RESUMO
In this study, we address the hypothesis that it is possible to exploit genetic/functional variation in parental Chinese hamster ovary (CHO) cell populations to isolate clonal derivatives that exhibit superior, heritable attributes for biomanufacturing--new parental cell lines which are inherently more "fit for purpose." One-hundred and ninety-nine CHOK1SV clones were isolated from a donor CHOK1SV parental population by limiting dilution cloning and microplate image analysis, followed by primary analysis of variation in cell-specific proliferation rate during extended deep-well microplate suspension culture of individual clones to accelerate genetic drift in isolated cultures. A subset of 100 clones were comparatively evaluated for transient production of a recombinant monoclonal antibody (Mab) and green fluorescent protein following transfection of a plasmid vector encoding both genes. The heritability of both cell-specific proliferation rate and Mab production was further assessed using a subset of 23 clones varying in functional capability that were subjected to cell culture regimes involving both cryopreservation and extended sub-culture. These data showed that whilst differences in transient Mab production capability were not heritable per se, clones exhibiting heritable variation in specific proliferation rate, endocytotic transfectability and N-glycan processing were identified. Finally, for clonal populations most "evolved" by extended sub-culture in vitro we investigated the relationship between cellular protein biomass content, specific proliferation rate and cell surface N-glycosylation. Rapid-specific proliferation rate was inversely correlated to CHO cell size and protein content, and positively correlated to cell surface glycan content, although substantial clone-specific variation in ability to accumulate cell biomass was evident. Taken together, our data reveal the dynamic nature of the CHO cell functional genome and the potential to evolve and isolate CHO cell variants with improved functional properties in vitro.
Assuntos
Biotecnologia/métodos , Células CHO/fisiologia , Heterogeneidade Genética , Proteínas Recombinantes/biossíntese , Animais , Anticorpos Monoclonais/biossíntese , Processos de Crescimento Celular/fisiologia , Tamanho Celular , Cricetinae , Cricetulus , Evolução Molecular Direcionada , Proteínas de Fluorescência Verde , Proteínas Recombinantes/análiseRESUMO
The HIF (hypoxia-inducible factor) hydroxylases [PHDs or EGLNs (prolyl hydroxylases), which in humans are PHD isoforms 1-3, and FIH (factor inhibiting HIF)] regulate HIF levels and activity. These enzymes are Fe(II)/2-oxoglutarate-dependent oxygenases, many of which are stimulated by ascorbate. We have investigated the ascorbate dependence of PHD2-catalysed hydroxylation of two prolyl hydroxylation sites in human HIF-1alpha, and of FIH-catalysed hydroxylation of asparaginyl hydroxylation sites in HIF-1alpha and in a consensus ankyrin repeat domain peptide. The initial rate and extent of hydroxylation was increased in the presence of ascorbate for each of these reactions. When ascorbate was replaced with structural analogues, the results revealed that the ascorbate side chain was not important in its contribution to HIF hydroxylase catalysis, whereas modifications to the ene-diol portion of the molecule negated the ability to promote hydroxylation. We investigated whether alternative reducing agents (glutathione and dithiothreitol) could be used to promote HIF hydroxylase activity, and found partial stimulation of hydroxylation in an apparently enzyme- and substrate-specific manner. The results raise the possibility of developing reducing agents targeted to specific HIF hydroxylase-catalysed reactions.
Assuntos
Repetição de Anquirina , Ácido Ascórbico/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Substâncias Redutoras/farmacologia , Asparagina/química , Asparagina/metabolismo , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia , Ácidos Cetoglutáricos/farmacologia , Fragmentos de Peptídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The calcium-sensing receptor (CaR) is expressed on intestinal epithelial serosal membrane and in Caco-2 cells. In renal epithelium, CaR expressed on the basolateral membrane acts to limit excess tubular Ca2+ reabsorption. Therefore, here we investigated whether extracellular calcium (Ca(o)2+) can regulate active or passive 45Ca2+ transport across differentiated Caco-2 monolayers via CaR-dependent or CaR-independent mechanisms. Raising the Ca(o)2+ concentration from 0.8 to 1.6 mM increased transepithelial electrical resistance (TER) and decreased passive Ca2+ permeability but failed to alter active Ca2+ transport. The Ca(o)2+ effect on TER was rapid, sustained and concentration-dependent. Increasing basolateral Mg2+ concentration increased TER and inhibited both passive and active Ca2+ transport, whereas spermine and the CaR-selective calcimimetic NPS R-467 were without effect. We conclude that small increases in divalent cation concentration elicit CaR-independent increases in TER and inhibit passive Ca2+ transport across Caco-2 monolayers, most probably through a direct effect on tight junction permeability. Whilst it is known that the complete removal of Ca(o)2+ lowers TER, here we show that Ca(o)2+ addition actually increases TER in a concentration-dependent manner. Therefore, such Ca(o)2+-sensitivity could modulate intestinal solute transport including the limiting of excess Ca2+ absorption.
Assuntos
Células CACO-2/metabolismo , Cálcio/metabolismo , Magnésio/metabolismo , Regulação Alostérica , Transporte Biológico/fisiologia , Células CACO-2/citologia , Colecalciferol/metabolismo , Humanos , Receptores de Detecção de Cálcio/metabolismoRESUMO
Ion transport activity in pancreatic alpha-cells was assessed by studying cell volume regulation in response to anisotonic solutions. Cell volume was measured by a video imaging method, and cells were superfused with either 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid-buffered or HCO(3)(-) -buffered solutions. alpha-Cells did not exhibit a regulatory volume increase (RVI) in response to cell shrinkage caused by hypertonic solutions. A RVI was observed, however, in cells that had first undergone a regulatory volume decrease (RVD), but only in HCO(3)(-)-buffered solutions. RVI was also observed in response to a HCO(3)(-) -buffered hypertonic solution in which the glucose concentration was increased from 4 to 20 mM. The post-RVD RVI and the glucose-induced RVI were both inhibited by 10 microM 5-(N-methyl-N-isobutyl) amiloride or 100 microM 2,2'-(1,2-ethenediyl) bis (5-isothio-cyanatobenzenesulfonic acid), but not by 10 microM benzamil nor 10 microM bumetanide. These data suggest that Na(+)-H(+) exchangers and Cl(-)-HCO(3)(-) exchangers contribute to volume regulation in alpha-cells.
Assuntos
Bicarbonatos/farmacologia , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/fisiologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Equilíbrio Hidroeletrolítico/efeitos dos fármacosRESUMO
The activation-induced deaminase/apolipoprotein B-editing catalytic subunit 1 (AID/APOBEC) family comprises four groups of proteins. Both AID, a lymphoid-specific DNA deaminase that triggers antibody diversification, and APOBEC2 (function unknown) are found in all vertebrates examined. In contrast, APOBEC1, an RNA-editing enzyme in gastrointestinal cells, and APOBEC3 are restricted to mammals. The function of most APOBEC3s, of which there are seven in human but one in mouse, is unknown, although several human APOBEC3s act as host restriction factors that deaminate human immunodeficiency virus type 1 replication intermediates. A more primitive function of APOBEC3s in protecting against the transposition of endogenous retroelements has, however, been proposed. Here, we focus on mouse APOBEC2 (a muscle-specific protein for which we find no evidence of a deaminating activity on cytidine whether as a free nucleotide or in DNA) and mouse APOBEC3 (a DNA deaminase which we find widely expressed but most abundant in lymphoid tissue). Gene-targeting experiments reveal that both APOBEC2 (despite being an ancestral member of the family with no obvious redundancy in muscle) and APOBEC3 (despite its proposed role in restricting endogenous retrotransposition) are inessential for mouse development, survival, or fertility.
Assuntos
Citidina Desaminase/genética , Citidina Desaminase/fisiologia , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Desaminases APOBEC , Desaminase APOBEC-3G , Animais , Southern Blotting , Sobrevivência Celular , Citidina/metabolismo , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Fertilidade , Citometria de Fluxo , Marcadores Genéticos , Técnicas Genéticas , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Modelos Genéticos , Músculos/metabolismo , Miocárdio/metabolismo , RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Recombinação Genética , Retroelementos/genética , Distribuição TecidualRESUMO
Pancreatic beta-cells increase in volume when exposed to elevated concentrations of extracellular glucose. This study has examined the effects of glucose on the volumes of pancreatic alpha-cells, which like beta-cells are regulated by glucose, and intestinal epithelial Caco-2 cells which are unresponsive to glucose. Cell volume changes were monitored by a video-imaging method. Increasing the extracellular glucose concentration caused a concentration-dependent increase in alpha-cell volume over the range 1-20mM. Glucose-induced swelling was not, however, observed in Caco-2 cells. The glucose-induced swelling in both alpha- and beta-cells was abolished by 0.5mM phloretin, an inhibitor of the GLUT proteins, indicating that GLUT mediated glucose transport is a pre-requisite for swelling. Glucose metabolism also appears to be essential, as islet cell swelling was not observed with 16 mM 3-O-methyl glucose. These data suggest that glucose-induced swelling may be a property exclusive to glucose-regulated cells.
Assuntos
Tamanho Celular/efeitos dos fármacos , Células Secretoras de Glucagon/citologia , Glucose/farmacologia , Edulcorantes/farmacologia , Animais , Células CACO-2 , Relação Dose-Resposta a Droga , Células Secretoras de Glucagon/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Floretina/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
We show that iterative antigen-mediated selection of B-cell lines that constitutively hypermutate their immunoglobulin V genes during culture can be exploited to generate antibodies in vitro. From Ramos, a hypermutating human B-cell line expressing IgM of unknown specificity, we derived descendants that exhibit stepwise improved binding to streptavidin. Binding is initially conferred by mutations in complementarity-determining regions (CDRs), but maturation is due to strategic framework mutations. A more powerful system is provided by a hypermutating chicken B-lymphoma line, owing to its rapid proliferation, high rate of mutation accumulation, and genetic tractability. Starting from a single cell, we selected parallel lineages of derivatives, making mutated antibodies of increasing affinity to independent test antigens. Selection is initiated at an exceedingly low affinity threshold, but antibodies can be delivered with nanomolar affinities. The strategy could prove useful for in vitro generation of antigen-specific monoclonal antibodies and may be extendable to the maturation of other protein-ligand interactions.
Assuntos
Anticorpos Monoclonais/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/imunologia , Evolução Molecular Direcionada/métodos , Imunoglobulinas Intravenosas/genética , Imunoglobulinas Intravenosas/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/genética , Afinidade de Anticorpos/imunologia , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/efeitos dos fármacos , Sítios de Ligação de Anticorpos/genética , Linfoma de Burkitt/metabolismo , Galinhas , Clonagem Molecular , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoglobulinas Intravenosas/biossíntese , Mutagênese , Ratos , Valores de Referência , Seleção Genética , Sensibilidade e Especificidade , Especificidade da Espécie , Proteína Estafilocócica A/imunologia , Proteína Estafilocócica A/metabolismo , Estreptavidina/administração & dosagem , Estreptavidina/imunologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Deriving and validating local adjusted calcium equations is important for ensuring appropriate calcium status classification. We investigated the impact on our local adjusted calcium equation of a change in calcium method by the manufacturer from cresolphthalein complexone to NM-BAPTA. METHODS: Calcium and albumin results from general practice requests were extracted from the Laboratory Information Management system for a three-month period. Results for which there was evidence of disturbance in calcium homeostasis were excluded leaving 13,482 sets of results for analysis. The adjusted calcium equation was derived following least squares regression analysis of total calcium on albumin and normalized to the mean calcium concentration of the data-set. The revised equation (NM-BAPTA calcium method) was compared with the previous equation (cresolphthalein complexone calcium method). RESULTS: The switch in calcium assay resulted in a small change in the adjusted calcium equation but was not considered to be clinically significant. The calcium reference interval differed from that proposed by Pathology Harmony in the UK. CONCLUSIONS: Local adjusted calcium equations should be re-assessed following changes in the calcium method. A locally derived reference interval may differ from the consensus harmonized reference interval.
Assuntos
Cálcio/análise , Homeostase , HumanosRESUMO
The expression of K+-Cl- cotransporters (KCC) was examined in pancreatic islet cells. mRNA for KCC1, KCC3a, KCC3b and KCC4 were identified by RT-PCR in islets isolated from rat pancreas. In immunocytochemical studies, an antibody specific for KCC1 and KCC4 revealed the expression of KCC protein in alpha-cells, but not pancreatic beta-cells nor delta-cells. A second antibody which does not discriminate among KCC isoforms identified KCC expression in both alpha-cell and beta-cells. Exposure of isolated alpha-cells to hypotonic solutions caused cell swelling was followed by a regulatory volume decrease (RVD). The RVD was blocked by 10 microM [dihydroindenyl-oxy] alkanoic acid (DIOA; a KCC inhibitor). DIOA was without effect on the RVD in beta-cells. NEM (0.2 mM), a KCC activator, caused a significant decrease of alpha-cell volume, which was completely inhibited by DIOA. By contrast, NEM had no effects on beta-cell volume. In conclusion, KCCs are expressed in pancreatic alpha-cells and beta-cells. However, they make a significant contribution to volume homeostasis only in alpha-cells.
Assuntos
Ilhotas Pancreáticas/química , Simportadores/genética , Animais , Tamanho Celular , Soluções Hipertônicas/farmacologia , Soluções Hipotônicas/farmacologia , Ilhotas Pancreáticas/citologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Simportadores/metabolismo , Simportadores/fisiologia , Distribuição Tecidual , Cotransportadores de K e Cl-RESUMO
Full-length Aß1-42 and Aß1-40, N-truncated pyroglutamate Aß3-42 and Aß4-42 are major variants in the Alzheimer brain. Aß4-42 has not been considered as a therapeutic target yet. We demonstrate that the antibody NT4X and its Fab fragment reacting with both the free N-terminus of Aß4-x and pyroglutamate Aß3-X mitigated neuron loss in Tg4-42 mice expressing Aß4-42 and completely rescued spatial reference memory deficits after passive immunization. NT4X and its Fab fragment also rescued working memory deficits in wild type mice induced by intraventricular injection of Aß4-42. NT4X reduced pyroglutamate Aß3-x, Aßx-40 and Thioflavin-S positive plaque load after passive immunization of 5XFAD mice. Aß1-x and Aßx-42 plaque deposits were unchanged. Importantly, for the first time, we demonstrate that passive immunization using the antibody NT4X is therapeutically beneficial in Alzheimer mouse models showing that N-truncated Aß starting with position four in addition to pyroglutamate Aß3-x is a relevant target to fight Alzheimer's disease.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Imunização Passiva/métodos , Fragmentos de Peptídeos/imunologia , Doença de Alzheimer/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , RatosRESUMO
Mice carrying human immunoglobulin transloci were immunised with HIV-1 gp140 antigen to gain insight into the range and nature of human monoclonal antibodies (mAbs) that can be elicited from such humanised mice. Using five-feature mice that harbour YAC-based germline-configuration human IgM, Igκ and Igλ transloci in a mouse background disrupted for endogenous mouse IgH and Igκ expression, gp140-specific human IgM mAbs were readily elicited following serial immunisation. These mAbs were converted to human IgG1 format and were found to bind diverse epitopes within gp140, exhibiting high functional affinity for the antigen-typically in the nanomolar or sub-nanomolar range. The number of specific, stable hybridomas per mouse was, however, low (typically around five) with the hybridomas within individual mice often being clonally related. Nevertheless, different mice used B cell clones expressing varied V(D)J combinations, with affinity maturation through somatic hypermutation making a critical contribution. Thus, a wide range of distinct high-affinity mAbs can be obtained by immunising multiple animals. The results confirm the utility of the translocus-mouse approach and give insight into strategies for possible future improvement.
Assuntos
Anticorpos Monoclonais/genética , Genes de Imunoglobulinas , Imunoglobulina M/genética , Translocação Genética/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/metabolismo , Síndrome da Imunodeficiência Adquirida/patologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Cromossomos Artificiais de Levedura/genética , Humanos , Hibridomas/citologia , Hibridomas/imunologia , Hibridomas/metabolismo , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Transgênicos , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
In this study, we systematically compare two vector design strategies for recombinant monoclonal antibody (Mab) synthesis by Chinese hamster ovary (CHO) cells; a dual open reading frame (ORF) expression vector utilizing separate cytomegalovirus (CMV) promoters to drive heavy chain (HC) and light chain (LC) expression independently, and a single ORF vector design employing a single CMV promoter to drive HC and LC polypeptide expression joined by a foot and mouth disease virus F2A polypeptide self-cleaving linker sequence. Initial analysis of stable transfectants showed that transfectants utilizing the single ORF vector designs exhibited significantly reduced Mab production. We employed an empirical modeling strategy to quantitatively describe the cellular constraints on recombinant Mab synthesis in all stable transfectants. In all transfectants, an intracellular molar excess of LC polypeptide over HC polypeptide was observed. For CHO cells transfected with the single ORF vectors, model-predicted, and empirical intracellular intermediate levels could only be reconciled by inclusion of nascent HC polypeptide degradation. Whilst a local sensitivity analysis showed that qMab of all transfectants was primarily constrained by recombinant mRNA translation rate, our data indicated that all single ORF transfectants exhibited a reduced level of recombinant gene transcription and that Mab folding and assembly reactions generically exerted greater control over qMab. We infer that the productivity of single ORF transfectants is limited by ER processing/degradation "capacity" which sets a limit on transcriptional input. We conclude that gene vector design for oligomeric recombinant proteins should be based on an understanding of protein-specific synthetic kinetics rather than polypeptide stoichiometry.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Vetores Genéticos/genética , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Animais , Células CHO , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Vetores Genéticos/metabolismo , Humanos , Fases de Leitura Aberta , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.
Assuntos
Anticorpos Monoclonais/biossíntese , Modelos Animais de Doenças , Produtos do Gene env/imunologia , HIV-1/imunologia , Imunoglobulinas/genética , Animais , Anticorpos Monoclonais/química , Humanos , Imunoglobulinas/biossíntese , Camundongos , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr(888) contributing significantly to this effect. To determine whether CaR(T888) is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaR(pT888)). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaR(T888A), phorbol ester (PMA) treatment increased CaR(pT888) immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca(2+) concentration from 0.5 to 2.5 mM increased CaR(T888) phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mM extracellular Ca(2+) and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaR(T888A) did exhibit increased apparent agonist sensitivity, by converting intracellular Ca(2+) (Ca(2+)(i)) oscillations to sustained plateau responses in some cells, we still observed Ca(2+)(i) oscillations in a significant number of cells. This suggests that CaR(T888) contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca(2+)(i) oscillations. Finally, dephosphorylation of CaR(T888) was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca(2+)(i) oscillations. In addition, calyculin/PMA cotreatment increased CaR(T888) phosphorylation in bovine parathyroid cells. Therefore, CaR(T888) is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.
Assuntos
Compostos de Anilina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glândulas Paratireoides/enzimologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Receptores de Detecção de Cálcio/metabolismo , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Sinalização do Cálcio/genética , Carcinógenos/farmacologia , Bovinos , Linhagem Celular , Expressão Gênica , Humanos , Mutação , Glândulas Paratireoides/citologia , Fosfoproteínas Fosfatases/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1 , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Receptores de Detecção de Cálcio/genética , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The Ca2+-sensing receptor (CaR) is a pleiotropic, type III G protein-coupled receptor (GPCR) that associates functionally with the cytoskeletal protein filamin. To investigate the effect of CaR signaling on the cytoskeleton, human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK) were incubated with CaR agonists in serum-free medium for up to 3 h. Addition of the calcimimetic NPS R-467 or exposure to high extracellular Ca2+ or Mg2+ levels elicited actin stress fiber assembly and process retraction in otherwise stellate cells. These responses were ablated by cotreatment with the calcilytic NPS 89636 and were absent in vector-transfected HEK-293 cells. Cotreatment with the Rho kinase inhibitors Y-27632 and H1152 attenuated the CaR-induced morphological change but not intracellular Ca2+ (Ca2+(i)) mobilization or ERK activation, although transfection with a dominant-negative RhoA-binding protein also inhibited calcimimetic-induced actin stress fiber assembly. CaR effects on morphology were unaffected by inhibition of G(q/11) or G(i/o) signaling, epidermal growth factor receptor, or the metalloproteinases. In contrast, CaR-induced cytoskeletal changes were not induced by the aromatic amino acids, treatments that also failed to potentiate CaR-induced ERK activation despite inducing Ca2+(i) mobilization. Together, these data establish that CaR can elicit Rho-mediated changes in stress fiber assembly and cell morphology, which could contribute to the receptor's physiological actions. In addition, this study provides further evidence that aromatic amino acids elicit differential signaling from other CaR agonists.