RESUMO
Skin-derived precursors (SKPs) have been described as multipotent dermal precursors. Here, we provide a review of the breadth and depth of scientific literature and studies regarding SKPs, accounting for a large number of scientific publications. Interestingly, these progenitors can be isolated from embryonic and adult skin, as well as from a population of dermal cells cultured in vitro in monolayer. Gathering information from different authors, this review explores different aspects of the SKP theme, such as the potential distinct origins of SKPs in rodents and in humans, and also their ability to differentiate in vitro and in vivo into multiple lineages of different progeny. This remarkable capacity makes SKPs an interesting endogenous source of precursors to explore in the framework of experimental and therapeutic applications in different domains. SKPs are not only involved in the skin's dermal maintenance and support as well as wound healing, but also in hair follicle morphogenesis. This review points out the interests of future researches on SKPs for innovative perspectives that may be helpful in many different types of scientific and medical domains.
Les cellules souches précurseurs dérivées de la peau (nommées SKPs) ont été décrites comme des progéniteurs dermiques multipotents. La présente revue permet d'avoir une vue d'ensemble des études et travaux portant sur le thème des SKPs, regroupant un nombre important de publications scientifiques. Il est intéressant de noter que ces cellules progénitrices peuvent être isolées de la peau embryonnaire et adulte, ainsi que d'une population de cellules dermiques cultivées in vitro en monocouche. Réunissant les travaux de plusieurs auteurs, cette revue explore différents thèmes autour des SKPs, tels que leur origine chez les rongeurs et chez l'homme ; mais aussi, leur capacité à se différencier in vitro et in vivo en un grand nombre de lignées cellulaires de descendances différentes. Cette capacité fondamentale des SKPs en fait une source de précurseurs endogènes intéressante à explorer dans le cadre d'applications expérimentales et thérapeutiques dans différents domaines. Les SKPs participent non seulement à l'homéostasie, au maintien du derme, mais aussi à la cicatrisation des plaies et à la morphogenèse du follicule pileux. Cette revue souligne l'intérêt des expérimentations en cours et à venir concernant les SKPs en vue de perspectives innovantes pouvant influencer la recherche dans de nombreux domaines scientifiques et médicaux.
Assuntos
Regeneração , Pele , Animais , Humanos , Roedores , Células-Tronco/citologia , CicatrizaçãoRESUMO
In human skin, melanogenesis is a tightly regulated process. Indeed, several extracellular signals are transduced via dedicated signalling pathways and mostly converge to MITF, a transcription factor integrating upstream signalling and regulating downstream genes involved in the various inherent mechanisms modulating melanogenesis. The synthesis of melanin pigments occurs in melanocytes inside melanosomes where melanogenic enzymes (tyrosinase and related proteins) are addressed with the help of specific protein complexes. The melanosomes loaded with melanin are then transferred to keratinocytes. A more elaborate level of melanogenesis regulation comes into play via the action of non-coding RNAs (microRNAs, lncRNAs). Besides this canonical regulation, melanogenesis can also be modulated by other non-specific intrinsic pathways (hormonal environment, inflammation) and by extrinsic factors (solar irradiation such as ultraviolet irradiation, environmental pollution). We developed a bioinformatic interaction network gathering the multiple aspects of melanogenesis and skin pigmentation as a resource to better understand and study skin pigmentation biology.
Assuntos
Poluentes Ambientais/toxicidade , Melaninas/biossíntese , Pigmentação da Pele , Pele/metabolismo , Luz Solar , Humanos , Melanócitos/metabolismo , Melanossomas/metabolismo , MicroRNAs/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , RNA Longo não Codificante/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Pele/efeitos dos fármacos , Pele/efeitos da radiaçãoRESUMO
OBJECTIVE: Human epidermis provides the body a barrier against environmental assaults. To assume this function, the epidermis needs the renewal of keratinocytes allowed by constant mitosis, which replace the exfoliating corneocytes. Keratinocyte stem cells (KSCs) located in the basal epidermis are mitotically active, self-renewing and govern the epithelial stratification by producing renewed source of keratinocytes. Protein complex such as the chromosomal passenger complex (CPC) allows the correct development of this process. The CPC is composed of four members: INCENP, survivin, borealin and aurora kinase B, and the disruption of the CPC during cell division induces mitotic spindle defects and improper repartition of chromosomes. The aim of our study was to investigate the implication of CRM1 and survivin in the progress of mitosis in skin keratinocytes. METHODS: Cultured human keratinocytes and skin biopsies were used in this study. KSCs-enriched population of keratinocytes was isolated from total keratinocytes by differential attachment to a type IV collagen matrix. Survivin and CRM1 expression levels were assessed by immunofluorescence and immunoblotting. Specific siRNAs for each CPC member and for CRM1 were used to determine the relationship between these proteins. Survivin-specific siRNA was used to induce the apparition of mitotic abnormalities in cultured keratinocytes. RESULTS: We demonstrated the ability of our compound 'IV08.009' to modulate the expression level of survivin and CRM1 in keratinocytes and in skin biopsies. We observed that members of the CPC are interdependent: siRNA-induced inhibition of one component caused a decrease in the expression of all other CPC members. Downregulation of survivin or CRM1 induced mitotic abnormalities in keratinocytes. However, decreased number of mitotic abnormalities was observed in keratinocytes after 'IV08.009' application. CONCLUSION: Basal keratinocytes may divide frequently during skin lifespan, and signs of deterioration could appear such as loss of protein factors required for correct mitosis. Our findings suggest that mitotic abnormalities can be prevented by the modulation of CRM1 and survivin. We demonstrated the ability of compound 'IV08.009' to efficiently protect cultured keratinocytes from mitotic abnormalities.
Assuntos
Proteínas Inibidoras de Apoptose/fisiologia , Carioferinas/fisiologia , Queratinócitos/citologia , Mitose/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Adulto , Células Cultivadas , Feminino , Humanos , Proteínas Inibidoras de Apoptose/genética , Carioferinas/genética , Receptores Citoplasmáticos e Nucleares/genética , Survivina , Proteína Exportina 1RESUMO
OBJECTIVE: Monitoring the chronic and subacute toxicity is essential in the development of new cosmetic ingredients. In response to the present lack of validated alternative methods, we developed an in vitro model for repeated dose cytotoxicity on THP-1 cells. METHODS: Cultured in suspension, cells were treated with chemicals for 14 days with a frequency of three applications per week, and cell viability was determined by MTT assay. We first investigated the long-term effects of chemicals that induce different kinds of cytotoxicity: Paraquat (PQ), 3-Nitropropanoic acid (3-NPA) and sodium dodecyl sulphate (SDS). From acute studies, doses between 1 and 10 µg ml(-1) were chosen to perform our subacute cytotoxicity assay. Comparative genotoxicity evaluations were made with H2 O2 or Paraquat treated TPH-1 cells. Comet assays were performed at 1 h (4°C); after a 24-h recovery period (37°C); and finally, after a long-term period of treatment (14 days, 37°C).Once adapted to plant extracts or highly diluted molecules, some of our cosmetic compounds were tested with this model. RESULTS: As expected, after 14 days of treatment with Paraquat, cell viability rates dramatically decreased for doses as low as 3 µg ml(-1) , whereas 10 µg ml(-1) of 3-NPA and SDS did not induce more than 44% of cell death. Surprisingly, after subacute treatment, comet assay results revealed a dose-dependent increase in tail moments for Paraquat, whereas those of H2 O2 remained low. Moreover, all our compounds tested at 0.5-5 µg ml(-1) were classified as safe, even with a cut-off at 90% of cell viability. CONCLUSION: In conclusion, this assay could be of interest for subacute cytotoxicity and genotoxic assessment of daily and topically applied products and suggests that PQ is a choice worthy positive control.
Assuntos
Testes de Toxicidade/métodos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Humanos , Nitrocompostos/toxicidade , Paraquat/toxicidade , Propionatos/toxicidade , Dodecilsulfato de Sódio/toxicidadeRESUMO
The stem cell factor (SCF) and its protein-tyrosine kinase receptor KIT are together implicated in the regulation of diverse biological processes and particularly in melanogenesis. Indeed, this signalling pathway controls melanoblast migration from the neural crest during embryogenesis and allows the communication between keratinocytes and melanocytes in the adult. In melanocytes, the binding of SCF to its transmembrane receptor leads to the activation of signalling pathways implicating protein kinases which finally control the expression of pigmentation-related genes. We have developed a biological compound called IV09.007, which we previously described as a modulator of the SCF/KIT signalling pathway with a pro-pigmenting effect. In the present work, we have studied the expression and localization of both SCF and KIT mRNAs and proteins in the skin or skin-derived cell lines. Then, we explored with a microarray approach the ability of IV09.007 to modulate the expression of genes in human keratinocytes and melanocytes in culture. Thereby, we observed the regulation of genes implicated in DNA repair, mainly related to base/nucleotides excision pathways. A modulated transcriptional response was also observed for some genes implicated in the response against oxidative stress, in apoptosis inhibition and in lowering inflammatory immune response. These microarray results predicted a conferred protective effect of IV09.007 and we verified this hypothesis by performing comet assays on UVB-irradiated keratinocytes or melanocytes, to demonstrate the efficacy of IV09.007 on preventing DNA damage.
Assuntos
Dano ao DNA , Perfilação da Expressão Gênica , Queratinócitos/efeitos da radiação , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Fator de Células-Tronco/metabolismo , Raios Ultravioleta , Adulto , Linhagem Celular , Feminino , Humanos , Queratinócitos/metabolismo , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Cotton honeydew extract is composed of a unique combination of oligosaccharides, including fructose, glucose, inositol, melezitose, saccharose, trehalose and trehalulose. Studies have shown that these oligosaccharides exhibit a protective effect. Therefore, we were interested in studying the effect of these oligosaccharides on normal and damaged human hair. Both clinical and scanning electron microscopy (SEM) studies were performed. Standardized human hair samples were used to determine the effect of a rinse-off mask with 1% cotton honeydew extract on the ultrastructure of hair. In addition, hair samples were submitted to different aggressions, following various experimental protocols. SEM showed that, without extra aggression, the cuticle scales appeared to lie more smoothly in the hair in cotton honeydew extract-treated samples than in untreated samples. The extract-treated hair samples were also less prone to chipping. In contrast, the control, untreated hair samples retained a dry and damaged appearance and were prone to chipping and progressive splitting. In a clinical study, 15 volunteers had half of their hair treated with a formula with 1% honeydew extract and the other half was left untreated as a control. Pictures and visual evaluation of the hair showed that the honeydew extract formula left the hair with a smoothness that was far superior to the control side and this result was confirmed by SEM. In addition, mRNA studies on epidermal cells were performed and confirmed the stimulating effect of honeydew extract on keratin synthesis. These results demonstrate that cotton honeydew extract can be of great use in hair care products and cosmetics.
Assuntos
Cosméticos , Gossypium/química , Preparações para Cabelo , Extratos Vegetais/farmacologia , Adulto , Linhagem Celular , Feminino , Cabelo/ultraestrutura , Humanos , Queratinas/biossíntese , Queratinas/genética , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
This work describes the molecular cloning of a variant isoform of the low-affinity levocabastine-sensitive neurotensin receptor isolated from mouse brain. Although the corresponding mRNA encodes for a 282 amino acid protein unable to bind neurotensin after transient transfection in COS-7 cells, this non-functional neurotensin receptor is expressed in cerebral neocortex, cerebellum, olfactory bulb, striatum and hypothalamus with a level similar to that of the full-length low-affinity neurotensin receptor. By contrast, this receptor form is very weakly expressed in mesencephalon and absent in the pituitary, but is the major product in the spinal cord.
Assuntos
Encéfalo/metabolismo , Expressão Gênica , Piperidinas/farmacologia , Receptores de Neurotensina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Clonagem Molecular , DNA Complementar , Variação Genética , Camundongos , Dados de Sequência Molecular , Neurotensina/metabolismo , Receptores de Neurotensina/metabolismoRESUMO
In order to identify charged amino-acid residues of the cloned rat brain neurotensin (NT) receptor (NTR) that are critical for NT binding, we performed site-directed mutagenesis on the cDNA encoding this protein, followed by transient expression into mammalian COS-7 cells and in Xenopus laevis oocytes. Point substitutions of charged residues in the N-terminal part and in the 2nd and 3rd extracellular loop of the receptor either did not affect (125)I-Tyr3-NT binding or resulted in a decrease in binding affinity by a factor of 2-3. Mutations of amino acids Asp113 in the second transmembrane domain (TM) and of Arg149 or Asp150 in TM III yielded receptors that bound NT as efficiently as the native receptor. By contrast, replacement of the Asp139 residue in the 1st extracellular loop, or of Arg143 or Arg327-Arg328 residues at the top of TM III and in TM VI, respectively, completely abolished ligand binding. Confocal and EM immunocytochemical studies of the expression of these affected receptors, tagged with the C-terminal sequence of the vesicular stomatitis virus glycoprotein (VSV-G), indicated that this loss of binding was not due to altered receptor expression or to their improper insertion into the plasma membrane. When these mutated forms of neurotensin receptor were expressed into Xenopus oocytes, Asp139-Gly- and Arg143-Gly-modified receptors remained functional in spite of a lowered response to NT whereas the Arg327-Arg328 mutant form was totally insensitive to NT at concentrations up to 10 microM. In the case of the Arg327-Arg328 mutation, the observed insensibility to NT could be the result of a drastic conformational alteration of this mutant protein. By contrast, it would appear that Asp139 and Arg143 residues located in the first extracellular loop of the receptor may be directly involved in the interaction of the receptor with neurotensin.
Assuntos
Aminoácidos/análise , Encéfalo/metabolismo , Neurotensina/metabolismo , Receptores de Neurotensina/química , Animais , Encéfalo/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica , Mutagênese Sítio-Dirigida , Ensaio Radioligante , Ratos , Receptores de Neurotensina/metabolismo , Receptores de Neurotensina/ultraestrutura , Relação Estrutura-AtividadeRESUMO
The effect of the drug SR 48692 on the Ca(2+)-activated Cl- current induced by neurotensin on Xenopus oocytes injected with cRNAs encoding rodent high and low affinity neurotensin receptors, was examined. In this receptor expression system, SR 48692 failed to antagonize electrophysiological measurement of neurotensin-evoked current via the rat high affinity neurotensin receptor, whereas its application onto oocytes expressing the mouse low affinity neurotensin receptor triggered an inward current, as well as neurotensin itself. However, no current activation was observed after application of the drug on oocytes expressing the rat high affinity neurotensin receptor. These observations in the oocyte expression system did not reflect typical antagonist properties of SR 48692 drug.
Assuntos
Cálcio/fisiologia , Cloretos/fisiologia , Neurotensina/farmacologia , Oócitos/fisiologia , Pirazóis/farmacologia , Quinolinas/farmacologia , Animais , Condutividade Elétrica , Feminino , Camundongos , Neurotensina/antagonistas & inibidores , Oócitos/efeitos dos fármacos , Piperidinas/farmacologia , Ratos , Receptores de Neurotensina/agonistas , Receptores de Neurotensina/antagonistas & inibidores , Receptores de Neurotensina/efeitos dos fármacosRESUMO
This work demonstrates the expression of two different proteins (47 and 44 kDa) from the rat high-affinity neurotensin receptor cDNA, observed after both translation in vitro and transient transfection into eukaryotic COS-7 cells. These two proteins are the consequence of two initiation sites of translation present in the corresponding mRNA. Site-directed mutagenesis indicated that the 47 kDa protein was generated from the first AUG codon (Met1). In contrast, suppression of the second AUG codon found in the sequence (Met27) did not modify the expression of the two proteins previously observed with the wild-type neurotensin receptor. Therefore this second translation site could correspond to a non-AUG codon. Moreover, when the 5' end untranslated region of the neurotensin receptor mRNA is deleted, the expression of the higher-molecular-mass protein is enhanced, indicating that this region could be involved in the regulation of expression of these two proteins.
Assuntos
DNA Complementar/genética , Regulação da Expressão Gênica , Biossíntese de Proteínas , Receptores de Neurotensina/genética , Sequências Reguladoras de Ácido Nucleico , Animais , Células COS , Peso Molecular , Mutagênese Sítio-Dirigida , RNA Mensageiro/genética , Ratos , Receptores de Neurotensina/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
This work describes the cloning and expression of the levocabastine-sensitive neurotensin (NT) receptor from mouse brain. The receptor protein comprises 417 amino acids and bears the characteristics of G-protein-coupled receptors. This new NT receptor (NTR) type is 39% homologous to, but pharmacologically distinct from, the only other NTR cloned to date from the rat brain and the human HT29 cell line. When the receptor is expressed in Xenopus laevis oocytes, the H1 antihistaminic drug levocabastine, like NT and neuromedin N, triggers an inward current. The pharmacological properties of this receptor correspond to those of the low-affinity, levocabastine-sensitive NT binding site described initially in membranes prepared from rat and mouse brain. It is expressed maximally in the cerebellum, hippocampus, piriform cortex, and neocortex of adult mouse brain.
Assuntos
Encéfalo/metabolismo , Neurotensina/metabolismo , Fragmentos de Peptídeos/metabolismo , Piperidinas/farmacologia , Receptores de Neurotensina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Antagonistas dos Receptores Histamínicos H1/farmacologia , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , Receptores de Neurotensina/genética , Relação Estrutura-Atividade , Distribuição Tecidual , Xenopus laevisRESUMO
In order to identify the amino acid sequences responsible for the internalization of the cloned rat brain neurotensin receptor, we carried out site-directed mutagenesis of the cDNA encoding the receptor followed by expression of the receptor into mammalian COS 7 cells. In cells transfected with the full-length neurotensin receptor, 56% of iodinated neurotensin specifically bound to the cells after 60 min of incubation at 37 degrees C was internalized. Deletions made in the third intracellular loop did not affect receptor internalization. By contrast, internalization was reduced to 5% of total in cells in which almost all the carboxyl-terminal tail of the receptor had been deleted (R392stop). In order to determine which part of the tail was responsible for this effect, several Ser and Thr residues were deleted in the carboxyl cytoplasmic sequence of the receptor. Almost all of these receptors were internalized as efficiently as the wild type. Only the form of the neurotensin receptor truncated at Glu-421 (deletion of the last three residues, TLY) produced a significant decrease in the amount of ligand internalized. Finally, point mutations of Thr-422 and Tyr-424 residues to Gly led to an almost complete loss of ligand internalization demonstrating the involvement of these 2 residues in the internalization process. Replacement of the last three amino acids by the cytoplasmic endocytosis signal of the vesicular stomatitis virus did not restore the efficiency of neurotensin receptor internalization. These biochemical results were confirmed by confocal microscopic analysis. Cell transfected with the wild type receptor showed a temperature-dependent intracellular accumulation of a fluorescent analog of neurotensin, whereas cells transfected with a receptor truncated at the carboxyl terminus showed a clustering of the fluorescent peptide at the cell surface.
Assuntos
Receptores de Neurotensina/metabolismo , Treonina/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Linhagem Celular , Cisteína/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ratos , Receptores de Neurotensina/genéticaRESUMO
The expression of sst2A and sst2B somatostatin (SRIH) receptor splice variants was examined by polymerase chain reaction (PCR) in rat brain and pituitary. Two sets of primers chosen to recognize either both sst2A and sst2B or only the sst2B transcript yielded bands of the size predicted from the cloned sst2A and sst2B sequences in both mouse and rat tissue extracts. Accordingly, experiments carried out on mouse brain extracts and on AtT-20 pituitary cells yielded sst2A/sst2B expression ratios comparable to those previously published. As in the mouse, rat pituitary extracts contained both sst2A and sst2B transcripts. By contrast, various cerebral regions in which both sst2A and sst2B forms were detected in the mouse contained only sst2A form in rat brain, with the exception of the cerebral cortex which also showed weak sst2B expression. No sst2B mRNA was detected in the arcuate nucleus/median eminence complex, indicating that the sst2A form is the one that is associated with growth-hormone-releasing-hormone-containing neurons documented to express sst2 receptors in this region. Finally, only sst2A transcripts were detected in rat astrocytes in culture, suggesting that this form of the receptor is responsible for sst2-mediated effects of SRIH on these cells.
Assuntos
Encéfalo/metabolismo , Expressão Gênica , Variação Genética , Hipófise/metabolismo , Splicing de RNA , Receptores de Somatostatina/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
The highly conserved aspartate residue in the second transmembrane domain of G protein-coupled receptors is present in position 113 in the type 1 neurotensin receptor (NTR1) but is replaced by an Ala residue in position 79 in the type 2 neurotensin receptor (NTR2). NTR1 couples to Galphaq to stimulate phospholipase C and its binding affinity for neurotensin is decreased by sodium ions and GTP analogs. By contrast, NTR2 does not seem to couple to any G protein in eukaryotic cells, and its binding of neurotensin is insensitive to sodium and GTP analogs. By using site-directed mutagenesis, we substituted Asp113 of the NTR1 by alanine and the homologous residue Ala79 of NTR2 by aspartate. Both mutant receptors display similar affinity for neurotensin as compared with their respective wild type. We demonstrate that the presence of the Asp residue determines by itself the occurrence of the sodium effect on neurotensin affinity for both wild-type and mutated NTR1 and -2. The introduction of an Asp in the second transmembrane domain of NTR2 is not enough to restore a functional coupling to G proteins. In contrast, replacement of Asp113 by Ala residue in NTR1 strongly decreases its ability to activate inositol turnover, indicating that the functionally active conformation of NTR1 is maintained by interaction of sodium ions with aspartate 113.
Assuntos
Ácido Aspártico/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Receptores de Neurotensina/metabolismo , Sódio/farmacologia , Sequência de Aminoácidos , Animais , Ácido Aspártico/genética , Ligação Competitiva/efeitos dos fármacos , Células COS , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Dados de Sequência Molecular , Mutação , Neurotensina/metabolismo , Fosfatidilinositóis/metabolismo , Receptores de Neurotensina/agonistas , Receptores de Neurotensina/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
The recently cloned new subtype of G protein-coupled neurotensin receptor (NTRL) was stably expressed in the HEK 293 cell line in order to investigate its binding and internalization properties. The expressed receptor exhibited the typical binding characteristics of the low affinity, levocabastine-sensitive binding site previously described in rat and mouse brain and was detected as a protein with an apparent MW of 45 kDa by photoaffinity labeling. Although intracellular modulation of adenylate cyclase, guanylate cyclase and phospholipase C was not detected after application of neurotensin or levocabastine on NTRL-transfected cells, this receptor was able to internalize iodinated neurotensin. The internalization process was followed by recycling of receptors to the cell membrane. By contrast, no recycling was observed with the high affinity neurotensin receptor (NTRH). The differential intracellular routing of NTRH and NTRL after internalization is most probably the consequence of their divergent carboxy-terminal sequences.
Assuntos
Regulação da Expressão Gênica/genética , Piperidinas/farmacologia , Receptores de Neurotensina/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Linhagem Celular , Endocitose/fisiologia , Humanos , Radioisótopos do Iodo/metabolismo , Cinética , Camundongos , Dados de Sequência Molecular , Neurotensina/metabolismo , Peptídeos/metabolismo , Marcadores de Fotoafinidade/metabolismo , Ligação Proteica , Receptores de Neurotensina/química , Transdução de Sinais/fisiologia , Transfecção/genéticaRESUMO
The expression of the 3 currently known neurotensin receptors was studied in human cancer cells of prostatic, colonic or pancreatic origin by means of RT-PCR analysis and binding experiments. All the cells selected for this work have been shown to exhibit a growth response to neurotensin. We found that the 7 transmembrane domain, levocabastine insensitive receptor (NTR1) is expressed in most but not all of the cells studied whereas the 7 transmembrane domain, levocabastine sensitive receptor (NTR2) is present in none of these cells. The 100 kDa-type I neurotensin receptor (NTR3) is expressed in all the cells assayed. Moreover, we demonstrated that neurotensin can stimulate the growth of CHO cells stably transfected with the NTR3. Taken together, our results strongly suggest that the NTR3 subtype could be involved in the growth response of human cancer cells to neurotensin.
Assuntos
Neurotensina/metabolismo , Neurotensina/farmacologia , Receptores de Neurotensina/biossíntese , Animais , Células CHO , Membrana Celular/metabolismo , Ácidos Cólicos/farmacologia , Neoplasias do Colo/metabolismo , Cricetinae , Resistencia a Medicamentos Antineoplásicos , Eletroforese em Gel de Poliacrilamida , Humanos , Cinética , Masculino , Neoplasias Pancreáticas/metabolismo , Piperidinas/farmacologia , Neoplasias da Próstata/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Receptores de Neurotensina/química , Receptores de Neurotensina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
Alpine Marmots (Marmota marmota) are a good model to study intraspecific chemical communication among mammals. This species has been subjected to several behavioural and biochemical studies regarding both their scent-marking behaviour by cheek-rubbing, and the chemical composition of their glandular secretions. However, no molecular study has been undertaken until today on proteins from the olfactory epithelium possibly implicated in chemical perception. In this study, we identified, to our knowledge for the first time, some olfatory receptors from this wild rodent. Starting with olfactory epithelium of an Alpine Marmot, and by mean of reverse transcriptase polymerase chain reaction technique (RT-PCR), we isolated fourteen partial sequences that exhibited a high degree of homology (45-92%) with olfactory receptors from other vertebrates. Conserved identities and structural features clearly defined these Alpine Marmot sequences as members of the seven transmembrane domain olfactory receptors. All sequences were observed as belonging to known olfactory receptor families and were classified into ten subfamilies of the tetrapods OR class. Finally, Northern blot analysis revealed specific expression of these sequences in the Alpine Marmot olfactory epithelium tissue.
Assuntos
Marmota/genética , Receptores Odorantes/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Dados de Sequência Molecular , Mucosa Olfatória/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Odorantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
The binding of [3H]SR 48692, a new potent and specific nonpeptide neurotensin (NT) receptor antagonist, was characterized in membranes from mouse fibroblast LTK- cells stably transfected with the G protein-coupled rat NT receptor. The binding of [3H]SR 48692 was specific, time dependent, reversible, and saturable. Scatchard analysis of saturation experiments indicated that [3H]SR 48692 bound to a single population of sites, with a Kd of 3.4 nM and a Bmax value that was 30-40% greater than that observed in saturation experiments with [125I]NT. Two SR 48692-related enantiomers, SR 48527 and SR 49711, were 10 and 1000 times less potent, respectively, than unlabeled SR 48692 in inhibiting [3H]SR 48692. Unlabeled NT inhibited [3H]SR 48692 binding in a complex manner that was best analyzed with a three-site model, with high (Ki = 0.22 nM) and low (Ki = 57 nM) affinity NT binding sites and a site insensitive to unlabeled NT (up to 10 microM), which represented 60, 20, and 20%, respectively, of the total number of [3h]SR 48692 binding sites. Digitonin (10 micrograms/ml) markedly reduced the proportion of NT-insensitive sites without affecting [3H]SR 48692 binding. Na+ and guanosine-5'-(gamma-thio)triphosphate differentially modulated [3H]SR 48692 and [125I]NT binding and inverted the proportions of the high and low affinity NT binding sites. A mutant rat NT receptor that contained a deletion in a region (amino acids 45-60) of the amino-terminal extracellular domain near the first transmembrane helix and was expressed in COS M6 cells retained the same affinity for [3H]SR 48692 and the same stereoselectivity for SR 48527 and SR 49711 as the wild-type receptor. In contrast, it bound NT with 3000-fold lower potency. In conclusion, the data indicate that [3H]SR 48692 represents a new, potent, nonpeptide antagonist radioligand of the NT receptor that differentiates between agonist- and antagonist-receptor interactions. Furthermore, the data demonstrate that the peptide agonist and the nonpeptide antagonist bind to distinct regions of the NT receptor.
Assuntos
Pirazóis/metabolismo , Pirazóis/farmacologia , Quinolinas/metabolismo , Quinolinas/farmacologia , Receptores de Neurotensina/antagonistas & inibidores , Receptores de Neurotensina/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Digitonina/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Cinética , Camundongos , Mutação , Ensaio Radioligante , Ratos , Receptores de Neurotensina/agonistas , Sódio/farmacologia , TransfecçãoRESUMO
The two neurotensin receptor subtypes known to date, NTR1 and NTR2, belong to the family of G-protein-coupled receptors with seven putative transmembrane domains (TM). SR 48692, a nonpeptide neurotensin antagonist, is selective for the NTR1. In the present study we attempted, through mutagenesis and computer-assisted modeling, to identify residues in the rat NTR1 that are involved in antagonist binding and to provide a tentative molecular model of the SR 48692 binding site. The seven putative TMs of the NTR1 were defined by sequence comparison and alignment of bovine rhodopsin and G-protein-coupled receptors. Thirty-five amino acid residues within or flanking the TMs were mutated to alanine. Additional mutations were performed for basic residues. The wild type and mutant receptors were expressed in COS M6 cells and tested for their ability to bind 125I-NT and [3H]SR 48692. A tridimensional model of the SR 48692 binding site was constructed using frog rhodopsin as a template. SR 48692 was docked into the receptor, taking into account the mutagenesis data for orienting the antagonist. The model shows that the antagonist binding pocket lies near the extracellular side of the transmembrane helices within the first two helical turns. The data identify one residue in TM 4, three in TM 6, and four in TM 7 that are involved in SR 48692 binding. Two of these residues, Arg327 in TM 6 and Tyr351 in TM 7, play a key role in antagonist/receptor interactions. The former appears to form an ionic link with the carboxylic group of SR 48692, as further supported by structure-activity studies using SR 48692 analogs. The data also show that the agonist and antagonist binding sites in the rNTR1 are different and help formulate hypotheses as to the structural basis for the selectivity of SR 48692 toward the NTR1 and NTR2.
Assuntos
Pirazóis/metabolismo , Quinolinas/metabolismo , Receptores de Neurotensina/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Bovinos , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ratos , Receptores de Neurotensina/antagonistas & inibidores , Receptores de Neurotensina/química , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
The neuropeptide neurotensin (NT) elicits hypothermic and naloxone-insensitive analgesic responses after brain injection. Recent pharmacological evidence obtained with NT agonists and antagonists suggests that these effects are mediated by a receptor distinct from the initially cloned high-affinity NT receptor (NTR1). The recent cloning of a second NT receptor (NTR2) prompted us to evaluate its role in NT-induced analgesia. Intracerebroventricular injections in mice of two different antisense oligodeoxynucleotides from the NTR2 markedly decreased NTR2 mRNA and protein and reduced NT-induced analgesia. This effect was specific, because NTR1 levels were unaffected, and sense or scramble oligodeoxynucleotides had no effect. Structure-activity studies revealed a close correlation between the analgesic potency of NT analogs and their affinity for the NTR2 and disclosed potent and selective agonists of this receptor. These data confirm that NTR1 is involved in the NT-elicited turning behavior and demonstrate that the NTR2 mediates NT-induced analgesia.