Brefeldin A detoxification in rat extraorbital lacrimal glands.
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Animals↗
Biomedical subjects
P Robin
Publications and source records attributed to P Robin.
PKA inhibitor, H-89, affects the intracellular transit of regulated secretory proteins in rat lacrimal glands.
We tested the effect of H-89, a protein kinase A (PKA) inhibitor, on the intracellular transit of the regulated secretory proteins in rat lacrimal glands. We show that H-89, by itself, induces the secretion of newly synthesized proteins trafficking in its presence but not of proteins already stored in the mature secretory granules. This secretion does not depend on the presence of extracellular Ca2+. The proteins released are identical to those secreted after cholinergic stimulation or under the action of the ionophore A-23187, but the secretion level is approximately 40% lower. The effect of H-89 seems to be due to PKA inhibition because other protein kinase inhibitors (calphostin C, chelerythrine, H-85) do not induce secretion. We further show that H-89 does not modify the rate of glycoprotein galactosylation but induces the secretion of newly galactosylated glycoproteins. Finally, we used a "20 degrees C block" procedure to show that H-89 affects a trans-Golgi network (TGN) or post-TGN step of the secretory pathway. Our results demonstrate that, in lacrimal cells, H-89 affects the intracellular trafficking of secretory proteins, suggesting a role for PKA in this process.
Animals↗
[Histone deacetylase and retinoblastoma protein].
The balance between cellular proliferation and differentiation is strictly controlled in the cell and the deregulation of this balance can lead to tumour formation. The tumour suppressor protein Rb plays a key role in this balance essentially by repressing progression through the cell cycle and thereby it blocks the cell in G1 phase. Rb represses S phase genes through the recruitment of an enzyme which modifies DNA structure, the histone deacetylase HDAC1. The Rb/HDAC1 complex is a key element in the control of cell proliferation and differentiation. Moreover, this complex is likely to be a target for transforming viral proteins.
Adenovirus E1A Proteins↗
The CREB-binding protein (CBP) cooperates with the serum response factor for transactivation of the c-fos serum response element.
The serum response element is one of the major promoter elements of the immediate early response to extracellular signals. The serum response element includes two main binding sites for proteins: the Ets box, which binds p62(TCF), and the CArG box, which binds p67(SRF). These two proteins are direct targets for signal transduction pathways; p62(TCF) is a nuclear end point of the Ras/mitogen-activated protein kinase pathway, and p67(SRF) is targeted by the Rho/Rac small G-proteins. The mechanism by which the signal is further transduced from the transcription factors to the basal transcriptional machinery is poorly understood. Recent data have suggested that the cAMP-responsive element-binding protein (CREB)-binding protein, a transcriptional adaptor involved in the transactivation through a wide variety of enhancer elements, participates in p62(TCF) activity. We here show that the CREB-binding protein also cooperates in the process of transactivation by p67(SRF). Cotransfections of expression vectors for the CREB-binding protein increased the expression, in response to serum, of reporters under the control of the c-fos serum response element. Interestingly, the C-terminal moiety of the CREB-binding protein was not necessary to observe this effect. The cooperation did not require the Ets box in the serum response element, and the CArG box was sufficient, indicating that the CREB-binding protein is able to cooperate with p67(SRF) in the absence of an Ets protein. Co-immunoprecipitation experiments using cell extracts showed that p67(SRF) could be retained with antibodies directed against the CREB-binding protein, suggesting that the two proteins form a multimolecular complex in live cells. The physical interaction between p67(SRF) and the CREB-binding protein was further confirmed by two-hybrid assays in mammalian cells. Our results indicate that the CREB-binding protein cooperates with p67(SRF) and, thus, suggest that the serum response element is regulated by a multimolecular complex, which includes the CREB-binding protein, p67(SRF), and p62(TCF), with multiple interactions between the components of the complex.
3T3 Cells↗
Activation of the c-fos SRE through SAP-1a.
TCFs, which are members of the Ets family of transcription factors, are recruited to the Serum Response Element (SRE) in the c-fos promoter by SRF. These Ets proteins, which are substrates for the MAP kinases, are direct targets of the Ras/MAP kinase signal transduction pathway. In this paper, we demonstrate that one of the TCFs, SAP-1a, displays a significant level of autonomous binding to the SRE Ets box. In contrast to previous observations, deletion of the SRF binding domain did not modulate the autonomous binding of SAP-1a. Also, the autonomous binding was not modulated by the phosphorylation of SAP-1a by MAP kinases. The autonomous binding was also detected in live cells: transfected SAP-1a was able to restore the response of a CArG-less SRE in PC12 cells. The response occurred in the absence of SRF recruitment since a mutant of SAP-1a in which the B-box, a domain required for interaction with SRF, had been deleted was still able to transactivate the CArG-less SRE. The transactivation was repressed by a Ras transdominant negative mutant, indicating the involvement of the Ras/MAP kinase pathway. Taken together, these data demonstrate that SAP-1a is capable of binding to the c-fos SRE in the absence of SRF.
Animals↗
Transcriptional and posttranscriptional mechanisms of glucocorticoid-mediated repression of phosphoenolpyruvate carboxykinase gene expression in adipocytes.
Glucocorticoids exert pleiotropic effects, among which negative regulation of transcription has been recognized as of crucial importance. While glucocorticoids induce phosphoenolpyruvate carboxykinase (PEPCK) gene expression in liver cells, it represses gene activity in adipose cells. We used the 3T3-F442A adipocytes to analyze the underlying mechanisms in these cells, the synthetic glucocorticoid dexamethasone exerts a dominant repression either on basal or on beta-agonist stimulation of PEPCK gene expression. To determine whether glucocorticoid action required protein synthesis, we employed cycloheximide, anisomycin, and puromycin, three different translation inhibitors. None of these affected induction by isoprenaline or repression by dexamethasone of isoprenaline stimulation. In contrast, dexamethasone inhibitory action on basal PEPCK mRNA was totally prevented by the three translation inhibitors. Time courses of glucocorticoid action on basal and on induction by beta-agonist were similar. Half-maximal effect of dexamethasone on isoprenaline-induced PEPCK mRNA was obtained at about 10 nM, a tenfold higher concentration than that observed for the reduction of basal mRNA. Using the transcription inhibitor DRB, we showed that dexamethasone did not alter mRNA half-life, while isoprenaline strongly stabilized mRNA. In a 3T3-F442A stable transfectant bearing -2,100 base pairs of the PEPCK promoter fused to the chloramphenicol acetyltransferase (CAT) gene, isoprenaline stimulated CAT activity, whereas dexamethasone reduced basal and isoprenaline-induced CAT expression. Hence, beta-agonists exert both transcriptional and posttranscriptional regulation, while glucocorticoid action is purely transcriptional. However, mechanisms of glucocorticoid repression of basal and of beta-agonist stimulation appear different.
Adipocytes↗
Recruitment of transcription factors to the target site by triplex-forming oligonucleotides.
Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a hairpin-TFO able to recruit transcription factors to a target DNA. The designed oligonucleotide contains a triplex-forming sequence, linked through a nucleotide loop to a double-stranded hairpin including the SRE enhancer of the c-fos gene promoter. We show here that this oligonucleotide can specifically recognise its DNA target at physiological salt and pH conditions. The stability of the triplex formed under these conditions is very high: >90% of the triplex remains intact after 24 h of incubation. Bound to the double-stranded target DNA, the oligonucleotide retains its ability to interact specifically with transcription factors, recruiting them to the proximity of the target DNA. Our results suggest that this type of oligonucleotide may prove useful in the design of new tools for artificial modulation of gene expression.
Base Sequence↗
Detoxification mechanism in the rat extraorbital lacrimal gland: conjugation of brefeldin A to glutathione.
In this work the existence of a glutathione based detoxification system in rat lacrimal glands is reported. We showed that brefeldin A, a drug used as a tool for the study of intracellular trafficking mechanisms, was inactivated by metabolization and converted into two derivatives. We purified them by high performance liquid chromatography and determined, by mass spectroscopy, that they correspond to glutathione and cysteine derivatives of BFA. The determination of the respective amounts of these derivatives in the medium and the tissue in different experimental conditions, revealed that glutathione-BFA is formed in the tissue, excreted from the cells, cleaved by gamma-glutamyl transpeptidase and finally converted to cysteine-BFA.
Animals↗
Calcifying fibrous pseudotumor of the mediastinum.
Calcifying fibrous pseudotumor has recently been described in the soft tissues. It is a rare benign lesion characterized by the presence of abundant hyalinized collagen with psammomatous or dystrophic calcifications and lymphoplasmacytic infiltrate. We report a case of a young woman with a mediastinal mass treated by a complete resection. The mass had all the pathologic features of calcifying fibrous pseudotumor.
Adult↗
Regulation of gene transcription by fatty acids, fibrates and prostaglandins: the phosphoenolpyruvate carboxykinase gene as a model.
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Adipocytes↗
Bilateral thoracoscopy for sympathectomy in the treatment of hyperhidrosis.
Thoracoscopic sympathectomy has been established as the least invasive technique with high success rates for treatment of palmary hyperhidrosis [1,4,5]. In our procedure both sides are treated during the same operation. A bilateral thoracoscopy was performed in 20 patients for incapacitating hyperhidrosis. Immediate complications at operation were minimal. All patients reported satisfaction with the procedure in spite of compensatory sweating. The short hospital stay has significant financial benefits and these are increased if both sides are treated at the same time. This procedure is more aggressive than the single side procedure but the morbidity is not increased.
Adolescent↗
SRE elements are binding sites for the fusion protein EWS-FLI-1.
EWS-FLI-1 is a chimeric protein produced in most Ewing's sarcomas. It results from the fusion of the N-terminal-encoding region of the EWS gene to the C-terminal DNA-binding domain (the ETS domain) encoded by the FLI-1 ets family gene. Both EWS-FLI-1 and FLI-1 proteins function as transcription factors that bind specifically to ets sequences (the ets boxes) present in promoter elements. EWS- FLI-1 is a powerful transforming protein, whereas FLI-1 is not. In a search for potential DNA binding sites for these two proteins, we have tested their ability to recognize the serum responsive element (SRE) in the c-fos promoter. This cis element contains an ets box which can be occupied by members of the ETS protein family which do not bind DNA autonomously but form a ternary complex with a second protein, p67SRF (serum responsive factor). We demonstrate here that EWS-FLI-1, but not FLI-1, is able to form a ternary complex on the c-fos SRE. Using a GST pull-down assay, we show that both FLI-1 and EWS-FLI-1 interact in vitro with SRF in the absence of DNA. In electromobility shift assays, EWS-FLI-1 binding to the SRE is detectable in the absence of SRF whereas the binding of FLI-1 is not, suggesting that the interaction with DNA is the step which limits ternary complex formation by FLI-1. Deletion of the N-terminal portion of FLI-1 resulted in a protein which behaved as EWS-FLI-1, suggesting the existence of an N- terminal inhibitory domain in the normal protein. Taken together, our data indicate that there are intrinsic differences in the binding of EWS-FLI-1 and FLI-1 proteins to distinct ets sequences.
Base Sequence↗
Physical interaction between the mitogen-responsive serum response factor and myogenic basic-helix-loop-helix proteins.
Terminal differentiation of muscle cells results in opposite effects on gene promoters: muscle-specific promoters, which are repressed during active proliferation of myoblasts, are turned on, whereas at least some proliferation-associated promoters, such as c-fos, which are active during cell division, are turned off. MyoD and myogenin, transcription factors from the basic-helix-loop-helix (bHLH) family, are involved in both processes, up-regulating muscle genes and down-regulating c-fos. On the other hand, the serum response factor (SRF) is involved in the activation of muscle-specific genes, such as c-fos, as well as in the up-regulation of a subset of genes that are responsive to mitogens. Upon terminal differentiation, the activity of these various transcription factors could be modulated by the formation of distinct protein-protein complexes. Here, we have investigated the hypothesis that the function of SRF and/or MyoD and myogenin could be modulated by a physical association between these transcription factors. We show that myogenin from differentiating myoblasts specifically binds to SRF. In vitro analysis, using the glutathione S-transferase pull-down assay, indicates that SRF-myogenin interactions occur only with myogenin-E12 heterodimers and not with isolated myogenin. A physical interaction between myogenin, E12, and SRF could also be demonstrated in vivo using a triple-hybrid approach in yeast. Glutathione S-transferase pull-down analysis of various mutants of the proteins demonstrated that the bHLH domain of myogenin and that of E12 were necessary and sufficient for the interaction to be observed. Specific binding to SRF was also seen with MyoD. In contrast, Id, a natural inhibitor of myogenic bHLH proteins, did not bind SRF in any of the situations tested. These data suggest that SRF, on one hand, and myogenic bHLH, on the other, could modulate each other's activity through the formation of a heterotrimeric complex.
3T3 Cells↗
Recovery of protein secretion after brefeldin A treatment of rat lacrimal glands: effect of cAMP.
In exocrine cells, the discharge of secretory granule contents in response to extracellular stimuli has been widely documented. However, few data are available concerning the effect of these stimuli on the steps of the secretory pathway preceding protein exocytosis. To obtain more data on this subject, we used brefeldin A (BFA) to perturb intracellular protein transit. When, after exposure of the lacrimal gland lobules to 10 microM BFA, which led to a complete dismantling of the Golgi apparatus and fully inhibited the secretion of newly synthesized proteins, the drug concentration was lowered to 100 nM, a restoration of protein secretion was observed in a secretagogue-dependent manner. Secretagogues increasing the adenosine 3',5'-cyclic monophosphate (cAMP) level facilitated the recovery of protein secretion and Golgi apparatus restructuring, whereas other secretagogues, involving the calcium pathway, did not. Furthermore, the cAMP effect was prevented by H-89, a specific protein kinase A inhibitor. These effects of cAMP are due to neither BFA degradation nor BFA excretion from the cells. We conclude from these results that in rat lacrimal glands the recovery from the dramatic damage caused by BFA is promoted by a cAMP-dependent mechanism and further suggest a role of cAMP in the regulation of the Golgi structure and/or function.
Animals↗
Fatty acids and fibrates are potent inducers of transcription of the phosphenolpyruvate carboxykinase gene in adipocytes.
Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) plays a critical role in adipose tissue glyceroneogenesis. We have previously shown that transcription of the PEPCK gene was stimulated by isoprenaline and retinoic acid in 3T3-F442A adipocytes. We also showed that oleate increased PEPCK mRNA. Here, we analysed the effect that fatty acids of various chain lengths and unsaturation degrees exerted on PEPCK gene expression in 3T3-F442A adipocytes. When maintained in serum-free, glucose-free medium, differentiated cells responded to unsaturated long-chain fatty acids by a large increase in PEPCK mRNA whereas saturated fatty acids were inefficient. A maximum fivefold stimulation by oleate was attained at 4 h of treatment with 1 mM fatty acid bound to albumin in a 6:1 ratio. The poly-unsaturated very long-chain fatty acid all-cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6) was even more potent and produced a tenfold increase. The expression of the genes encoding glycerol-3-phosphate dehydrogenase, hormone-sensitive lipase or actin remained unaffected by oleate exposure. A 4-h treatment by the hypolipidemic drug clofibrate, 0.5-2 mM, also produced a large (3-9-fold) increase in PEPCK mRNA. When used at non-saturating concentrations, oleate and clofibrate acted in an additive manner. At maximally effective concentrations, additivity was lost, suggesting that fatty acids and fibrates might act through similar mechanisms. Nuclear transcription experiments showed that oleate and clofibrate stimulated the transcription rate of the gene. 3T3-F442A cells were stably transfected with a plasmid containing the base pairs -2100 to +69 of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase gene. These differentiated stable transfectants responded to oleate and clofibrate by a specific increase in chloramphenicol acetyltransferase activity. Adipocytes express various isoforms of peroxisome-proliferator-activated receptors that can be activated by fibrates and fatty acids. Potential recognition sequences for peroxisome-proliferator-activated receptors are present in the -2100 to +69 fragment of the PEPCK gene promoter. Thus, this gene represents an ideal molecular target for understanding the complex transcriptional control exerted by fatty acids and peroxisome proliferators.
3T3 Cells↗
Myogenin binds to and represses c-fos promoter.
Myogenin (a member of the myogenic basic helix-loop-helix transcription factor family) seems to be the main effector of proliferation repression, a crucial step which precedes muscle cell terminal differentiation during muscle development. Proliferation repression most likely occurs through inhibition of proliferation-associated genes such as the proto-oncogene, c-fos. Here, we demonstrate that myogenin binds to an E-box located in the main element of the c-fos promoter, the serum response element (SRE). Results from co-transfection experiments indicate that myogenin acts as a repressor for the SRE. Our data suggest that myogenin could play a role in c-fos inhibition at the onset of muscle cell terminal differentiation.
3T3 Cells↗
Expression of the phosphoenolpyruvate carboxykinase gene in 3T3-F442A adipose cells: opposite effects of dexamethasone and isoprenaline on transcription.
The enzyme phosphoenolpyruvate carboxykinase (PEPCK) plays a key role in gluconeogenesis in liver and in glyceroneogenesis in adipose tissue. These processes, and PEPCK, are regulated by a number of hormones, some of which have different effects on the enzyme in liver and adipose tissue. To explore this phenomenon, PEPCK gene expression was studied in 3T3-F442A adipocytes maintained in a serum-free medium. The beta-adrenergic agonist isoprenaline (isoproterenol) and a cyclic AMP analogue (8-CPT-cAMP) increased PEPCK mRNA. A maximal 3-fold induction occurred in 2 h. Dexamethasone decreased PEPCK mRNA by 80% in 4 h. Dexamethasone also counteracted the inductive effects of isoprenaline and 8-CPT-cAMP. Run-on transcription experiments showed that the isoprenaline and dexamethasone actions were, at least in part, exerted at the level of PEPCK gene transcription. These effects were further analysed by using transient and stable transfection of adipocytes with a plasmid containing bp -2100 to 69 of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase (CAT) gene. In such cells isoprenaline stimulated CAT expression, an effect that was prevented if the cells were also exposed to dexamethasone.
3T3 Cells↗
Effect of microtubule network disturbance by nocodazole and docetaxel (Taxotere) on protein secretion in rat extraorbital lacrimal and parotid glands.
The role of microtubules in the exocrine secretory process is not yet well established. Contradictory effects of anti-microtubule drugs on intracellular transit and protein secretion have been reported. In this work we used microscopic techniques and pulse-chase experiments to compare the involvement of microtubules in the regulated secretory process of two rat exocrine glands: parotid and extraorbital lacrimal glands. In our experiments microtubules were either disrupted by nocodazole or stabilized by a taxoid, docetaxel (Taxotere). We show that the effect of nocodazole and docetaxel on the release of newly synthesized proteins is radically different in the two tissues; in parotid gland they only weakly affect protein release, triggered by stimulation of either muscarinic or beta-adrenergic receptors, but in lacrimal gland, they strongly inhibit protein secretion. This effect or lack of effect of the drug is independent of the signal transduction pathways involved by the different secretagogues used to trigger exocytosis. Furthermore, in lacrimal glands, studies on protein galactosylation (which occurs in the trans-Golgi compartment) indicate that postgalactosylation events are more sensitive to both drugs than pregalactosylation events. On the other hand, we show that the effect of nocodazole and docetaxel on the microtubule network is comparable on the two tissues. Finally, in lacrimal cells, we observed a scattering of the Golgi apparatus concomitant with the disruption of microtubules by nocodazole. We conclude from this study that microtubule network integrity is essential for protein secretion in lacrimal glands but not in parotid glands. This result implies that for the same physiological function, i.e. protein secretion, different mechanisms may be involved.
Animals↗