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PubMed2000

The peroxisome proliferator perfluorodecanoic acid inhibits the peripheral-type benzodiazepine receptor (PBR) expression and hormone-stimulated mitochondrial cholesterol transport and steroid formation in Leydig cells.

The peroxisome proliferator perfluordecanoic acid (PFDA) has been shown to exert an antiandrogenic effect in vivo by acting directly on the interstitial Leydig cells of the testis. The objective of this study was to examine the in vitro effects of PFDA and identify its site of action in steroidogenesis using as model systems the mouse tumor MA-10 and isolated rat Leydig cells. PFDA inhibited in a time- and dose-dependent manner the hCG-stimulated Leydig cell steroidogenesis. This effect was localized at the level of cholesterol transport into the mitochondria. PFDA did not affect either the total cell protein synthesis or the mitochondrial integrity. Moreover, it did not induce any DNA damage. Morphological studies indicated that PFDA induced lipid accumulation in the cells, probably due to the fact that cholesterol mobilized by hCG did not enter the mitochondria to be used for steroidogenesis. In search of the target of PFDA, we examined its effect on key regulatory mechanisms of steroidogenesis. PFDA did not affect the hCG-induced steroidogenic acute regulatory protein (StAR) levels. However, it was found to inhibit the mitochondrial peripheral-type benzodiazepine receptor (PBR) ligand binding capacity, 18-kDa protein, and messenger RNA (mRNA) levels. Further studies indicated that PFDA did not affect PBR transcription, but it rather accelerated PBR mRNA decay. Taken together, these data suggest that PFDA inhibits the Leydig cell steroidogenesis by affecting PBR mRNA stability, thus inhibiting PBR expression, cholesterol transport into the mitochondria, and the subsequent steroid formation. Moreover, this action of PFDA on PBR mRNA stability indicates a new mechanism of action of peroxisome proliferators distinct from the classic transcription-mediated regulation of target genes.

Animals↗
PubMed2004

Lack of an inducible effect of dietary soy isoflavones on the mRNA abundance of hepatic cytochrome P-450 isozymes in rats.

Modulation of the activity and content of cytochrome P-450 (CYP) in hepatic microsomes may be important to human health since these enzymes activate and inactivate a wide range of xenobiotics and food components. Regulation of the inducibility of most CYPs involves transcriptional regulation and post-transcriptional mRNA stabilization. We examined in the present study the effect of dietary soy isoflavone (0-300 mg of isoflavone/kg of diet) on the mRNA abundance of rat hepatic CYP1A1, CYP1A2, CYP2B1/2, CYP2C11, CYP2E1, CYP3A1, CYP3A2 and CYP4A1 by quantitative competitive RT-PCR and real-time monitored RT-PCR. A fermented soy extract containing 155 mg/g of genistein, 127 mg/g of daidzein, and other minor isoflavones was used as the isoflavone source. The dietary soy isoflavone had no affect on the hepatic mRNA abundance of these CYPs. The results by both methods were well matched and indicate that the dietary soy isoflavone did not cause the induction of CYPs by transcriptional step-up regulation or post-transcriptional mRNA stabilization.

Animals↗
PubMed1993

Differential regulation of carcinoembryonic antigen and biliary glycoprotein by gamma-interferon.

Carcinoembryonic antigen (CEA), biliary glycoprotein (BGP), and non-specific cross-reacting antigen (NCA) are three closely related cell surface glycoproteins induced by gamma-interferon (IFN-gamma) in colonic epithelial cells. Maximal induction of CEA by IFN-gamma and tumor necrosis factor alpha (TNF-alpha) in the colon carcinoma cell line HT-29 occurs at 5-6 days with maximal secreted levels at 14 ng/ml for IFN-gamma and 20 ng/ml for TNF-alpha. Cell viability was reduced to 67% of controls for TNF-alpha and to 36% for IFN-gamma. Dose-response curves showed maximal induction of CEA at 500 units/ml for TNF-alpha and at 200 units/ml for IFN-gamma. Combinations of the two lymphokines revealed that the CEA induction effects were additive and the cytotoxicity effects were synergistic. Northern blot analysis of HT-29 cells treated with IFN-gamma and probed with specific probes for BGP, CEA, and NCA showed a 2-fold increase in mRNA level for BGP, and a greater than 10-fold induction for CEA and NCA. Similar results were obtained for the SW403 cell line, but in the case of the LS174T cell line, CEA mRNA levels remained constant before and after IFN-gamma treatment, while BGP and NCA mRNA levels increased by 2-5-fold. Polymerase chain reaction analysis of the four alternatively spliced transcripts of BGP revealed no differential induction of one transcript over another by IFN-gamma. A comparison of the kinetics of induction of the mRNA levels for BGP and CEA by IFN-gamma in the HT29 cell line revealed a half-time of < 6 h for BGP and 48 h for CEA. The induction of CEA mRNA was completely inhibited with either cycloheximide (protein synthesis inhibitor) or actinomycin D (RNA synthesis inhibitor), but the induction of BGP mRNA was superinduced by cycloheximide. The difference in the kinetics of induction and effect of cycloheximide on CEA and BGP mRNAs suggest that the two genes are regulated differently in the same cell line. We conclude that the regulation occurs mainly at the posttranscriptional level for CEA and involves mRNA stability. BGP regulation may be more complex, involving transcriptional and posttranscriptional regulation, and more closely resembles the regulation of MHC class II mRNA by IFN-gamma in epithelial cells. The mRNA stability effects may be mediated by the dramatically different sequences present in the 3'-untranslated regions of CEA and BGP.

Antigens, CD↗
PubMed2003

Inherent instability of plasminogen activator inhibitor type 2 mRNA is regulated by tristetraprolin.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that is subject to regulation at the post-transcriptional level. At least two mRNA instability elements reside within the PAI-2 transcript; one in the coding region and another within the 3'-untranslated region (UTR). For the latter, a functional AU-rich motif (ARE) has been identified that provides a binding site for a number of cellular proteins, including the mRNA stability protein, HuR. In this study, we used the yeast three-hybrid system to screen a human leukocyte cDNA library to identify other proteins that associate with the PAI-2 ARE. This screen identified tristetraprolin (TTP) as a PAI-2 mRNA ARE-binding protein. UV cross-linking and immunoprecipitation experiments showed that TTP expressed in HEK293 cells could associate with the PAI-2 ARE in vitro. Co-transfection of plasmids expressing TTP and PAI-2 in HEK293 cells resulted in an increase in the decay rate of PAI-2 mRNA and loss of PAI-2 protein in a process that was dependent upon the PAI-2 3'-UTR. The 29-nt PAI-2 AU-rich element alone was also capable of conferring TTP-dependent mRNA instability to a reporter transcript. The extent of PAI-2 mRNA stability was remarkably sensitive to TTP since TTP-dependent PAI-2 mRNA decay occurred at TTP levels that were below Western blot detection limits. This study identifies TTP as a functional PAI-2 ARE-binding protein that modulates the post-transcriptional regulation of the PAI-2 gene.

3' Untranslated Regions↗
PubMed1995

Histone H3 transcript stability in alfalfa.

The stability of histone H3 transcripts in alfalfa for replication-dependent and -independent gene variants was measured by northern analysis under conditions of inhibition of transcription and/or translation. Replication-dependent histone H3.1 transcripts were about three-fold less stable than the equally polyadenylated mRNA for replacement variant H3.2 histone. In actively growing suspension cultures treated with dactinomycin half-lives of 2 and 7 h were observed for H3.1 and H3.2 mRNAs, respectively. mRNA stabilities were also measured indirectly by histone protein synthesis. The translation inhibitor cycloheximide strongly increased mRNA levels for both histone H3 variants. The dependence of histone mRNA turnover on translation in animals also appears to exist in plants. The combination of inhibition of transcription and translation by dactinomycin and cycloheximide was used in an indirect assessment of H3 mRNA stability throughout the cell cycle in partially synchronized and cycle-arrested cultures. Destabilization of replication-dependent histone H3.1 mRNA was detected in non-S phase cells.

Cell Cycle↗
PubMed1997

Role of posttranscriptional processes in the regulation of glutathione S-transferase P1 gene expression in human breast cancer cells.

Human glutathione S-transferase P1 (GSTP1) is normally expressed in estrogen receptor negative (ER-) but not receptor positive (ER+) cultured breast cancer cells. Previous results indicated that posttranscriptional mechanisms may contribute to this differential expression of GSTP1 (J. Biol. Chem. 267, 10544-10550, 1992). Here, we have tested the hypothesis that differences in posttranscriptional processing of primary transcripts to mature mRNA or differences in mRNA stability influence the levels of GSTP1 in ER- versus ER+ breast cancer cells. We examined the expression both of the endogenous GSTP1 gene and of uniquely designed GSTP1 minigenes that were stably transfected into HS578T (ER-) and MCF7 (ER+) cells. In both cell lines, GSTP1 transcripts are processed to mature, functional mRNAs. However, GSTP1 mRNA is considerably less stable in MCF7 than in HS578T cells. These results indicate that for a given level of GSTP1 gene transcription, differential mRNA stability will result in higher steady state levels of GSTP1 mRNA in ER-, HS578T than in ER+, MCF7 cells.

Breast Neoplasms↗
PubMed1998

Compensatory responses to inhibition of hepatic squalene synthase.

The mechanism by which depletion of hepatic cholesterol levels, achieved by inhibition of squalene synthase, alters hepatic LDL receptor, HMG-CoA reductase, and cholesterol 7alpha-hydroxylase gene expression was investigated by measuring transcription rates, mRNA stability, rates of translation, translational efficiency, and levels of sterol response element binding proteins. It was found that the transcription of both hepatic LDL receptor and HMG-CoA reductase were increased about twofold. The increase in LDL receptor transcription occurred within 2 h after giving 2 mg/kg zaragozic acid A, a potent inhibitor of squalene synthase. This preceded the increase in transcription of HMG-CoA reductase that occurred at 4 h. Increases in the stability of both of these mRNAs were also observed. These changes account for the increases in LDL receptor and HMG-CoA reductase mRNA levels previously observed. The rate of transcription of hepatic cholesterol 7alpha-hydroxylase was decreased to about 25% of control within 3 h after administration of zaragozic acid A, which correlates with the decrease in this mRNA. The rates of translation, as determined by pulse labeling, of both hepatic HMG-CoA reductase and LDL receptor were increased two- to threefold. The translational efficiency of these two mRNAs was also increased as judged by polysome profile analysis. There was an increase in mRNA associated with the heaviest polysome fraction and a decrease in that associated with monosomes. No significant change was observed in the levels of sterol response element binding protein 2, the form that mediates induced transcription, in response to zaragozic acid A treatment, indicating that this protein might not be involved in mediating the observed transcriptional changes. An increase in sterol response element binding protein -1 was observed 30 min after giving zaragozic acid A. The results suggest that compensatory responses to depletion of squalene-derived products involve alterations in the rates of transcription, mRNA stability, and translational of key proteins involved in cholesterol homeostasis.

Animals↗
PubMed1989

Two distinct mechanisms of interleukin-2 gene expression in human T lymphocytes.

Interleukin-2 (IL-2) gene regulation was investigated in primary cultures of highly purified human peripheral blood CD28+ T cells. Two discrete mechanisms for induction of T-cell proliferation could be distinguished by examining cell cycle progression and the expression of the IL-2 gene. Stimulation of cells by CD3 MoAb induced only transiently expressed, small amounts of IL-2 mRNA that was completely suppressed by cyclosporine. Costimulation of T cells with CD3 MoAb and either CD28 MoAb or PMA, but not calcium ionophore, induced a 50-100-fold increased in IL-2 gene expression and secretion. High levels of IL-2 gene expression could also be achieved by stimulation with calcium ionophore and PMA or CD28 MoAb and PMA, but not by CD28 MoAb plus calcium ionophore. IL-2 gene expression and T-cell proliferation induced by CD3 MoAb plus PMA or calcium ionophore plus PMA were completely suppressible by cyclosporine. In contrast, IL-2 gene expression and T-cell proliferation induced by CD28 MoAb plus PMA were unaffected by cyclosporine. The CD28 signal was dependent on new protein synthesis. Nuclear run-on transcription assays showed that anti-CD28 did not affect lymphokine transcription. A major effect of CD28 stimulation on mRNA stability was shown by studies using actinomycin D; CD28 stimulation substantially increased the half-life of IL-2 and TNF-alpha mRNA. The effects of anti-CD28 stimulation were specific for growth factors, and thus differ from previously described effects of cycloheximide on mRNA stability. These studies suggest the existence of two biochemical pathways for the induction of IL-2 production, one that occurs at the transcriptional level and is mediated by intracellular calcium release and protein kinase C and is cyclosporine-sensitive, and one that acts post-transcriptionally, is mediated by CD28 stimulation, and is cyclosporine-resistant.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2006

Transcript stabilization by mRNA sequences from hrpA of Pseudomonas syringae.

Production of heterologous proteins in bacteria is one of the main applications of biotechnology. Although several high-efficiency expression systems have been developed, different steps in protein production may become rate-limiting depending on the production system and the protein being produced. One bottle neck can be the instability of the mRNA. We have used fragments of the unusually long-living mRNA hrpA from the plant pathogenic bacteria Pseudomonas syringae pathovars tomato and phaseolicola to increase the half-lives of heterologous transcripts. The stabilizing effect was extended to Escherichia coli, as half-lives of several heterologous transcripts were increased from a few minutes to up to 19min. Production of heterologous proteins was also increased manifold by the addition of the stabilizing hrpA elements. We have mapped the regions of the hrpA transcript necessary and sufficient for the stabilization process.

Bacterial Proteins↗
PubMed1997

In vivo kinetics of a redox-regulated transcriptional switch.

SoxR is a transcription activator governing a cellular response to superoxide and nitric oxide in Escherichia coli. SoxR protein is a homodimer, and each monomer has a redox-active [2Fe-2S] cluster. Oxidation and reduction of the [2Fe-2S] clusters can reversibly activate and inactivate SoxR transcriptional activity. Here, we use electron paramagnetic resonance spectroscopy to follow the redox-switching process of SoxR protein in vivo. SoxR [2Fe-2S] clusters were in the fully reduced state during normal aerobic growth, but were completely oxidized after only 2-min aerobic exposure of the cells to superoxide-generating agents such as paraquat. The oxidized SoxR [2Fe-2S] clusters were rapidly re-reduced in vivo once the oxidative stress was removed. The in vivo kinetics of SoxR [2Fe-2S] cluster oxidation and reduction exactly paralleled the increase and decrease of transcription of soxS, the target gene for SoxR. The kinetic analysis also revealed that an oxidative stress-linked decrease in soxS mRNA stability contributes to the rapid attainment of a new steady state after SoxR activation. Such a redox stress-related change in soxS mRNA stability may represent a new level of biological control.

Bacterial Proteins↗
PubMed2006

The polypyrimidine tract-binding protein (PTB) is involved in the post-transcriptional regulation of human inducible nitric oxide synthase expression.

Human inducible nitric oxide synthase (iNOS) expression is regulated by transcriptional and post-transcriptional mechanisms. We have recently shown that the multifunctional RNA-binding proteins KH-type splicing regulatory protein and tristetraprolin are critically involved in the post-transcriptional regulation of human iNOS expression. Several reports have shown that KH-type splicing regulatory protein colocalizes with the polypyrimidine tract-binding protein (PTB), and both RNA-binding proteins seem to interact with the same mRNAs. Therefore we analyzed the involvement of PTB in human iNOS expression. In human DLD-1 cells, cytokine incubation necessary to induce iNOS expression did not change PTB localization or expression. However, intracellular binding of PTB to the human iNOS mRNA increased after cytokine stimulation. Overexpression of PTB resulted in enhanced cytokine-induced iNOS expression. Accordingly, small interfering RNA-mediated knock down of PTB reduced cytokine-dependent iNOS expression. Recombinant PTB displayed binding to an UC-rich sequence in the 3'-untranslated region of the human iNOS mRNA. Transfection experiments showed that PTB mediates its effect on iNOS expression via binding to this region. The underlying mechanism is based on a modulation of iNOS mRNA stability. In summary, human iNOS is the first example of a human pro-inflammatory gene regulated by PTB on the level of mRNA stability.

Carcinoma↗
PubMed1996

Extracellular matrix alters PDGF regulation of fibroblast integrins.

Extracellular matrix (ECM) and growth factors are potent regulators of cell phenotype. These biological mediators of cellular responses are potentially interactive and as such could drive cells through progressive phenotypes to create new tissue as in morphogenesis and wound repair. In fact, ECM composition changes during tissue formation accompanied by alterations in cell growth and migration. How alterations in the ECM regulate cell activities is poorly defined. To address this question in wound repair, we cultured normal human dermal skin fibroblasts in relaxed collagen gels, fibronectin-rich cultures or stressed fibrin gels, and stressed collagen gels to model normal dermis, early wound provisional matrix, and late granulation tissue, respectively. Integrin subunits, alpha 2, alpha 3, and alpha 5, that define receptor specificity for collagen and provisional matrix, respectively, were measured at mRNA steady-state level before and after stimulation with platelet-derived growth factor-BB (PDGF-BB), a potent mitogen and chemoattractant for fibroblasts. Fibronectin-rich cultures and fibrin gels supported PDGF-BB induction of alpha 3 and alpha 5 mRNA. In contrast, both stressed and relaxed collagen attenuated these responses while promoting maximal alpha 2 mRNA expression. Posttranscriptional regulation was an important mechanism in this differential response. Together PDGF-BB and collagen gels promoted alpha 2, but not alpha 3 and alpha 5, mRNA stability. Conversely, when fibroblasts were in fibronectin-rich cultures, PDGF-BB promoted alpha 3 and alpha 5, but not alpha 2, mRNA stability. We suggest that ECM alterations during wound healing or any new tissue formation cause cells to respond differently to repeated growth factor stimuli. An ordered progression of cell phenotypes results, ultimately consummating tissue repair or morphogenesis.

Animals↗
PubMed1996

Impaired interleukin-3 mRNA decay in autocrine mast cell tumors after transient calcium ionophore stimulation.

Autocrine interleukin-3 (IL-3) expression of v-H-ras transformed mast cell tumors involves either IL-3 mRNA stabilization (class-I tumors) or enhanced IL-3 transcription (class-II tumors). Since calcium ionophores induce IL-3 expression in untransformed PB-3c cells by transcript stabilization, we asked whether class-I tumor could still respond to calcium ionophores. We found that ionomycin treatment further increased IL-3 mRNA expression of class-I tumor cells. Following ionomycin wash-out, IL-3 mRNA decay was slower in class-I tumor cells than in class-II tumor or precursor cell lines (t1/2 > 50 min versus < 20 min, respectively). Somatic cell fusion of the class-I tumor cells with the non-tumorigenic PB-3c cells resulted in reversion to rapid decay after ionomycin wash-out. The data indicate that a recessive defect of IL-3 mRNA degradation can be revealed in class-I tumor cells by transient calcium ionophore stimulation. However, IL-3 mRNA stabilization operating constitutively in class-I tumor cells appears to be distinct from the ionomycin induced process.

Animals↗
PubMed2004

The protein phosphatase calcineurin determines basal parathyroid hormone gene expression.

Calcium and phosphate regulate PTH mRNA stability through differences in binding of parathyroid (PT) proteins to a minimal 63-nucleotide (nt) cis-acting instability element in its 3'-untranslated region. One of these proteins is adenosine-uridine-rich binding factor (AUF1), whose levels are not regulated in PT extracts from rats fed the different diets. However, two-dimensional gels showed posttranslational modification of AUF1 that included phosphorylation. There is no PT cell line, but in HEK 293 cells the 63-nt element is recognized as an instability element, and RNA interference for AUF1 decreased human PTH secretion in cotransfection experiments. Stably transfected cells with a chimeric GH gene containing the PTH 63-nt cis-acting element were used to study the signal transduction pathway that regulates AUF1 modification and chimeric gene mRNA stability. Cyclosporine A, the calcineurin inhibitor, regulated AUF1 posttranslationally, and this correlated with an increase in the stability of GH-PTH 63-nt mRNA but not of the control GH mRNA. Mice with genetic deletion of the calcineurin Abeta gene had markedly increased PTH mRNA levels that were still regulated by low calcium and phosphorus diets. Therefore, calcineurin regulates AUF1 posttranslationally in vitro and PTH gene expression in vivo but still allows its physiological regulation by calcium and phosphate.

3' Untranslated Regions↗
PubMed1998

Regulatory signals in messenger RNA: determinants of nutrient-gene interaction and metabolic compartmentation.

Nutrition has marked influences on gene expression and an understanding of the interaction between nutrients and gene expression is important in order to provide a basis for determining the nutritional requirements on an individual basis. The effects of nutrition can be exerted at many stages between transcription of the genetic sequence and production of a functional protein. This review focuses on the role of post-transcriptional control, particularly mRNA stability, translation and localization, in the interactions of nutrients with gene expression. The effects of both macronutrients and micronutrients on regulation of gene expression by post-transcriptional mechanisms are presented and the post-transcriptional regulation of specific genes of nutritional relevance (glucose transporters, transferrin, selenoenzymes, metallothionein, lipoproteins) is described in detail. The function of the regulatory signals in the untranslated regions of the mRNA is highlighted in relation to control of mRNA stability, translation and localization and the importance of these mRNA regions to regulation by nutrients is illustrated by reference to specific examples. The localization of mRNA by signals in the untranslated regions and its function in the spatial organization of protein synthesis is described; the potential of such mechanisms to play a key part in nutrient channelling and metabolic compartmentation is discussed. It is concluded that nutrients can influence gene expression through control of the regulatory signals in these untranslated regions and that the post-transcriptional regulation of gene expression by these mechanisms may influence nutritional requirements. It is emphasized that in studies of nutritional control of gene expression it is important not to focus only on regulation through gene promoters but also to consider the possibility of post-transcriptional control.

Gene Expression Regulation↗
PubMed1993

Follicle-stimulating hormone increases the turnover of G-protein alpha i-1- and alpha i-2-subunit messenger RNA in Sertoli cells by a mechanism that is independent of protein synthesis.

We have shown previously that FSH differentially regulated G-protein alpha i-subunit mRNA levels in primary cultures of rat Sertoli cells. The mechanism by which FSH regulated the steady state levels of these mRNAs was investigated by assessing the effect of FSH on G-protein alpha-subunit mRNA stability and the potential involvement of newly synthesized proteins. The half-life (t 1/2) of alpha-subunit mRNA was determined by inhibiting Sertoli cell transcription with actinomycin-D and quantifying alpha-subunit mRNA levels by Northern blot analyses. Transcripts for alpha i-2 and alpha 3 were extremely stable, possessing t1/2 of 44 and 51 h, respectively. In contrast, the t1/2 of alpha i-3 mRNA was only 3.6 h. Turnover of alpha i-1 mRNA occurred as a two-phase decay, with an initial t1/2, fast of 0.8 h, followed by a second phase with t1/2, slow of 11.1 h. Treatment of Sertoli cells with FSH in the presence of actinomycin-D destabilized the alpha i-1 mRNA to a single phase decay with a t1/2 of 5.7 h. FSH also decreased the stability of the alpha i-2 mRNA from a t1/2 of 51 h to a t1/2, fast of 16.5 h. These effects of FSH on alpha i-1 and alpha i-2 mRNA stability may contribute to the ability of FSH to decrease steady state alpha i-1 and alpha i-2 mRNA levels. The effect of FSH on alpha i-3 mRNA stability was not significant, suggesting an alternative mechanism regulating the FSH-mediated increase of alpha i-3 mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Gonadotropin subunit messenger RNA concentrations after blockade of gonadotropin-releasing hormone action: testosterone selectively increases follicle-stimulating hormone beta-subunit messenger RNA by posttranscriptional mechanisms.

Regulation of gonadotropin gene expression by sex steroids may occur via direct effects on the pituitary and/or indirect effects of steroids mediated through hypothalamic GnRH. We aimed to define the effects of testosterone (T) on alpha, LH beta, and FSH beta mRNA expression in the male rat after blockade of GnRH action on the gonadotrope. A water-soluble GnRH antagonist was administered iv to castrate male rats (increased endogenous GnRH secretion) and to castrate T-replaced rats in which gonadotropin subunit mRNAs had been increased by prior treatment with exogenous GnRH pulses. In castrate male rats, GnRH antagonist resulted in a fall in all three subunit mRNAs. Alpha and LH beta declined at slower rates (half-disappearance after 50 and 65 h, respectively), and neither fell to values present in intact rats over 84 h. In contrast, FSH beta mRNA declined more rapidly, with a half-disappearance after 20 h. In castrate T-replaced rats, alpha mRNA declined at a rate similar to that in castrates (half-disappearance after 50 h). LH beta declined more slowly, and the rate of FSH beta decline was markedly prolonged in the presence of T (half-disappearance time increased from 20 to 50 h). These results suggest that T exerts direct effects on FSH beta transcription or mRNA stability which are independent of GnRH action. To assess these possibilities, a long-acting GnRH antagonist (Detirelix) was administered to castrate male rats, which also received T or sham implants 4 days after castration. FSH beta mRNA levels fell during the 4 days of Detirelix alone, but the addition of T on day 4 resulted in a 2-fold rise in FSH beta mRNA, restoring FSH beta mRNA to levels present in intact rats. Serum FSH closely paralleled FSH beta mRNA concentrations. Alpha mRNA was reduced by 25%, and LH beta mRNA concentrations were unchanged in the presence of T. The rate of alpha mRNA transcription was markedly reduced and that of LH beta tended to fall in T-treated rats, but T had no significant effect on the FSH beta transcription rate. Thus, the action of T to increase concentrations of cytosolic FSH beta mRNA appears to be exerted at a posttranscriptional level, possibly via effects of T on FSH beta mRNA stability. This may represent a mechanism by which T can effect differential regulation of gonadotropin subunit mRNA concentrations.

Amino Acid Sequence↗
PubMed2002

Identification of a 16-nucleotide sequence that mediates post-transcriptional regulation of rat CYP2E1 by insulin.

Insulin directly down-regulates the gene expression of the rat CYP2E1 by altering its mRNA stability (De Waziers, I., Garlatti, M., Bouguet, J., Beaune, P. H., and Barouki, R. (1995) Mol. Pharmacol. 47, 474-479). Because the regulation of CYP mRNA stability was poorly understood, the molecular mechanisms involved in this regulation in the rat hepatoma H4IIEC3 cell line were studied. By using RNase T1 protection methods, the formation of a major CYP2E1 RNA-protein complex was observed. By competition experiments, the binding site of this complex was located on a 16-nucleotide sequence in the 5'-proximal region of the CYP2E1-coding sequence. Insulin did not modify the binding pattern of proteins to this sequence. and transfections of expression vectors or antisense oligonucleotides were undertaken to demonstrate the actual functionality of the 16-mer sequence. The insertion of this sequence in a luciferase gene was sufficient to render the chimeric mRNA sensitive to insulin. Furthermore, transfection of H4IIEC3 cells with antisense oligonucleotide complementary to this sequence blocked the insulin effect on the CYP2E1 mRNA expression, i.e. its rapid degradation. All these results demonstrate that this 16-nucleotide sequence is implicated in the CYP2E1 post-transcriptional regulation by insulin.

Animals↗