosíntesisRESUMEN
Chromaffin cells were isolated from bovine adrenal glands and fractionated into two distinct subpopulations by density gradient centrifugation on Percoll. Cells in the more dense fraction stored epinephrine (E) as their predominant catecholamine (81% of total catecholamines), contained high levels of phenylethanolamine N-methyltransferase (PNMT) activity, and exhibited intense PNMT immunoreactivity. This population of chromaffin cells was termed the E-rich cell population. Cells in the less dense fraction, the norepinephrine (NE)-rich cell population, stored predominantly NE (75% of total catecholamines). Although the NE-rich cells had only 3% as much PNMT activity as did the E-rich cells, 20% of the NE-rich cells were PNMT immunoreactive. This suggested that the PNMT-positive cells in the NE-rich cell cultures contained less PNMT per cell than did E-rich cells and may not be typical adrenergic cells. The regulation of PNMT mRNA levels and PNMT activity in primary cultures of E-rich and NE-rich cells was compared. At the time the cells were isolated, PNMT mRNA levels in NE-rich cells were approximately 20% of those in E-rich cells; within 48 h in culture, PNMT mRNA in both populations declined to almost undetectable levels. Treatment with dexamethasone increased PNMT mRNA levels and PNMT activity in both populations. In E-rich cells, dexamethasone restored PNMT mRNA to the level seen in freshly isolated cells and increased PNMT activity twofold. In NE-rich cells, dexamethasone increased PNMT mRNA to levels twice those found in freshly isolated cells and increased PNMT activity sixfold. Cycloheximide blocked the effects of dexamethasone on PNMT mRNA expression in NE-rich cells but had little effect in E-rich cells. Angiotensin II, forskolin, and phorbol 12,13-dibutyrate elicited large increases in PNMT mRNA levels in E-rich cells but had no effect in NE-rich cells. Our data suggest that PNMT expression is regulated differently in the two chromaffin cell subpopulations.
Asunto(s)
Sistema Cromafín/citología , Feniletanolamina N-Metiltransferasa/metabolismo , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/enzimología , Animales , Especificidad de Anticuerpos , Bovinos , Separación Celular , Células Cultivadas/química , Células Cultivadas/citología , Células Cultivadas/enzimología , Centrifugación , Sistema Cromafín/química , Sistema Cromafín/enzimología , Coloides , Cicloheximida/farmacología , Dexametasona/farmacología , Epinefrina/análisis , Epinefrina/metabolismo , Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Norepinefrina/análisis , Norepinefrina/metabolismo , Feniletanolamina N-Metiltransferasa/genética , Feniletanolamina N-Metiltransferasa/inmunología , Povidona , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/análisis , Dióxido de Silicio , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
We have established the presence of at least two large chondroitin sulfate proteoglycans in the developing chick brain, one that reacts exclusively with HNK-1, a carbohydrate epitope found on several neural specific molecules, and one that reacts with S103L, a defined peptide epitope in the CS-2 domain of the cartilage-specific chondroitin sulfate proteoglycan (CSPG), aggrecan. In order to determine the relationships between the two distinct S103L-reactive CSPGs from cartilage (chondrocytes) and brain (neurons), as well as among the three large CSPGs expressed in brain, S103L, HNK-1 and versican, we studied the expression of these multiple proteoglycan species in the brain of nanomelic chicks. We have previously shown that homozygous embryos expressing the nanomelic phenotype exhibit a single point mutation in the aggrecan gene. In the present study, the S103L CSPG is not accumulated or synthesized by embryonic chick CNS tissue or E8CH neuronal cultures derived from nanomelic chick embryo cerebral hemispheres. In contrast, expression of both versican and the HNK-1 CSPG was normal in the mutant embryo CNS. Pulse chase experiments demonstrated the presence of the 380 kDa precursor in normal neurons and the 300 kDa truncated precursor in nanomelic neurons. Northern blot analysis revealed normal-sized mRNA but reduced levels of expression of the S103L CSPG message in nanomelic neurons, while expression of the versican message was comparable in normal and nanomelic neurons. Most conclusively, the point mutation previously identified in nanomelic cartilage mRNA was also identified in nanomelic brain mRNA. Together these results provide evidence that a single aggrecan gene is expressed in both cartilage and CNS tissue leading to the production of identical core proteins which then undergo differential and tissue-specific post-translation processing, resulting in the characteristic tissue-specific proteoglycans. Furthermore, versican and the HNK-1 CSPG, although structurally and chemically similar to the S103L CSPG, are the products of separate genes.
Asunto(s)
Cartílago/metabolismo , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/metabolismo , Mutación/fisiología , Neuronas/metabolismo , Animales , Autorradiografía , Northern Blotting , Western Blotting , Química Encefálica/genética , Química Encefálica/fisiología , Antígenos CD57/biosíntesis , Antígenos CD57/genética , Cartílago/citología , Cartílago/enzimología , Embrión de Pollo , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , ARN Mensajero/biosíntesisRESUMEN
During development, the extracellular matrix (ECM) is a complex dynamic structure whose components and organization help to establish the requisite position and state of differentiation. Until recently, the large chondroitin sulfate proteoglycan, aggrecan, has been localized predominantly to skeletal tissue and considered a hallmark of cartilage differentiation. We have identified the presence of aggrecan in two other highly differentiated systems, brain and notochord, with clearly distinct expression patterns. In chick cartilage, aggrecan starts to be expressed at embryonic day 5 in limb rudiments, continues through the entire period of chondrocyte development, and remains a biochemical marker of the cartilage phenotype thereafter. In brain, aggrecan has a very low level of expression beginning at day 7, increases up to day 13, markedly decreases after day 16, and is not expressed posthatching. This pattern coincides with migration and establishment of neuronal nuclei in the chick telencephalon and has been proposed to be a component of the migration arrest mechanism. In very primitive embryos, aggrecan is detected as early as stage 16 in the notochord, long before chondrogenesis occurs, is then expressed up to day 5 and decreases thereafter. The expression of aggrecan occurs during the time of active neural crest migration and through the onset of sclerotomal differentiation, and correlates with the notochords' ability to inhibit neural crest cell migration. Animal models defective in aggrecan biosynthesis have been invaluable in delineating these functions. In addition we have characterized these proteoglycans by chemical, biosynthetic, and molecular analyses. Although significant post-translation differences distinguish the cell-specific aggrecan species, their core proteins are the products of a single gene. Our findings of the expression of the same gene (aggrecan) in multiple ontogenously unrelated differentiating tissue systems and at different times over the developmental life of an organism provide an elegant model system to study the regulation and interplay in expression of that gene, as well as the effect of alterations in that single gene simultaneously in several developing programs.
Asunto(s)
Encéfalo/metabolismo , Proteínas de la Matriz Extracelular , Proteoglicanos/metabolismo , Agrecanos , Animales , Cartílago/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Humanos , Lectinas Tipo C , Notocorda/metabolismoRESUMEN
Resistance to thyroid hormone (RTH) is a dominantly inherited syndrome characterized by hyposensitivity to thyroid hormone caused by mutations in the thyroid hormone receptor-beta (TR beta) gene. Replication-defective recombinant adenoviruses were constructed that express the human wild-type (WT) TR beta, a human mutant TR beta identified in a family with RTH, and luciferase under the control of thyroid hormone (Luc). The efficient introduction and expression of these recombinant genes into adult mouse liver were confirmed by immunocytochemistry. Hypothyroid mice were infected with Luc alone and in combination with the WT TR beta or mutant TR beta. Half of the mice from each group were then treated with T3. Compared with mice infected with Luc alone, T3 treatment of mutant TR beta infected mice showed no changes in liver luciferase, weight, or 5'-deiodinase and spot 14 messenger RNA, and the decrease in the serum cholesterol concentration was blunted as in patients with RTH. The effects of T3 in mice infected with WT TR beta were comparable to those in mice infected with Luc alone. However, overexpression of the WT TR beta tended to further increase serum cholesterol in the hypothyroid state and decrease it in response to T3, suggesting that the unliganded TR has a constitutive effect in vivo and that higher TR levels can aggravate the manifestations of hypothyroidism and enhance the action of thyroid hormone. Transient somatic transfer of mutant TR genes provides a model for the study of RTH. It allows evaluation of the effect of genetic factors interacting with mutant TRs that modify the phenotype of RTH, without animal back-crossing.
Asunto(s)
Resistencia a Medicamentos/genética , Técnicas de Transferencia de Gen , Modelos Biológicos , Mutación , Receptores de Hormona Tiroidea/genética , Hormonas Tiroideas/farmacología , Animales , Línea Celular , Colesterol/sangre , Humanos , Hipotiroidismo/metabolismo , Hígado/metabolismo , Masculino , Ratones , Proteínas Recombinantes de Fusión , Triyodotironina/farmacologíaRESUMEN
Primary astrocytic cultures derived from day-15 chick embryo (E15) cerebral hemispheres (CH) or cerebellum (CB) express a calcium/phospholipid-dependent isoform as the major protein kinase C (PKC-alpha/beta). PKC was activated (translocation of activity from cytosol to membrane) following stimulation with carbachol, so we tested for activation of phospholipase C (PLC) as the source of diacylglycerol released from polyphosphoinositide (PIP2) hydrolysis. Carbachol activated PLC (inositol phosphate release) 4-fold in a time- and dose-dependent manner in cortical (CH) astrocytes, but there was no activation of PLC in astrocytes from cerebellum (CB). Pirenzepine, but not gallamine, attenuated both carbachol-induced PKC translocation and PIP2 hydrolysis in E15CH astrocytes, arguing for contribution of M1 subtype. The phorbol ester TPA completely inhibited PIP2 hydrolysis, both basal and carbachol-stimulated, and elicited a stronger, but shorter (10 min) activation of PKC than that observed with carbachol. We investigated phospholipase D (PLD) activation as an alternate source of diacylglycerol in astrocytes, since the ratio of PLC to PKC activation by carbachol was lower in astrocytes than observed in neurons. We observed a dramatic (10-fold) time- and dose-dependent activation of PLD by TPA in CH and a 3-fold increase in CB. The duration of TPA-dependent PLD activation correlated well with increased cell proliferation and changes in astrocytic phenotype markers. Carbachol-stimulated PLD activation was observed in CH but not in CB astrocytes, being mostly dependent on the M3 receptor subtype in the former. In contrast, glutamate elicited a greater PLD activation in CB astrocytes, than in CH astrocytes. TPA activation of PLD was totally blocked by staurosporine (PKC inhibitor) and genistein (a tyrosine kinase inhibitor) in cerebellar (CB) astrocytes; however, total inhibition of TPA-dependent PLD activation was only achieved in cortical (CH) astrocytes after addition of EGTA. Thapsigargin activated PLD in both populations, further emphasizing the PLD activation dependency on [Ca2+]i. Taken together with our previous observations that TPA induces proliferation, cytoskeleton changes, and decreases of glutamine synthetase activity, these data suggest that phospholipase D is a differential but important participant in the regulation of the signalling of mitosis and differentiation in astrocytes during their development.
Asunto(s)
Astrocitos/efectos de los fármacos , Carbacol/farmacología , Carcinógenos/farmacología , Ácido Glutámico/farmacología , Agonistas Muscarínicos/farmacología , Ésteres del Forbol/farmacología , Fosfolipasa D/fisiología , Fosfolipasas de Tipo C/fisiología , Animales , Astrocitos/fisiología , Calcio/farmacología , Células Cultivadas/citología , Células Cultivadas/fisiología , Cerebelo/citología , Corteza Cerebral/citología , Embrión de Pollo , Morfogénesis/fisiología , Transducción de Señal/fisiologíaRESUMEN
We have recently shown that expression of specific protein kinase C (PKC) isoforms correlates with cell fate in neural chicken embryo cells. Therefore we investigated the effects of PKC activation by phorbol esters on acquisition of the astrocytic phenotype, using cultured embryonic cortical astrocytes, derived from 15-day-old chick embryos (E15CH), as a model. Short term treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA), which activates PKC-alpha/beta in E15CH, caused association of PKC with the cytoskeleton. In vitro kinase assays of cytoskeleton-associated PKC demonstrated phosphorylation of many cytoskeletal proteins. Phosphorylation was blocked by protein kinase inhibitors (H8), and enhanced by phosphatase inhibitors (calyculin A). Among these PKC substrates, a most prominent 60-kDa protein was identified as vimentin. Assembly of vimentin into the cytoskeleton depends on cell type and state of differentiation. To establish that TPA (PKC) regulates assembly of vimentin into the cytoskeleton of astrocytes, we used pulse-chase (20/5 min) labeling with [35S]methionine, and immunoprecipitations with an anti-vimentin mAb from extractable and cytoskeletal fractions. These studies revealed that 20 min treatment with TPA leads to a 3-fold increase in the rate of newly synthesized full-length vimentin assembly (posttranslational assembly). Furthermore, TPA increased cotranslational assembly of vimentin. The protein kinase A activator forskolin, did not have such effects on vimentin assembly. Long-term TPA treatment, which correlates with a prolonged phospholipase D (PLD) activation, was mitogenic and caused dramatic changes in the morphology of astrocytes. In addition these fibrous, polarized astrocytes had decreased activity of the astrocyte specific enzyme, glutamine synthetase, but had increased abundance of vimentin protein. These studies provide biochemical evidence on acquisition of a different astrocytic phenotype after activation of the PKC/PLD pathway, in the chick embryo. Therefore PKC and PLD activation is pivotal for the acquisition and maintenance of phenotypes in chick embryonic astrocytes.
Asunto(s)
Carcinógenos/farmacología , Citoesqueleto/efectos de los fármacos , Glutamato-Amoníaco Ligasa/efectos de los fármacos , Ésteres del Forbol/farmacología , Proteína Quinasa C/farmacología , Vimentina/efectos de los fármacos , Animales , Astrocitos/citología , Astrocitos/enzimología , Biomarcadores , Calcio/farmacología , División Celular/efectos de los fármacos , Tamaño de la Célula , Células Cultivadas/citología , Células Cultivadas/enzimología , Corteza Cerebral/citología , Embrión de Pollo , Citoesqueleto/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Isoenzimas/fisiología , Fenotipo , Fosforilación , Proteína Quinasa C/fisiología , Proteína Quinasa C beta , Proteína Quinasa C-alfa , Transducción de Señal/fisiología , Especificidad por Sustrato , Factores de TiempoRESUMEN
Protein kinase C (PKC) is expressed as many isoforms and in high quantities in the central nervous system (CNS), which suggests an important role for this enzyme in neuronal development and function. We used specific antibodies to investigate the expression of the known PKC isoforms in extracts from chick major CNS areas during embryogenesis, from day 3 (E3) of incubation to day 1 post-hatching (P1). PKC-epsilon was the predominant isoform and was expressed from E6 onward in all brain regions, except retina (E12 and on). PKC-alpha/beta and -zeta isoforms were expressed at lower levels prior to PKC-epsilon expression and throughout embryogenesis. No other isoforms were detected in neural tissue preparations. We then used neural culture systems derived from the chick CNS to study the expression of PKC isoforms in neuroblasts, cortical neurons, and cortical glial cells. Western blotting and immunostaining of neuroblast-enriched cultures, derived from E3 CNS, showed only the Ca(2+)-dependent PKC-alpha/beta to be present. Studies on neuronal cultures derived from E6 cerebral hemispheres revealed only the Ca(2+)-independent PKC-epsilon to be expressed in neurons, as predicted by the developmental studies on tissue homogenates. PKC-epsilon immunoreactivity was seen intracellularly in differentiating neurons, regardless of their neurotransmitter phenotypes, and it correlated well with the level of neuronal activity. Furthermore, PKC-alpha/beta immunoreactivity was verified on glia cells, as the glial lineage emerges in E15 cortical cultures. These data suggest that PKC-epsilon expression is associated with the final neuroblast division in neurons, and the correlation of PKC isoform expression and neural cell lineage is discussed.
Asunto(s)
Encéfalo/enzimología , Isoenzimas/metabolismo , Neuroglía/enzimología , Neuronas/enzimología , Proteína Quinasa C/metabolismo , Médula Espinal/enzimología , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Encéfalo/embriología , Células Cultivadas , Embrión de Pollo , Desarrollo Embrionario y Fetal , Immunoblotting , Inmunohistoquímica , Isoenzimas/análisis , Microscopía Inmunoelectrónica , Neuritas/enzimología , Neuritas/ultraestructura , Neuroglía/citología , Neuroglía/ultraestructura , Neuronas/citología , Neuronas/ultraestructura , Especificidad de Órganos , Proteína Quinasa C/análisis , Retina/embriología , Retina/enzimología , Médula Espinal/embriologíaRESUMEN
Chondroitin sulfate proteoglycans (CSPG) of the extracellular matrix may play regulatory roles in central nervous system (CNS) development. We have examined the expression of two large CSPGs of the embryonic chick brain, which can be differentiated using the monoclonal antibodies HNK-1 and S103L, in cultures of embryonic day 6 chick cerebral hemisphere neurons. Western blot analysis following immunoprecipitation and endoglycosidase treatment revealed that these cultures produce S103L- and HNK-1-reactive proteoglycans which are biochemically indistinguishable from the CSPGs (previously) identified in homogenized chick embryo brain extracts. The HNK-1-reactive CSPG accumulated in the medium throughout the course of cultures. In contrast, the S103L-reactive CSPG was found in a neuron-associated form during the period of aggregate establishment in culture, as well as in a soluble form secreted into the medium. Immunocytochemical staining of cultures with the S103L antibody localized reactivity to most neurons during the period of aggregate formation, while neuronal processes and the few flat cells present (presumably neuroblasts and early glia) were negative. Cell selection experiments confirmed that neurofilament-positive cells were the source of the S103L-reactive CSPG. The use of differential fixation techniques suggested that the cell-associated S103L reactivity may be intracellular. Because of this pattern of expression and localization, we propose that the developmentally regulated S103L-reactive CSPG may play a role in neuronal migration arrest and organization of neurons into functional aggregates.