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PubMed2001

Inhibition of histone deacetylation induces constitutive derepression of the beta interferon promoter and confers antiviral activity.

The induction of alpha/beta interferon (IFN-alpha/beta) genes constitutes one of the first responses of the cell to virus infection. The IFN-beta gene is constitutively repressed in uninfected cells and is transiently activated after virus infection. In this work we demonstrate that histone deacetylation regulates the silent state of the murine IFN-beta gene. Using chromatin immunoprecipitation (ChIP) assays, we show a direct in vivo correlation between the transcriptionally silent state and a state of hypoacetylation of histone H4 on the IFN-beta promoter region. Trichostatin A (TSA), a specific inhibitor of histone deacetylases, induced strong, constitutive derepression of the murine IFN-beta promoter stably integrated into a chromatin context, as well as the hyperacetylation of histone H4, without requiring de novo protein synthesis. We also show in this work that TSA treatment strongly enhances the endogenous IFN level and confers an antiviral state to murine fibroblastic L929 cells. Inhibition of histone deacetylation with TSA protected the cells against the lost of viability induced by vesicular stomatitis virus (VSV) and inhibited VSV multiplication. Using antibodies neutralizing IFN-alpha/beta, we show that the antiviral state induced by TSA is due to TSA-induced IFN production. The demonstration of the predominant role of histone deacetylation during the regulation of the constitutive repressed state of the IFN-beta promoter constitutes an interesting advance on the understanding of the negative regulation of this gene and opens up the possibility of new therapeutic perspectives.

Acetylation↗
PubMed1981

Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid.

Mutants of Kluyveromyces lactis with elevated uninduced levels of beta-galactosidase (EC 32.1.2.3) activity, constitutive mutants (lac10c), were isolated and characterized to determine the basis for their constitutiveness. These lesions are not operator-type regulatory mutants because they are not closely linked to the beta-galactosidase structural gene. In a constitutive strain having a 7-fold increase in beta-galactosidase activity, the concentration of beta-galactosidase messenger ribonucleic acid (mRNA) was 8- to 10-fold higher than uninduced wild type. The half-life of beta-galactosidase mRNA was the same in the mutant strain (t1/2 = 4.5 +/- 0.2 min) as in uninduced wild-type cells (t1/2 = 4.8 +/- 0.1 min), indicating that the elevated mRNA level in the mutant was not due to a decreased rate of mRNA degradation. Consequently, we hypothesize that the LAC10 product regulates transcription of the beta-galactosidase gene; it probably affects the rate of transcription initiation. Parallel increases in enzyme protein, in constitutive levels of beta-galactosidase activity, and in mRNA further support this position, making translational or posttranslational control by LAC10 unlikely. Several types of data suggest that the LAC10 product functions as a negative regulatory element to prevent transcription. Other data demonstrate that lac10c mutations have pleiotrophic effects, there being constitutive levels not only of beta-galactosidase activity, but also the other lactose-inducible activities of galactokinase (EC 2.7.5.1), galactose-1-phosphate uridyl transferase (EC 2.7.7.10), and lactose transport. It would appear that LAC10 regulates lactose-inducible proteins.

Chromosome Mapping↗
PubMed1993

Transcriptional regulation of the rat tissue type plasminogen activator gene: localization of DNA elements and nuclear factors mediating constitutive and cyclic AMP-induced expression.

We have characterized tissue type plasminogen activator (tPA) promoter elements and nuclear factors required for follicle-stimulating hormone (FSH)-induced transcription of the rat tPA gene in granulosa cells and constitutive expression of the gene in the rat neuroblastoma cell line B103. Run-on transcription analysis of isolated nuclei revealed that B103 cells transcribe the tPA gene at a high and constitutive level, while FSH was found to induce tPA gene transcription in a rapid and transient manner in granulosa cells. The maximal FSH-induced transcription rate was obtained after 20 min and was similar in the absence or presence of the protein synthesis inhibitor cycloheximide. However, in the presence of cycloheximide, tPA transcription was not turned off but continued at a high rate for several hours. This phenomenon may at least partly explain the earlier finding that tPA mRNA is superinduced by FSH in the presence of cycloheximide. DNase I footprinting analysis of the first 621 bp of the tPA promoter revealed a total of six regions that interact with nuclear factors from B103 and granulosa cells. Deletion of the promoter region from positions -269 to -621, a region that includes the two most-upstream footprints, had no effect on constitutive or FSH-induced transcription in transient expression experiments. Nuclear extracts from both granulosa cells and B103 cells showed strong binding to a consensus cyclic AMP-responsive element (CRE) at positions -178 to -185 and a neighboring binding site for nuclear factor 1 (NF1) at positions -145 to -158. The factors binding to these two regions were identified as members of the CRE-binding protein and NF1 families of transcription factors, respectively. Footprints were also obtained over two GC boxes at positions -64 to -71 and -41 to -49. These footprints were more pronounced with nuclear extracts from B103 cells than with extracts from untreated or FSH-treated granulosa cells, but gel shift assays indicate that similar amounts of two distinct factors bind to the two GC boxes in both cell types. Transfection experiments using promoter constructs with inactivated promoter elements indicate that both the CRE and NF1 sites contribute to the FSH responsiveness of the rat tPA gene in granulosa cells, while only the NF1 site is important for constitutive expression in B103 cells. The two GC boxes were found to be necessary both for constitutive expression in B103 cells and for FSH-induced expression in granulosa cells, and inactivation of both GC boxes essentially eliminated the tPA promoter activity in both cell types.

Animals↗
PubMed1993

Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements.

The calcitonin/calcitonin gene-related peptide (CGRP) primary transcript is alternatively spliced in thyroid C cells and neurons, resulting in the tissue-specific production of calcitonin and CGRP mRNAs. Analyses of mutated calcitonin/CGRP transcription units in permanently transfected cell lines have indicated that alternative splicing is regulated by a differential capacity to utilize the calcitonin-specific splice acceptor. The analysis of an extensive series of mutations suggests that tissue-specific regulation of calcitonin mRNA production does not depend on the presence of a single, unique cis-active element but instead appears to be a consequence of suboptimal constitutive splicing signals. While only those mutations that altered constitutive splicing signals affected splice choices, the action of multiple regulatory sequences cannot be formally excluded. Further, we have identified a 13-nucleotide purine-rich element from a constitutive exon that, when placed in exon 4, entirely switches splice site usage in CGRP-producing cells. These data suggest that specific exon recruitment sequences, in combination with other constitutive elements, serve an important function in exon recognition. These results are consistent with the hypothesis that tissue-specific alternative splicing of the calcitonin/CGRP primary transcript is mediated by cell-specific differences in components of the constitutive splicing machinery.

Alternative Splicing↗
PubMed1997

Constitutive activation of gene expression by thyroid hormone receptor results from reversal of p53-mediated repression.

Thyroid hormone receptor (T3R) is a member of the steroid hormone receptor gene family of nuclear hormone receptors. In most cells T3R activates gene expression only in the presence of its ligand, L-triiodothyronine (T3). However, in certain cell types (e.g., GH4C1 cells) expression of T3R leads to hormone-independent constitutive activation. This activation by unliganded T3R occurs with a variety of gene promoters and appears to be independent of the binding of T3R to specific thyroid hormone response elements (TREs). Previous studies indicate that this constitutive activation results from the titration of an inhibitor of transcription. Since the tumor suppresser p53 is capable of repressing a wide variety of gene promoters, we considered the possibility that the inhibitor is p53. Evidence to support this comes from studies indicating that expression of p53 blocks T3R-mediated constitutive activation in GH4C1 cells. In contrast with hormone-independent activation by T3R, p53 had little or no effect on T3-dependent stimulation which requires TREs. In addition, p53 mutants which oligomerize with wild-type p53 and interfere with its function also increase promoter activity. This enhancement is of similar magnitude to but is not additive with the stimulation mediated by unliganded T3R, suggesting that they target the same factor. Since p53 mutants are known to target wild-type p53 in the cell, this suggests that T3R also interacts with p53 in vivo and that endogenous levels of p53 act to suppress promoter activity. Evidence supporting both functional and physical interactions of T3R and p53 in the cell is presented. The DNA binding domain (DBD) of T3R is important in mediating constitutive activation, and the receptor DBD appears to functionally interact with the N terminus of p53 in the cell. In vitro binding studies indicate that the T3R DBD is important for interaction of T3R with p53 and that this interaction is reduced by T3. These findings are consistent with the in vivo studies indicating that p53 blocks constitutive activation but not ligand-dependent stimulation. These studies provide insight into mechanisms by which unliganded nuclear hormone receptors can modulate gene expression and may provide an explanation for the mechanism of action of the v-erbA oncoprotein, a retroviral homolog of chicken T3R alpha.

Animals↗
PubMed1997

Repression of the heat shock factor 1 transcriptional activation domain is modulated by constitutive phosphorylation.

Heat shock transcription factor 1 (HSF1) is constitutively expressed in mammalian cells and negatively regulated for DNA binding and transcriptional activity. Upon exposure to heat shock and other forms of chemical and physiological stress, these activities of HSF1 are rapidly induced. In this report, we demonstrate that constitutive phosphorylation of HSF1 at serine residues distal to the transcriptional activation domain functions to repress transactivation. Tryptic phosphopeptide analysis of a collection of chimeric GAL4-HSF1 deletion and point mutants identified a region of constitutive phosphorylation encompassing serine residues 303 and 307. The significance of phosphorylation at serines 303 and 307 in the regulation of HSF1 transcriptional activity was demonstrated by transient transfection and assay of a chloramphenicol acetyltransferase reporter construct. Whereas the transfected wild-type GAL4-HSF1 chimera is repressed for transcriptional activity and derepressed by heat shock, mutation of serines 303 and 307 to alanine results in derepression to a high level of constitutive activity. Similar results were obtained with mutation of these serine residues in the context of full-length HSF1. These data reveal that constitutive phosphorylation of serines 303 and 307 has an important role in the negative regulation of HSF1 transcriptional activity at control temperatures.

3T3 Cells↗
PubMed1997

Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth.

Breast cancers often progress from a hormone-dependent, nonmetastatic, antiestrogen-sensitive phenotype to a hormone-independent, antiestrogen- and chemotherapy-resistant phenotype with highly invasive and metastatic growth properties. This progression is usually accompanied by altered function of the estrogen receptor (ER) or outgrowth of ER-negative cancer cells. To understand the molecular mechanisms responsible for metastatic growth of ER-negative breast cancers, the activities of the transcription factor NF-kappaB (which modulates the expression of genes involved in cell proliferation, differentiation, apoptosis, and metastasis) were compared in ER-positive (MCF-7 and T47-D) and ER-negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines. NF-kappaB, which is usually maintained in an inactive state by protein-protein interaction with inhibitor IkappaBs, was found to be constitutively active in ER-negative breast cancer cell lines. Constitutive DNA binding of NF-kappaB was also observed with extracts from ER-negative, poorly differentiated primary breast tumors. Progression of the rat mammary carcinoma cell line RM22-F5 from an ER-positive, nonmalignant phenotype (E phenotype) to an ER-negative, malignant phenotype (F phenotype) was also accompanied by constitutive activation of NF-kappaB. Analysis of individual subunits of NF-kappaB revealed that all ER-negative cell lines, including RM22-F5 cells of F phenotype, contain a unique 37-kDa protein which is antigenically related to the RelA subunit. Cell-type-specific differences in IkappaB alpha, -beta, and -gamma were also observed. In transient-transfection experiments, constitutive activity of an NF-kappaB-dependent promoter was observed in MDA-MB-231 and RM22-F5 cells of F phenotype, and this activity was efficiently repressed by cotransfected ER. Since ER inhibits the constitutive as well as inducible activation function of NF-kappaB in a dose-dependent manner, we propose that breast cancers that lack functional ER overexpress NF-kappaB-regulated genes. Furthermore, since recent data indicate that NF-kappaB protects cells from tumor necrosis factor alpha-, ionizing radiation-, and chemotherapeutic agent daunorubicin-mediated apoptosis, our results provide an explanation for chemotherapeutic resistance in ER-negative breast cancers.

Animals↗
PubMed2004

Persistent activation by constitutive Ste7 promotes Kss1-mediated invasive growth but fails to support Fus3-dependent mating in yeast.

Mitogen-activated protein kinase kinase kinase-Ste11 (MAPKKK-Ste11), MAPKK-Ste7, and MAPK-Kss1 mediate pheromone-induced mating differentiation and nutrient-responsive invasive growth in Saccharomyces cerevisiae. The mating pathway also requires the scaffold-Ste5 and the additional MAPK-Fus3. One contribution to specificity in this system is thought to come from stimulus-dependent recruitment of the MAPK cascade to upstream activators that are unique to one or the other pathway. To test this premise, we asked if stimulus-independent signaling by constitutive Ste7 would lead to a loss of biological specificity. Instead, we found that constitutive Ste7 promotes invasion without supporting mating responses. This specificity occurs because constitutive Ste7 activates Kss1, but not Fus3, in vivo and promotes filamentation gene expression while suppressing mating gene expression. Differences in the ability of constitutive Ste7 variants to bind the MAPKs and Ste5 account for the selective activation of Kss1. These findings support the model that Fus3 activation in vivo requires binding to both Ste7 and the scaffold-Ste5 but that Kss1 activation is independent of Ste5. This scaffold-independent activation of Kss1 by constitutive Ste7 and the existence of mechanisms for pathway-specific promoter discrimination impose a unique developmental fate independently of any distinguishing external stimuli.

Adaptor Proteins, Signal Transducing↗
PubMed1988

Constitutive expression of the maltose fermentative enzymes in Saccharomyces carlsbergensis is dependent upon the mutational activation of a nonessential homolog of MAL63.

Maltose fermentation in Saccharomyces carlsbergensis is dependent upon the MAL6 locus. This complex locus is composed of the MAL61 and MAL62 genes, which encode maltose permease and maltase, respectively, and a third gene, MAL63, which codes for a trans-acting positive regulatory product. In wild-type strains, expression of the MAL61 and MAL62 mRNAs and proteins is induced by maltose and induction is dependent upon the MAL63 gene. Mutants constitutively expressing the MAL61 and MAL62 gene products have been isolated in mal63 backgrounds, and the mutations which have been analyzed map to a fourth MAL6-linked gene, MAL64. Cloning and characterization of this new gene are described in this report. The results revealed that the MAL64-C alleles present in constitutive strains encode a trans-acting positive function required for constitutive expression of the MAL61 and MAL62 gene products. In inducible strains, the MAL64 gene is dispensable, as deletion of the gene had no effect on maltose fermentation or maltose-regulated induction. MAL64 encoded transcripts of 2.0 and 1.4 kilobase pairs. While both MAL64 mRNAs were constitutively expressed in constitutive strains, they were maltose inducible in wild-type strains and induction was dependent upon the MAL63 gene. The MAL63 and MAL64 genes are at least partially structurally homologous, suggesting that they control MAL61 and MAL62 transcript accumulation by similar mechanisms.

Cloning, Molecular↗
PubMed1989

Isolation of constitutive mutations affecting the proline utilization pathway in Saccharomyces cerevisiae and molecular analysis of the PUT3 transcriptional activator.

The enzymes of the proline utilization pathway (the products of the PUT1 and PUT2 genes) in Saccharomyces cerevisiae are coordinately regulated by proline and the PUT3 transcriptional activator. To learn more about the control of this pathway, constitutive mutations in PUT3 as well as in other regulators were sought. A scheme using a gene fusion between PUT1 (S. cerevisiae proline oxidase) and galK (Escherichia coli galactokinase) was developed to select directly for constitutive mutations affecting the PUT1 promoter. These mutations were secondarily screened for their effects in trans on the promoter of the PUT2 (delta 1-pyrroline-5-carboxylate dehydrogenase) gene by using a PUT2-lacZ (E. coli beta-galactosidase) gene fusion. Three different classes of mutations were isolated. The major class consisted of semidominant constitutive PUT3 mutations that caused PUT2-lacZ expression to vary from 2 to 22 times the uninduced level. A single dominant mutation in a new locus called PUT5 resulted in low-level constitutive expression of PUT2-lacZ; this mutation was epistatic to the recessive, noninducible put3-75 allele. Recessive constitutive mutations were isolated that had pleiotropic growth defects; it is possible that these mutations are not specific to the proline utilization pathway but may be in genes that control several pathways. Since the PUT3 gene appears to have a major role in the regulation of this pathway, a molecular analysis was undertaken. This gene was cloned by functional complementation of the put3-75 mutation. Strains carrying a complete deletion of this gene are viable, proline nonutilizing, and indistinguishable in phenotype from the original put3-75 allele. The PUT3 gene encodes a 2.8-kilobase-pair transcript that is not regulated by proline at the level of RNA accumulation. The presence of the gene on a high-copy-number plasmid did not alter the regulation of one of its target genes, PUT2-lacZ, suggesting that the PUT3 gene product is not limiting and that a titratable repressor is not involved in the regulation of this pathway.

Cloning, Molecular↗
PubMed2001

Studies on the psychosomatic functioning of ill-health according to Eastern and Western medicine 5. Psychosomatic characteristics of anxiety and anxiety-affinitive constitution.

In successive studies of the psychosomatic functioning of ill-health according to Oriental and Western medicine in medical students, we established the existence of the psychosomatic characteristics we have provisionally termed the anxiety-affinitve constitution at the core of ill-health. Therefore, we conducted this research because our previous investigation showed this constitution included a high complexity of respiratory movement and eye movement with a significant correlation to the State Trait Anxiety Inventory (STAI). We examined the correlation between the STAI and somatic function of 88 medical students to identify the psychosomatic characteristics of anxiety and the anxiety-affinitive constitution. These tests included STAI, fractal (EEG, EOG, plethysmogram, respiratory curves, and EMG) and non-fractal (accelerated plethysmogram) dimension analyses, and malocclusion (based on Angle's classification). In particular, EOG, plethysmogram, and respiratory curves are known to have close association with trait anxiety. We were able to discover the correlation between (1) trait anxiety and thoracic and abdominal respiratory movements, and malocclusion (Class III), and (2) the correlation of state anxiety with thoracic respiratory movement, horizontal eye movement, a plethysmogram and an EEG-Pz (in males only). In subsequent study the relation between thoracic dominance and state-trait anxiety and between abdominal dominance and state-trait anxiety should be assessed to develop this research regarding the psychosomatic characteristics of anxiety and the anxiety-affinitive constitution. Further, it is essential to create an anxiety-affinitve constitution index based on multi-regression analysis.

Adult↗
PubMed2007

Pharmacogenomic and structural analysis of constitutive g protein-coupled receptor activity.

G protein-coupled receptors (GPCRs) respond to a chemically diverse plethora of signal transduction molecules. The notion that GPCRs also signal without an external chemical trigger, i.e., in a constitutive or spontaneous manner, resulted in a paradigm shift in the field of GPCR pharmacology. The discovery of constitutive GPCR activity and the fact that GPCR binding and signaling can be strongly affected by a single point mutation drew attention to the evolving area of GPCR pharmacogenomics. For a variety of GPCRs, point mutations have been convincingly linked to human disease. Mutations within conserved motifs, known to be involved in GPCR activation, might explain the properties of some naturally occurring, constitutively active GPCR variants linked to disease. In this review, we provide a brief historical introduction to the concept of constitutive receptor activity and the pharmacogenomic and structural aspects of constitutive receptor activity.

Humans↗
PubMed1995

Substitution of an aspartic acid results in constitutive activation of c-kit receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3.

The c-kit protooncogene encodes a receptor tyrosine kinase that mediates signals required for differentiation, proliferation and survival of mast cells. We have already shown the constitutive activation of c-kit receptor tyrosine kinase (KIT) in a human mast cell leukemia line (HMC-1) and a murine mastocytoma cell line (P-815). We here examined whether such constitutive activation of KIT occurred in the rat tumor mast cell line RBL-2H3 as well, which is frequently used as a tool for studying functions of mast cells. In RBL-2H3 cells, KIT was constitutively phosphorylated on tyrosine and activated in the absence of autocrine production of its ligand, stem cell factor (SCF). Sequencing analysis revealed that one of c-kit genes of RBL-2H3 cells had a point mutation, resulting in amino acid substitution of Tyr for Asp in codon 817. When rat wild-type c-kit cDNA and mutant-type c-kit cDNA encoding KITTyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KITTyr817 was constitutively phosphorylated on tyrosine and activated in the absence of SCF. Since mutations at the same Asp codon constitutively activated KIT in all the human HMC-1, murine P-815, and rat RBL-2H3 cell lines, and since the incorporation of antisense oligonucleotides of c-kit messenger RNA significantly suppressed the proliferation of RBL-2H3 cells, the activating mutations in the Asp codon of the c-kit gene appeared to be involved in neoplastic growth of mast cells.

Amino Acid Sequence↗
PubMed2001

Constitutive and inducible expression of Cyp1a1 and Cyp1b1 in vascular smooth muscle cells: role of the Ahr bHLH/PAS transcription factor.

Ahr is a ligand-activated bHLH/PAS transcription factor involved in cytochrome P450 (CYP) gene regulation and murine susceptibility to atherogenic stimuli. The present studies were conducted to examine constitutive and inducible expression of Cyp1a1 and Cyp1b1 in vascular smooth muscle cells (VSMCs) from Ahr(+/+) and Ahr(-/-) mice. Cyp1a1 mRNA was not expressed constitutively in VSMCs irrespective of Ahr phenotype. Although Cyp1a1 was inducible in Ahr(+/+) by 3 micromol/L benzo(a)pyrene, a known hydrocarbon inducer, the protein was uninducible. In contrast, Cyp1b1 mRNA and protein were expressed under constitutive and inducible conditions irrespective of Ahr phenotype or growth status. CYP-encoded aryl hydrocarbon hydroxylase activity was higher in Ahr(-/-) VSMCs under constitutive conditions and induced by benzo(a)pyrene in Ahr(+/+) and Ahr(-/-) VSMCs. CYP expression was influenced by mitogenic status, because randomly cycling cells consistently exhibited higher levels than growth-arrested counterparts. Actinomycin D (2 microgram/mL) or cycloheximide (10 micromol/L) did not inhibit constitutive or hydrocarbon-inducible aryl hydrocarbon hydroxylase activity in VSMCs. These data indicate that in murine VSMCs, expression of Cyp1al and Cyp1b1 is differentially influenced by Ahr phenotype and mitogenic status, with patterns that may dictate inherent susceptibility to atherogenic stimuli.

Animals↗
PubMed2003

Constitutive p40 promoter activation and IL-23 production in the terminal ileum mediated by dendritic cells.

IL-12 p40-related cytokines such as IL-12 p35/p40 heterodimer and IL-23 (p19/p40) are potent regulators of adaptive immune responses. Little is known, however, about the transcriptional regulation of the p40 gene in vivo. In an attempt toward this goal, we have generated transgenic mice expressing firefly luciferase under the control of the IL-12 p40 promoter. High constitutive transgene expression was found in the small intestine only, whereas little reporter gene activity was observed in other tissues. Within the small bowel, constitutive promoter activity was restricted to the terminal ileum and associated with high expression of p40 mRNA as well as p40 and IL-23 p19/p40 proteins. The cells constitutively producing IL-12 p40 were identified as CD8alpha and CD11b double-negative CD11c+ lamina propria dendritic cells (LPDCs) that represent a major cell population in the lamina propria of the small intestine, but not in the colon. FISH directly demonstrated the uptake of bacteria by a subset of LPDCs in the terminal ileum that was associated with p40 expression. Furthermore, little or no p40 protein expression in LPDCs was found in the terminal ileum of germfree mice, indicating a key role of the intestinal flora for constitutive p40 expression. In addition, analysis of transgenic mice with a mutated NF-kappaB target site in the p40 promoter showed a critical role of NF-kappaB for constitutive transgene expression. Our data reveal important functional differences between the mucosal immune systems of the small and large bowel in healthy mice and suggest that the high bacterial load in the terminal ileum activates p40 gene transcription in LPDCs through NF-kappaB. These data suggest a predisposition of the terminal ileum to develop chronic inflammatory responses through IL-23 and thus may provide a molecular explanation for the preferential clinical manifestation of Crohn disease in this part of the gut.

Animals↗
PubMed2004

Constitutive activity of the melanocortin-4 receptor is maintained by its N-terminal domain and plays a role in energy homeostasis in humans.

The melanocortin-4 receptor (MC4R), a centrally expressed G protein-coupled receptor (GPCR), is essential for the maintenance of long-term energy balance in humans. Mutations in MC4R are the most common genetic cause of obesity. Since activation of this receptor leads to a decrease in food intake, MC4R is also a major therapeutic target for the treatment of obesity. Control of MC4R activity in vivo is modulated by the opposing effects of the anorexigenic agonist alpha-melanocyte-stimulating hormone (alpha-MSH) and the orexigenic antagonist agouti-related protein (AGRP). In addition, experiments in vitro have demonstrated that the human MC4R has an intrinsic constitutive activity on which AGRP also acts as an inverse agonist. The physiological role of this constitutive activity in the control of energy balance as well as the domain of the protein implicated in its maintenance are unknown. By systematically studying functional defects in naturally occurring MC4R mutations from obese patients, we defined a cluster of N-terminal mutations that selectively impair the constitutive activity of the receptor. Further characterization of this domain demonstrated that it functions as a tethered intramolecular ligand that maintains the constitutive activity of MC4R and may provide novel avenues for the design of drugs targeting this receptor. Our results also suggest that the tonic satiety signal provided by the constitutive activity of MC4R may be required for maintaining long-term energy homeostasis in humans.

Agouti-Related Protein↗
PubMed2005

Elevated expression of CD30 in adult T-cell leukemia cell lines: possible role in constitutive NF-kappaB activation.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) is associated with the development of adult T-cell leukemia (ATL). HTLV-1 encoded Tax1 oncoprotein activates the transcription of genes involved in cell growth and anti-apoptosis through the NF-kappaB pathway, and is thought to play a critical role in the pathogenesis of ATL. While Tax1 expression is usually lost or minimal in ATL cells, these cells still show high constitutive NF-kappaB activity, indicating that genetic or epigenetic changes in ATL cells induce activation independent of Tax1. The aim of this study was to identify the molecules responsible for the constitutive activation of NF-kappaB in ATL cells using a retroviral functional cloning strategy. RESULTS: Using enhanced green fluorescent protein (EGFP) expression and blasticidin-resistance as selection markers, several retroviral cDNA clones exhibiting constitutive NF-kappaB activity in Rat-1 cells, including full-length CD30, were obtained from an ATL cell line. Exogenous stable expression of CD30 in Rat-1 cells constitutively activated NF-kappaB. Elevated expression of CD30 was identified in all ATL lines examined, and primary ATL cells from a small number of patients (8 out of 66 cases). CONCLUSION: Elevated CD30 expression is considered one of the causes of constitutive NF-kappaB activation in ATL cells, and may be involved in ATL development.

Animals↗
PubMed2005

Constitutive degradation of IkappaBalpha in human T lymphocytes is mediated by calpain.

BACKGROUND: Activation-induced induction of transcription factor NFkappaB in T lymphocytes is regulated by its inhibitor IkappaBalpha. NFkappaB activation has been demonstrated to occur either by phosphorylation on serine residues 32 and 36 of the inhibitor, IkappaBalpha, followed by ubiquitination and degradation of the inhibitor by the 26S proteasome, or by a proteasome-independent mechanism involving tyrosine phosphorylation, but not degradation. However, the mechanism underlying constitutive regulation of the levels of the inhibitor, IkappaB, in primary human T lymphocytes, remains to be fully delineated. RESULTS: We demonstrate here, the involvement of a proteasome-independent pathway for constitutive regulation of IkappaBalpha levels in primary human T lymphocytes. Pretreatment with a cell permeable calpain inhibitor, E64D, but not with a proteasome specific inhibitor, lactacystin, blocks stimulus-independent IkappaBalpha degradation in primary human T cells. However, E64D pre-treatment fails to impact on IkappaBalpha levels following stimulation with either TNFalpha or pervanadate. Other isoforms of the inhibitor, IkappaBbeta, and IkappaBgamma, appear not to be subject to a similar ligand-independent regulation. Unlike the previously reported decline in ligand-induced degradation of IkappaBalpha in T cells from the elderly, constitutive degradation does not exhibit an age-associated decline, demonstrating proteasome-independent regulation of the activity. CONCLUSION: Our studies support a role for an E64D sensitive protease in regulating constitutive levels of IkappaBalpha in T cells, independent of the involvement of the 26S proteasome, and suggests a biological role for constitutive degradation of IkappaBalpha in T cells.

Journal Article↗