RESUMO
In the search for a metallic material showing moderate and uniform degradation for application as degradable cardiovascular stents, electroformed iron (E-Fe) was evaluated by in vitro degradation and cell viability tests. Static immersion and dynamic degradation were used to evaluate degradation rate and mechanism, while cell viability assay was used to assess cytotoxicity. The results were compared with those of iron fabricated by casting and thermomechanical treatment previously investigated as a stent material. Electroformed iron showed faster degradation than iron fabricated by casting (0.25 vs. 0.14 mm year(-1)), with a uniform degradation mechanism. Cell viability results showed that E-Fe did not result in a decrease in metabolic activity when exposed to primary rat smooth muscle cells. However, it caused a decrease in cell proliferation activity which could be beneficial for the inhibition of in-stent restenosis.
Assuntos
Implantes Absorvíveis , Materiais Biocompatíveis/farmacologia , Eletroquímica/métodos , Ferro/farmacologia , Teste de Materiais/métodos , Stents , Animais , Sobrevivência Celular/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos , Aço Inoxidável/farmacologia , Difração de Raios XRESUMO
Originally described in the 1950s caveolae are morphologically identifiable as small omega-shaped plasma membrane invaginations present in most cell types. Caveolae are particularly abundant in adipocytes, fibroblasts, type 1 pneumocytes, endothelial and epithelial cells as well as in smooth and striated muscle cells. The first proposed function for caveolae was that of mediating the internalisation and transendothelial trafficking of solutes. Caveolae have been the object of intense research since the discovery of a biochemical marker protein, caveolin, in the early 1990s. Three genes encoding for caveolins have been characterised in mammals. Caveolins (18-24 kDa) are integral membrane proteins that constitute the major protein component of caveolar membrane in vivo. In addition to a structural role of caveolins in the formation of caveolae, caveolin protein interacts directly, and in a regulated manner, with a number of signalling molecules. We present here a general overview of the current knowledge on the structural role of caveolin in caveolae formation, and implication of caveolin in the control of cell signalling.
Assuntos
Cavéolas/fisiologia , Caveolinas/fisiologia , Animais , Membrana Celular/fisiologia , Humanos , Transdução de Sinais/fisiologiaRESUMO
Caveolae are plasma membrane microdomains that have been implicated in organizing and concentrating certain signaling molecules. Caveolins, constitute the main structural proteins of caveolae. Caveolae are abundant in terminally differentiated cell types. However, caveolin-1 is down-regulated in transformed cells and may have a potential tumor suppressor activity. In the lung, caveolae are present in the endothelium, smooth muscle cells, fibroblasts as well as in type I pneumocytes. The presence of caveolae and caveolin expression in the bronchial epithelium, although probable, has not been investigated in human. We were interested to see if the bronchial epithelia express caveolins and if this expression was modified in cancer cells. We thus tested for caveolin-1 and -2 expression several bronchial epithelial primary cell lines as well as eight lung cancer cell lines and one larynx tumor cell line. Both caveolin-1 and -2 are expressed in all normal bronchial cell lines. With the exception of Calu-1 cell line, all cancer cell lines showed very low or no expression of caveolin-1 while caveolin-2 expression was similar to the one observed in normal bronchial epithelial cells.
Assuntos
Caveolinas , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Caveolina 1 , Caveolina 2 , Regulação para Baixo/genética , Humanos , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Células Tumorais CultivadasRESUMO
Caveolae and their principal component caveolin have been implicated in playing a major role in G protein-mediated transmembrane signaling. We examined whether caveolin interacts with adenylyl cyclase, an effector of G protein signaling, using a 20-mer peptide derived from the N-terminus scaffolding domain of caveolin-1. When tissue adenylyl cyclases were examined, cardiac adenylyl cyclase was inhibited more potently than other tissue adenylyl cyclases. The caveolin-1 peptide inhibited type V, as well as type III adenylyl cyclase, overexpressed in insect cells, whereas the same peptide had no effect on type II. The caveolin-3 scaffolding domain peptide similarly inhibited type V adenylyl cyclase. In contrast, peptides derived from the caveolin-2 scaffolding domain and a caveolin-1 nonscaffolding domain had no effect. Kinetic studies showed that the caveolin-1 peptide decreased the maximal rate (Vmax) value of type V without changing the Michaelis constant (Km) value for the substrate ATP. Studies with various truncations and point mutations of this peptide revealed that a minimum of 16 amino acid residues and intact aromatic residues are important for the inhibitory effect. The potency of inhibition was greater when adenylyl cyclase was in stimulated condition vs. basal condition. Thus, caveolin may be another cellular component that regulates adenylyl cyclase catalytic activity. Our results also suggest that the caveolin peptide may be used as an isoform-selective inhibitor of adenylyl cyclase.
Assuntos
Inibidores de Adenilil Ciclases , Caveolinas , Inibidores Enzimáticos/farmacologia , Proteínas de Membrana/farmacologia , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Caveolina 1 , Colforsina/farmacologia , Deleção de Genes , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Isoenzimas/antagonistas & inibidores , Cinética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Mutagênese , Mutação Puntual , Relação Estrutura-AtividadeRESUMO
Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We and others have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting signaling molecules within caveolae membranes. In this regard, it has been shown that a 20-amino acid membrane-proximal region of the cytosolic NH2-terminal domain of caveolin is sufficient to mediate the interaction of caveolin with signaling proteins, namely G-proteins, Src-like kinases, eNOS, and H-Ras. This caveolin-derived protein domain has been termed the caveolin-scaffolding domain. Binding of the caveolin-scaffolding domain functionally suppresses the activity of G-protein alpha subunits, eNOS, and Src-like kinases, suggesting that caveolin binding may also play a negative regulatory role in signal transduction. Here, we report the direct interaction of caveolin with a growth factor receptor, EGF-R, a known caveolae-associated receptor tyrosine kinase. Two consensus caveolin binding motifs have been previously defined using phage display technology. One of these motifs is present within the conserved kinase domains of most known receptor tyrosine kinases (termed region IX). We now show that this caveolin binding motif within the kinase domain of the EGF-R can mediate the interaction of the EGF-R with the scaffolding domains of caveolins 1 and 3 but not with caveolin 2. In addition, the scaffolding domains of caveolins 1 and 3 both functionally inhibit the autophosphorylation of the EGF-R kinase in vitro. Importantly, this caveolin-mediated inhibition of the EGF-R kinase could be prevented by the addition of an EGF-R-derived peptide that (i) contains a well conserved caveolin binding motif and (ii) is located within the kinase domain of the EGF-R and most known receptor tyrosine kinases. Similar results were obtained with protein kinase C, a serine/threonine kinase, suggesting that caveolin may function as a general kinase inhibitor. The implications of our results are discussed within the context of caveolae-mediated signal transduction. In this regard, caveolae-coupled signaling might explain how linear signaling pathways can branch and interconnect extensively, forming a signaling module or network.
Assuntos
Caveolinas , Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Alanina , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Células CHO , Caveolina 1 , Linhagem Celular , Membrana Celular/metabolismo , Cricetinae , Humanos , Dados de Sequência Molecular , Receptor ErbB-2/metabolismo , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. Recent evidence has shown that eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo. Incubation of endothelial cell lysates or purified eNOS with glutathione S-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Utilizing a series of GST-caveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61-101) and to a lesser extent the C-terminal tail (amino acids 135-178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells. Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.
Assuntos
Caveolinas , Proteínas de Membrana/metabolismo , Óxido Nítrico Sintase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Caveolina 1 , Células Cultivadas , Endotélio Vascular/enzimologia , Escherichia coli , Microcirculação , Dados de Sequência Molecular , Ligação Proteica , Circulação Pulmonar , Proteínas Recombinantes/metabolismoRESUMO
Caveolae are microdomains of the plasma membrane that have been implicated in organizing and compartmentalizing signal transducing molecules. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membrane in vivo. Recently, we and other laboratories have identified a family of caveolin-related proteins; caveolin has been re-termed caveolin-1. Here, we examine the cell-type and tissue-specific expression of caveolin-2. For this purpose, we generated a novel mono-specific monoclonal antibody probe that recognizes only caveolin-2, but not caveolins-1 and -3. A survey of cell and tissue types demonstrates that the caveolin-2 protein is most abundantly expressed in endothelial cells, smooth muscle cells, skeletal myoblasts (L6, BC3H1, C2C12), fibroblasts, and 3T3-L1 cells differentiated to adipocytes. This pattern of caveolin-2 protein expression most closely resembles the cellular distribution of caveolin-1. In line with these observations, co-immunoprecipitation experiments with mono-specific antibodies directed against either caveolin-1 or caveolin-2 directly show that these molecules form a stable hetero-oligomeric complex. The in vivo relevance of this complex was further revealed by dual-labeling studies employing confocal laser scanning fluorescence microscopy. Our results indicate that caveolins 1 and 2 are strictly co-localized within the plasma membrane and other internal cellular membranes. Ultrastructurally, this pattern of caveolin-2 localization corresponds to caveolae membranes as seen by immunoelectron microscopy. Despite this strict co-localization, it appears that regulation of caveolin-2 expression occurs independently of the expression of either caveolin-1 or caveolin-3 as observed using two different model cell systems. Although caveolin-1 expression is down-regulated in response to oncogenic transformation of NIH 3T3 cells, caveolin-2 protein levels remain unchanged. Also, caveolin-2 protein levels remain unchanged during the differentiation of C2C12 cells from myoblasts to myotubes, while caveolin-3 levels are dramatically induced by this process. These results suggest that expression levels of caveolins 1, 2, and 3 can be independently up-regulated or down-regulated in response to a variety of distinct cellular cues.
Assuntos
Caveolinas , Proteínas de Membrana/metabolismo , Células 3T3 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Biopolímeros , Caveolina 1 , Caveolina 2 , Linhagem Celular Transformada , Membrana Celular/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Proteínas de Membrana/biossíntese , Camundongos , Microscopia Imunoeletrônica , Dados de Sequência MolecularRESUMO
Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting proteins within caveolae membranes. In this regard, caveolin co-purifies with a variety of lipid-modified signaling molecules, including G-proteins, Src-like kinases, Ha-Ras, and eNOS. Using several independent approaches, it has been shown that a 20-amino acid membrane proximal region of the cytosolic amino-terminal domain of caveolin is sufficient to mediate these interactions. For example, this domain interacts with G-protein alpha subunits and Src-like kinases and can functionally suppress their activity. This caveolinderived protein domain has been termed the caveolin-scaffolding domain. However, it remains unknown how the caveolin-scaffolding domain recognizes these molecules. Here, we have used the caveolin-scaffolding domain as a receptor to select random peptide ligands from phage display libraries. These caveolin-selected peptide ligands are rich in aromatic amino acids and have a characteristic spacing in many cases. A known caveolin-interacting protein, Gi2alpha, was used as a ligand to further investigate the nature of this interaction. Gi2alpha and other G-protein alpha subunits contain a single region that generally resembles the sequences derived from phage display. We show that this short peptide sequence derived from Gi2alpha interacts directly with the caveolin-scaffolding domain and competitively inhibits the interaction of the caveolin-scaffolding domain with the appropriate region of Gi2alpha. This interaction is strictly dependent on the presence of aromatic residues within the peptide ligand, as replacement of these residues with alanine or glycine prevents their interaction with the caveolin-scaffolding domain. In addition, we have used this interaction to define which residues within the caveolin-scaffolding domain are critical for recognizing these peptide and protein ligands. Also, we find that the scaffolding domains of caveolins 1 and 3 both recognize the same peptide ligands, whereas the corresponding domain within caveolin-2 fails to recognize these ligands under the same conditions. These results serve to further demonstrate the specificity of this interaction. The implications of our current findings are discussed regarding other caveolin- and caveolae-associated proteins.
Assuntos
Caveolinas , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Caveolina 1 , Sistema Livre de Células , Citoplasma/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Ligantes , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Transdução de SinaisRESUMO
The thyrotropin (TSH) receptor in human-thyroid glands is cleaved into an extracellular alpha subunit and a transmembrane beta subunit held together by disulfide bridges. An excess of the latter component relative to the former suggests shedding of the ectodomain. Indeed we observed such shedding in cultures of human thyrocytes and permanently transfected L or Chinese hamster ovary cells. Shedding was increased by inhibitors of endocytosis, recycling and lysosomal degradation suggesting that it was dependent on receptor residency at the cell surface. It was slightly increased by TSH and phorbol esters whereas forskolin and 8 bromo cAMP were without effect. The complete inhibition of soluble TSH receptor shedding by the specific inhibitor BB-2116 indicated that the cleavage reaction is catalyzed probably at the cell surface by a matrix metalloprotease-like enzyme. Shedding of the TSH receptor alpha domain is the consequence of two events: cleavage of the proreceptor into alpha and beta subunits and reduction of the disulfide bridge(s). The use of different specific inhibitors including monoclonal antibodies allowed us to implicate the enzyme protein disulfide isomerase in the reduction of TSHR disulfide bounds. The shed alpha subunit probably results in circulating TSH receptor ectodomain detected in human blood. This shedding mechanism might be implicated in the development of autoimmune diseases.
Assuntos
Metaloendopeptidases/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Receptores da Tireotropina/metabolismo , Glândula Tireoide/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Membrana Celular/metabolismo , Cricetinae , Ácido Ditionitrobenzoico/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Células L , Metaloendopeptidases/antagonistas & inibidores , Camundongos , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Receptores da Tireotropina/química , Receptores da Tireotropina/efeitos dos fármacos , Reagentes de Sulfidrila/farmacologia , TransfecçãoRESUMO
Caveolae are 50-100 nm invaginations that represent an appendage or subcompartment of the plasma membrane. They are found in most cell types but are abundant in fibroblasts, adipocytes, endothelial cells, type I pneumocytes, epithelial cells, and smooth and striated muscle cells. Functionally, caveolae have been implicated in three major processes: endothelial transcytosis, potocytosis, and signal transduction. Caveolin, a 21-24 kD integral membrane protein, is a principal component of the caveolar membrane in vivo. Within caveolar microdomains, caveolin functions as a multivalent docking site for recruiting and sequestering signaling molecules. More specifically, caveolin interacts directly in a regulated manner with multiple lipid-modified signaling molecules (such as Src-tyrosine kinases, Gα subunits, and H-Ras), preferring the inactive conformation of these molecules. Here, we present a general overview of our current knowledge of caveolae and caveolin functioning and possible implications for treatment of human disease. (Trends Cardiovasc Med 1997;7:103-110). © 1997, Elsevier Science Inc.
RESUMO
Caveolar localization of protein kinase C and the regulation of caveolar function by protein kinase C are well known. This study was undertaken to examine whether caveolin subtypes interact with various protein kinase C isoenzymes using the caveolin scaffolding domain peptide. When protein kinase C-alpha, -epsilon, and -zeta were overexpressed in COS cells followed by subcellular fractionation using the sucrose gradient method, all the isoenzymes (alpha, epsilon, and zeta) were detected in the same fraction as caveolin. The scaffolding domain peptide of caveolin-1 and -3, but not -2, inhibited the kinase activity and autophosphorylation of protein kinase C-alpha and -zeta, but not of protein kinase C-epsilon, overexpressed in insect cells. Truncation mutation studies of the caveolin-1 and -3 peptides demonstrated that a minimum of 16 or 14 amino acid residues of the peptide were required for the inhibition or direct binding of protein kinase C. Thus, the caveolin peptide physically interacted with protein kinase C and regulated its function. Further, this regulation occurred in a protein kinase C isoenzyme-dependent manner. Our results may provide a new mechanism regarding the regulation of protein kinase C isoenzyme activity and the molecular interaction of protein kinase C with its putative binding proteins.
Assuntos
Caveolinas , Proteínas de Membrana/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Caveolina 1 , Isoenzimas/metabolismo , Dados de Sequência Molecular , Dibutirato de 12,13-Forbol/metabolismo , Fosforilação , Proteínas Recombinantes/metabolismo , Frações Subcelulares/enzimologiaRESUMO
Caveolae are plasma membrane specializations present in most cell types. Caveolin, a 22-kDa integral membrane protein, is a principal structural and regulatory component of caveolae membranes. Previous studies have demonstrated that caveolin co-purifies with lipid modified signaling molecules, including Galpha subunits, H-Ras, c-Src, and other related Src family tyrosine kinases. In addition, it has been shown that caveolin interacts directly with Galpha subunits and H-Ras, preferentially recognizing the inactive conformation of these molecules. However, it is not known whether caveolin interacts directly or indirectly with Src family tyrosine kinases. Here, we examine the structural and functional interaction of caveolin with Src family tyrosine kinases. Caveolin was recombinantly expressed as a glutathione S-transferase fusion. Using an established in vitro binding assay, we find that caveolin interacts with wild-type Src (c-Src) but does not form a stable complex with mutationally activated Src (v-Src). Thus, it appears that caveolin prefers the inactive conformation of Src. Deletion mutagenesis indicates that the Src-interacting domain of caveolin is located within residues 82-101, a cytosolic membrane-proximal region of caveolin. A caveolin peptide derived from this region (residues 82-101) functionally suppressed the auto-activation of purified recombinant c-Src tyrosine kinase and Fyn, a related Src family tyrosine kinase. We further analyzed the effect of caveolin on c-Src activity in vivo by transiently co-expressing full-length caveolin and c-Src tyrosine kinase in 293T cells. Co-expression with caveolin dramatically suppressed the tyrosine kinase activity of c-Src as measured via an immune complex kinase assay. Thus, it appears that caveolin structurally and functionally interacts with wild-type c-Src via caveolin residues 82-101. Besides interacting with Src family kinases, this cytosolic caveolin domain (residues 82-101) has the following unique features. First, it is required to form multivalent homo-oligomers of caveolin. Second, it interacts with G-protein alpha-subunits and down-regulates their GTPase activity. Third, it binds to wild-type H-Ras. Fourth, it is membrane-proximal, suggesting that it may be involved in other potential protein-protein interactions. Thus, we have termed this 20-amino acid stretch of caveolin residues the caveolin scaffolding domain.
Assuntos
Proteínas de Transporte , Caveolinas , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Proteínas ras/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Caveolina 1 , Linhagem Celular , Citosol/metabolismo , Cães , Ativação Enzimática , Proteínas de Membrana/química , Dados de Sequência Molecular , Ligação Proteica , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
In human thyroid glands the TSH receptor undergoes a cleavage reaction which yields to an extracellular alpha subunit and a membrane spanning beta subunit linked together by disulfide bridges. A similar reaction is observed in transfected L cells although some uncleaved monomers persist in these cells. We have recently shown that the alpha subunit of the TSH receptor undergoes partial shedding in human thyroid cells and heterologous cells permanently transfected with an expression vector encoding the receptor. This shedding is a two-step process. The first step consists in the cleavage of the proreceptor at the cell surface probably by a matrix metalloprotease and the second step in the reduction of the disulfide bridge(s) (Couet, J., Sar, S., Jolivet, A., Vu Hai, M. T., Milgrom, E., & Misrahi, M. 1996, J. Biol. Chem. 271, 4545-4552). We have used the transfected L cells to study the second step involved in sTSHR shedding. The membrane impermeant sulfhydryl reagent DTNB (5,5'-dithiobis(2-nitrobenzoic acid) allowed us to confirm that the reduction of the TSH receptor disulfide bonds occurred at the cell surface. The antibiotic bacitracin even at low concentrations also elicited a marked inhibition of TSH receptor shedding. This led us to implicate the enzyme protein disulfide isomerase (PDI, EC 5.3.4.1) in this process. We thus tested the inhibitory activity of specific monoclonal antibodies raised against PDI. All antibodies elicited a marked inhibition of sTSHR shedding. This confirmed that cell surface PDI is involved in the shedding of the TSH receptor ectodomain. The shed alpha subunit may be at the origin of circulating TSH receptor ectodomain detected in human blood.
Assuntos
Isomerases/metabolismo , Receptores da Tireotropina/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Bacitracina/farmacologia , Membrana Celular/metabolismo , Células Cultivadas , Ácido Ditionitrobenzoico/farmacologia , Humanos , Isomerases/imunologia , Células L , Camundongos , Isomerases de Dissulfetos de Proteínas , Receptores da Tireotropina/efeitos dos fármacos , Receptores da Tireotropina/genética , Reagentes de Sulfidrila/farmacologia , Timo/citologia , TransfecçãoAssuntos
3-Hidroxiesteroide Desidrogenases/genética , Esteroide Isomerases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Isoenzimas , Macaca , Camundongos , Dados de Sequência Molecular , Ratos , Alinhamento de Sequência , Esteroide Isomerases/metabolismo , Relação Estrutura-Atividade , TrutaRESUMO
Until recently, neonatal hyperthyroidism has been considered to be related to the transplacental passage of thyroid-stimulating Ig present in the serum of the mother. We report here the case of a newborn who presented with severe hyperthyroidism, diffuse goiter, and important ocular signs (eyelid retraction and possibly proptosis). However, the absence of thyroid pathology in the parents and the lack of antithyroid antibodies in the mother and in the patient led us to suspect a nonimmune aetiology. Direct genomic sequencing of the last exon of the TSH receptor in the patient revealed a T-->C transversion yielding to a Met453-->Thr heterozygous substitution in the second transmembrane domain of the receptor. The mutation was absent in both parents. Eukaryotic expression analysis in COS-7 cells yielded a mutated receptor that produced constitutive activation of adenylate cyclase without enhancement of phospholipase C activity.
Assuntos
Hipertireoidismo/genética , Mutação , Tireotropina/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular/metabolismo , AMP Cíclico/metabolismo , Humanos , Hipertireoidismo/diagnóstico por imagem , Hipertireoidismo/fisiopatologia , Recém-Nascido , Masculino , Sondas Moleculares/genética , Dados de Sequência Molecular , Receptores da Tireotropina/metabolismo , Tireotropina/farmacologia , Tomografia Computadorizada por Raios X , TransfecçãoRESUMO
The thyrotropin (TSH) receptor in human thyroid glands has been shown to be cleaved into an extracellular alpha subunit and a transmembrane beta subunit held together by disulfide bridges. An excess of the latter component relative to the former suggested the shedding of the ectodomain. Indeed we observed such a shedding in cultures of human thyrocytes and permanently transfected L or Chinese hamster ovary cells. The shedding was increased by inhibitors of endocytosis, recycling, and lysosomal degradation, suggesting that it was dependent on receptor residency at the cell surface. It was slightly increased by TSH and phorbol esters, whereas forskolin and 8-bromo-cyclic AMP were without effect. Decreasing the serum concentration in cell culture medium enhanced the shedding by an unknown mechanism. The shedding of the TSH receptor alpha domain is the consequence of two events: cleavage of the receptor into alpha and beta subunits and reduction of the disulfide bridge(s). The complete inhibition of soluble TSH receptor shedding by the specific inhibitor BB-2116 indicated that the cleavage reaction is catalyzed probably at the cell surface by a matrix metalloprotease. This shedding mechanism may be responsible for the presence of soluble TSH receptor alpha subunit in human circulation.
Assuntos
Matriz Extracelular/enzimologia , Metaloendopeptidases/metabolismo , Receptores da Tireotropina/metabolismo , Glândula Tireoide/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Células CHO , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colforsina/farmacologia , Cricetinae , Endocitose , Humanos , Ácidos Hidroxâmicos/farmacologia , Cinética , Células L , Lisossomos/metabolismo , Substâncias Macromoleculares , Camundongos , Inibidores de Proteases/farmacologia , Receptores da Tireotropina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
We have investigated 17 alpha-hydroxylase and C17,20-lyase activities and the presence of cytochrome P450c17 mRNA in the esophagus, stomach, duodenum, and colon of adult rats of both sexes. All tissues converted [4-14C]pregnenolone mainly to dehydroepiandrosterone (DHEA) through the 5-ene-3 beta-hydroxysteroid route as opposed to the 4-ene-3-ketosteroid pathway in a control testicular incubate. Synthesis of dehydroepiandrosterone was particularly high in the duodenum and was found to be lower in the stomach, colon and esophagus, in decreasing order. 20 alpha-Hydroxypregnenolone and progesterone were also formed primarily by the esophagus and colon, respectively. P450c17 mRNA was demonstrated by ribonuclease protection assay in the stomach and duodenum, but not in esophagus and colon. However, a 335 bp-long cDNA fragment, whose sequence corresponded to that of rat P450c17 cDNA, was amplified by reverse transcription (RT) and polymerase chain reaction (PCR) from the poly(A)+ RNAs of all four tissues. This result was further confirmed by Southern blotting using a 794-bp testicular probe. The complete sequence of P450c17 cDNA in the stomach and duodenum was identical to that reported for rat testis P450c17 cDNA. No amplification and no positive signal in Southern blotting were observed with the total RNAs from adult male adrenal and spleen, which were taken as negative controls since they had been previously found unable to form androgens from pregnenolone. Although the levels of transcription in gonads, duodenum and stomach were found to be equivalent, as indicated by the RNase protection assay and semiquantitative RT-PCR assay, P450c17 enzyme activity was much higher in the testis, pointing at a possible dissimilarity in the respective rates of mRNA translation. Thus, P450c17 is differentially expressed in the rat gastrointestinal tract, where it leads to the synthesis of the sex steroid precursor DHEA, especially in the duodenum and stomach.
Assuntos
Desidroepiandrosterona/biossíntese , Sistema Digestório/enzimologia , RNA Mensageiro/metabolismo , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Aldeído Liases/metabolismo , Animais , Sequência de Bases , Colo/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/química , Duodeno/enzimologia , Esôfago/enzimologia , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Pregnenolona/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Estômago/enzimologiaRESUMO
In rat skin, type IV is the major 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) isoenzyme expressed. Although types I and II 3 beta-HSD mRNAs are also present in the skin, their level of expression is about two orders of magnitude lower than that of type IV. In this study, we have investigated the control of type IV 3 beta-HSD mRNA levels as well as 3 beta-HSD enzymatic activity in hypophysectomized adult rats of both sexes. Skin 3 beta-HSD activity was measured by the conversion of [14C]-dehydroepiandrosterone into [14C]-androstenedione, whereas ribonuclease protection assay using a specific type IV cRNA probe was used to assess mRNA levels. Intact male and female rats show a similar level of skin 3 beta-HSD activity, although hypophysectomy caused opposite effects, a decrease being observed in males while an increase was observed in hypophysectomized female animals. We next studied the effects of hyperprolactinemia, corticosterone and 1-thyroxine in hypophysectomized animals. L-thyroxine was found to stimulate 3 beta-HSD expression and activity in male rats whereas no significant effect was observed on the already elevated levels in hypophysectomized female rats. Corticosterone caused an inhibition of type IV 3 beta-HSD mRNA levels and activity in both male and female animals. Hyperprolactinemia achieved by pituitary implants inserted under the kidney capsule stimulated the expression of type IV mRNA as well as 3 beta-HSD enzymatic activity in hypophysectomized male and female animals. The present data demonstrate the multihormonal regulation of 3 beta-HSD/isomerase expression and activity in the rat skin.
Assuntos
Corticosterona/farmacologia , Isomerases/fisiologia , Complexos Multienzimáticos/fisiologia , Progesterona Redutase/fisiologia , Prolactina/farmacologia , Pele/enzimologia , Esteroide Isomerases/fisiologia , Animais , Feminino , Regulação Enzimológica da Expressão Gênica , Hiperprolactinemia/sangue , Isomerases/análise , Isomerases/genética , Masculino , Complexos Multienzimáticos/análise , Complexos Multienzimáticos/genética , Progesterona Redutase/análise , Progesterona Redutase/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Pele/química , Esteroide Isomerases/análise , Esteroide Isomerases/genética , Tiroxina/farmacologiaRESUMO
Hydroxysteroid sulfotransferases (DHEA ST) represent a family of enzymes that catalyze the conversion of dehydroepiandrosterone and other 3 beta-hydroxysteroids into more hydrophilic water-soluble sulfate conjugates. The present study was designed to investigate the regulation of hepatic DHEA ST expression by sex steroids and pituitary hormones, namely GH and PRL, in both male and female rats. DHEA ST mRNA levels were measured by dot blot hybridization using a 332-basepair fragment of rat DHEA ST (ST-20) cDNA as a probe. Hepatic DHEA ST mRNA levels were 2.8-fold higher in females than in males. A 15-day gonadectomy did not affect DHEA ST mRNA levels in the male liver; in females, a 35% decrease in DHEA ST mRNA levels compared with those in intact controls was observed. Administration of 17 beta-estradiol (E2; 1 microgram/kg, twice daily) alone had no effect on the accumulation of DHEA ST mRNA, but the same treatment completely reversed the marked inhibitory effect of dihydrotestosterone (DHT; 400 micrograms/kg, twice daily) on this parameter in both gonadectomized males and females. In female rats, 24-day hypophysectomy (HYPOX) decreased DHEA ST mRNA levels by 62%, whereas no change was detected in males. In HYPOX animals, treatment with E2 or DHT for 9 days starting 15 days after surgery had no effect on hepatic DHEA ST mRNA levels in rats of both sexes. In intact males, the presence of pituitary implants under the kidney capsule increased DHEA ST mRNA levels by 190% (above the control), which reached levels similar to those in intact females, whereas pituitary implants exerted no effect on this parameter in intact females. However, treatment of HYPOX rats with ovine PRL (oPRL; 4 mg/kg, twice daily) had no significant effect on the accumulation of DHEA ST mRNA in male and female animals. In HYPOX females, administration of rat GH (80 micrograms/kg, twice daily) had no effect on DHEA ST mRNA levels, whereas continuous infusion of rat GH (3.6 micrograms/h), using osmotic minipumps to mimic the female GH secretory pattern, increased this parameter by 290% (above the control value), thus completely reversing the effect of hypophysectomy. In HYPOX males, both modes of GH administration increased DHEA ST mRNA levels by 70-90% (above the control). The present study demonstrates that in both gonadectomized male and female rats, administration of E2 completely blocks the marked inhibitory effect of DHT on hepatic DHEA ST mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Hormônios/fisiologia , Fígado/metabolismo , RNA Mensageiro/metabolismo , Sulfotransferases/genética , Animais , Sequência de Bases , Castração , Di-Hidrotestosterona/farmacologia , Estradiol/farmacologia , Feminino , Hipofisectomia , Masculino , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , RNA Mensageiro/antagonistas & inibidores , Ratos , Ratos Sprague-DawleyRESUMO
In order to better understand the role of prolactin (PRL) and luteinizing hormone (LH) on progesterone biosynthesis in the ovary, we have investigated the time course (1-9 days) of the effect of PRL and human chorionic gonadotropin (hCG) on ovarian 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) expression in the hypophysectomized rat. As evaluated by quantitative in situ hybridization using a 35S labelled type I 3 beta-HSD cDNA probe, the administration of hCG for 2, 3 and 9 days induced increases of 63%, 145% and 146% above control, respectively, in 3 beta-HSD mRNA levels in ovarian interstitial cells. The absence of apparent effect of the gonadotropin in other ovarian cell types could explain the small modulation of ovarian 3 beta-HSD protein content and enzymatic activity observed in total ovarian tissue. On the other hand, treatment with PRL caused a rapid decrease in 3 beta-HSD mRNA levels in corpus luteum by 23%, 63%, 76% and 78% (P < 0.01) following 1, 2, 5 and 9 days of treatment, respectively. The short-term inhibitory effect of PRL was also observed on ovarian immunoreactive 3 beta-HSD protein, as measured by Western blot analysis, and on 3 beta-HSD activity measured by the conversion of [14C]dehydroepiandrosterone into [14C]androstenedione.(ABSTRACT TRUNCATED AT 250 WORDS)