Biomedical subjects

C Lemercier

Publications and source records attributed to C Lemercier.

At least 19 recordsLinked to original sources

PubMed2001

Functional significance of histone deacetylase diversity.

Nucleocytoplasmic shuttling of histone deacetylases is emerging as a major step in determining the composition, and hence the activity, of the corresponding nuclear regulatory complexes. This shuttling process is one of the distinctive characteristics of these enzymes, themselves belonging to structurally and functionally different classes. Considering the specific features of each class of deacetylases, it is possible to determine how each member can contribute to particular cellular functions.

Animals↗

PubMed2001

Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways.

The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.

Acetylation↗

PubMed2000

Active maintenance of mHDA2/mHDAC6 histone-deacetylase in the cytoplasm.

The intracellular localization, and thereby the function, of a number of key regulator proteins tagged with a short leucine-rich motif (the nuclear export signal or NES) is controlled by CRM1/exportin1, which is involved in the export of these proteins from the nucleus [1]. A common characteristic of these regulators is their transient action in the nucleus during either a specific phase of the cell cycle or in response to specific signals [1]. Here, we show that a particular member of the class II histone-deacetylases mHDA2/mHDAC6 [2] belongs to this family of cellular regulators that are present predominantly in the cytoplasm, but are also capable of shuttling between the nucleus and the cytoplasm. A very potent NES present at the amino terminus of mHDAC6 was found to play an essential role in this shuttling process. The sub-cellular localization of mHDAC6 appeared to be controlled by specific signals, since the arrest of cell proliferation was found to be associated with the translocation of a fraction of the protein into the nucleus. Data presented here suggest that mHDAC6 might be the first member of a functionally distinct class of deacetylases, responsible for activities not shared by other known histone deacetylases.

Amino Acid Sequence↗

PubMed2000

mHDA1/HDAC5 histone deacetylase interacts with and represses MEF2A transcriptional activity.

Recently we identified a new family of histone deacetylases in higher eukaryotes related to yeast HDA1 and showed their differentiation-dependent expression. Data presented here indicate that HDAC5 (previously named mHDA1), one member of this family, might be a potent regulator of cell differentiation by interacting specifically with determinant transcription factors. We found that HDAC5 was able to interact in vivo and in vitro with MEF2A, a MADS box transcription factor, and to strongly inhibit its transcriptional activity. Surprisingly, this repression was independent of HDAC5 deacetylase domain. The N-terminal non-deacetylase domain of HDAC5 was able to ensure an efficient repression of MEF2A-dependent transcription. We then mapped protein domains involved in the HDAC5-MEF2A interaction and showed that MADS box/MEF2-domain region of MEF2A interacts specifically with a limited region in the N-terminal part of HDAC5 which also possesses a distinct repressor domain. These data show that two independent class II histone deacetylases HDAC4 and HDAC5 are able to interact with members of the MEF2 transcription factor family and regulate their transcriptional activity, thus suggesting a critical role for these deacetylases in the control of cell proliferation/differentiation.

Blotting, Western↗

PubMed2000

The rat Mist1 gene: structure and promoter characterization.

Transcription factors of the basic Helix-Loop-Helix (bHLH) protein family play key roles in several developmental processes. Mist1 belongs to this group of proteins and shares several properties with the other family members. For example, Mist1 is capable of dimerization with the ubiquitously expressed E2A bHLH proteins and exhibits a strong DNA-binding activity to the core E-box sequence. Using in-situ hybridization and Northern blot hybridization, Mist1 mRNA has been detected in a variety of embryonic and adult rodent tissues. To understand the molecular mechanisms involved in the expression of the gene, we have cloned the rat Mist1 gene and analyzed 2.5 kb of its 5' flanking region. The Mist1 gene spans over 5 kilobases and is composed of two exons separated by a unique intron. The entire coding region is localized in the second exon. Sequence analysis of the promoter region indicated an absence of TATA-box or CAAT-box sequence, but several consensus Sp1-binding sites were present near the transcription start site. Deletion analysis of the promoter region identified a 272 bp proximal fragment to be sufficient to drive expression of a reporter gene in NIH3T3 fibroblasts. Subsequent deletion of potential Sp1 sites results in a marked decrease in promoter activity. Electrophoretic mobility shift assays revealed that Sp1 binds to two different regions in the proximal promoter, a typical Sp1 site located at (-38; -33) and a G/C-rich region between (-67; -62). These data suggest that the basal expression of this TATA-less gene might be driven by general transcription factors, such as Sp1.

3T3 Cells↗

PubMed2000

Expression of Flt3-ligand by the endothelial cell.

Flt3-ligand (FL) is a cytokine that is of paramount importance in the proliferation of primitive hematopoietic progenitors. In this study, we show that endothelial cells (EC) produce large amounts of soluble FL and express a membrane-bound form of the molecule. Bone marrow microvascular EC also produce FL, suggesting that EC are an important source of FL in the bone marrow. High concentrations of FL in EC supernatants contrast with its undetectable levels in long-term bone marrow cultures. A single mRNA for FL is detected, suggesting that soluble FL derives from the membrane-bound species by proteolytic release. FL mRNA is stable with a half-life of about 3 h. II-1alpha increases FL mRNA levels and membrane and soluble FL expression. Glucocorticoids, known inhibitors for many hematopoietic growth factors do not down-regulate the expression of FL. On the contrary, GC increase the expression of both species of FL. The neutralization of FL in cocultures EC/ hematopoietic progenitors results in an acceleration of the maturation of the progenitors. IFN-alpha, MIP-1 alpha and TGF-beta stimulate production of membrane-bound and soluble FL. This stimulation is essential to explain their modulatory effect on the generation of clonogenic cells in cocultures EC/hematopoietic progenitors. Leukemia (2000) 14, 153-162.

Base Sequence↗

PubMed2000

Involvement of retinoblastoma protein and HBP1 in histone H1(0) gene expression.

The histone H1(0)-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H1(0) promoter, a specific regulatory element, which we named the H4 box, exhibits features which implicate a role in mediating H1(0) gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H1(0) H4 box were therefore expected to link differentiation-dependent expression of H1(0) to the cell cycle control machinery. The aim of this work was to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, our cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacted specifically with the H1(0) H4 box and moreover was expressed in a differentiation-dependent manner. We also showed that the HBP1-encoding gene is able to produce different forms of HBP1. Finally, we demonstrated that both HBP1 and RB were involved in the activation of H1(0) gene expression. We therefore propose that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H1(0), HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells.

Adult↗

PubMed1999

Leukaemia inhibitory factor expression is inhibited by glucocorticoids through post-transcriptional mechanisms.

Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine which is involved in the regulation of the immune response and haematopoiesis. The authors investigated the regulation of the expression of LIF by glucocorticosteroids (GC). Endothelial cells (EC) constitutively produce LIF and this production is enhanced by interleukin 1 (IL-1). GC were found to inhibit the basal production of LIF by EC and to suppress its IL-1-induced augmentation. Whether corticosteroids suppress LIF production by blocking transcription of LIF mRNA, or by blocking LIF synthesis at a post-transcriptional level was examined. Northern blot hybridization analysis demonstrated that GC act mainly by decreasing the LIF mRNA level. In the presence of translation inhibitors a superinduction of LIF mRNA was observed. Dexamethasone (DEX) at a concentration of 1 microM was responsible for a rapid increase in the degradation rate of LIF mRNA which resulted in reducing its level by more than 50% within 2 h, whereas the transcription rate of LIF gene was not significantly altered in these conditions. These results demonstrated that GC inhibit LIF mRNA expression mainly by increasing the turnover rate of the LIF mRNA. The early LIF mRNA destabilizing activity of GC was translation dependent as shown by experiments with protein translation inhibitors. The results indicate that corticosteroids are inhibitors of LIF expression and that this inhibition mainly occurs through post-transcriptional mechanisms.

Actins↗

PubMed1999

Cloning of the murine Mist1 gene and assignment to mouse chromosome band 5G2-5G3.

Mist1 is a basic helix-loop-helix (bHLH) transcription factor that is highly expressed in the adult pancreas. In this study, we isolated the mouse Mist1 gene and established its primary DNA sequence and complete genomic structure. Fluorescence in situ hybridization mapping located the Mist1 gene to the telomere of mouse chromosome 5 at position 5G2-5G3, an area which is syntenic to human chromosome 13q and which contains several additional pancreatic regulatory genes including IPF1 and CDX2.

Amino Acid Sequence↗

PubMed1998

The basic helix-loop-helix transcription factor Mist1 functions as a transcriptional repressor of myoD.

A good model system to examine aspects of positive and negative transcriptional regulation is the muscle-specific regulatory factor, MyoD, which is a basic helix-loop-helix (bHLH) transcription factor. Although MyoD has the ability to induce skeletal muscle terminal differentiation in a variety of non-muscle cell types, MyoD activity itself is highly regulated through protein-protein interactions involving several different co-factors. Here we describe the characterization of a novel bHLH protein, Mist1, and how it influences MyoD function. We show that Mist1 accumulates in myogenic stem cells (myoblasts) and then decreases as myoblasts differentiate into myotubes. Mist1 functions as a negative regulator of MyoD activity, preventing muscle differentiation and the concomitant expression of muscle-specific genes. Mist1-induced inhibition occurs through a combination of mechanisms, including the formation of inactive MyoD-Mist1 heterodimers and occupancy of specific E-box target sites by Mist1 homodimers. Mist1 lacks a classic transcription activation domain and instead possesses an N-terminal repressor region capable of inhibiting heterologous activators. Thus, Mist1 may represent a new class of repressor molecules that play a role in controlling the transcriptional activity of MyoD, ensuring that expanding myoblast populations remain undifferentiated during early embryonic muscle formation.

Amino Acid Sequence↗

PubMed1997

Mist1: a novel basic helix-loop-helix transcription factor exhibits a developmentally regulated expression pattern.

Basic helix-loop-helix (bHLH) proteins often belong to a family of transcription factors that bind to the DNA target sequence -CANNTG- (E-box) that is present in the promoter or enhancer regions of numerous developmentally regulated genes. In this study, we report the isolation and initial characterization of a novel bHLH factor, termed Mist1, that was identified by virtue of its ability to interact with E-box regulatory elements in a yeast "one-hybrid" screening procedure. Northern analysis revealed that Mist1 transcripts are expressed in several adult tissues, including stomach, liver, lung, and spleen but no expression is detected in the heart, brain, kidney, or testis. During mouse embryogenesis, Mist1 mRNA is first observed at E10.5 in the primitive gut and in the developing lung bud. Expression persists through E16.5 and remains restricted primarily to the epithelial lining. Mist1 also is detected in skeletal muscle tissues beginning at E12.5, persisting throughout all embryonic stages examined although in older embryos and in the adult expression becomes severely reduced. At later developmental times, Mist1 transcripts also are found in the pancreas, submandibular gland, and adult spleen. As predicted, the Mist1 protein is nuclear and binds efficiently to E-box sites as a homodimer. Mist1 also is capable of binding to E-box elements when complexed as a heterodimer with the widely expressed E-proteins, E12 and E47. Surprisingly, although Mist1 binds to E-boxes in vivo, the Mist1 protein lacks a functional transcription activation domain. These observations suggest that Mist1 may function as a unique regulator of gene expression in several different embryonic and postnatal cell lineages.

Amino Acid Sequence↗

PubMed1996

Examination of mammalian basic helix-loop-helix transcription factors using a yeast one-hybrid system.

Basic helix-loop-helix (bHLH) transcription factors play diverse roles in controlling many developmental events. Although a great deal is understood about how bHLH factors activate gene transcription via E-box DNA consensus sequences, studies of bHLH factor function in higher eukaryotes often have been hindered by the presence of multiple family members. As a first step in developing a simplified in vivo system to examine bHLH factor activities, we examined whether the bHLH muscle regulatory factors MRF4 and MyoD function appropriately in yeast. We show that Gal4-MRF4 fusion proteins, or native MRF4 proteins, activate expression of an E-box HIS3 reporter gene whereas MyoD proteins remain inactive. Deletion of the MRF4 transcription activation domain (TAD) or point mutations that abolish MRF4 DNA interactions inhibit HIS3 expression. Substitution of the MRF4 TAD with the Gal4 TAD also produces a functional protein, demonstrating that these transcription activation domains are functionally equivalent in yeast. Replacement of the MRF4 TAD with the related MyoD TAD, however, generates an inactive protein, suggesting that some specificity exists between bHLH family members. Using this experimental system, we also demonstrate that mammalian cDNA libraries can be screened successfully for cDNAs encoding novel bHLH proteins that interact with E-box targets. Thus, this in vivo yeast system provides a novel approach to facilitate functional studies of bHLH factor regulation.

Animals↗

PubMed1995

Activation of the endothelium by IL-1 alpha and glucocorticoids results in major increase of complement C3 and factor B production and generation of C3a.

Constitutive secretion of complement C3 and factor B by the endothelial cell (EC) is lowered by therapeutic concentrations of glucocorticoids such as hydrocortisone or dexamethasone, whereas regulatory protein factor H production is increased by these hormones. In contrast, the proinflammatory cytokine IL-1 alpha has a stimulatory effect on C3 and factor B secretion by the endothelium and an inhibitory effect on factor H secretion. In this study, we examined the combined effect of IL-1 alpha and glucocorticoids on C3 and factor B expression by the endothelial cell. When dexamethasone or hydrocortisone were added to IL-1 alpha, significant potentialization of IL-1 alpha-induced stimulation of C3 and factor B production was observed, occurring at various concentrations of either stimuli. Dose-response experiments indicate that, in vitro, optimal concentrations are in the range of 10(-7) to 10(-5) M for dexamethasone and 50-200 U for IL-1 alpha. In contrast, dexamethasone counteracts, in an additive way, the inhibitory effect of IL-1 alpha on regulatory complement protein factor H production by EC. Such a potentialization between glucocorticoids and IL-1 alpha was not observed for another marker of endothelial activation, IL-1 alpha-induced stimulation of coagulation tissue factor expression. The association of glucocorticoids and IL-1 alpha therefore appears to be a specific and major stimulus for the secretion of complement C3 and factor B, two acute-phase proteins, by the endothelium. As a result of the in vitro endothelium stimulation by glucocorticoids and IL-1 alpha, C3a is generated in the vicinity of the endothelial cell. This study further suggests that complement activation, with its deleterious consequences, may result from the stimulation of endothelium in situations where high levels of IL-1 alpha and endogenous glucocorticoids coexist, such as in septic shock.

Cells, Cultured↗

PubMed1994

Expression of the components and regulatory proteins of the classical pathway of complement in normal and diseased synovium.

We studied the synthesis of the classical pathway complement components in synovial membrane. Ribonucleic acid was extracted from the synovial membranes of patients with rheumatoid arthritis (RA) or osteoarthritis (OA), as well as from normal synovial membrane. Northern blot and dot blot analysis showed that the mRNAs for all classical pathway complement components (C1qA chain, C1qB chain, C1qC chain, C1r, C1s, C4 and C2) and the fluid-phase regulatory components (C1-inhibitor, C4-bp and factor I) were present in all three types of synovial membrane. Thus, all the components of the classical pathway were expressed in normal and diseased synovium. In an attempt to determine which components were synthesised by each cell type, monocytes (mononuclear phagocytes), human umbilical vein endothelial cells (HUVEC), synovial membrane fibroblasts (from normal, OA and RA synovial membrane) and peripheral blood lymphocytes were cultured in vitro and secretion rates of individual components were measured and total cellular RNA was analysed by Northern blotting. Monocytes secreted C1q, C1r, C1s, C4, C2, C1-inhibitor and C4-bp but not factor I. Fibroblasts secreted C1r, C1s, C2, C3, C1-inhibitor and factor I but not C1q, C4 or C4-bp. HUVEC secreted C1s, C2, C1-inhibitor and factor I but not C1q, C1r, C4 or C4-bp. Lymphocytes did not secrete any of these components. In three instances mRNA was detected in the absence of secreted protein: mRNAs for the C1qA and C1qC chains were detected in HUVEC, whereas the mRNA for the C1qB chain was not, and C4 mRNA was detected in both fibroblasts and HUVEC.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthritis, Rheumatoid↗

PubMed1993

Expression of the components and regulatory proteins of the alternative complement pathway and the membrane attack complex in normal and diseased synovium.

We have studied synthesis of the complement components and regulatory proteins of the alternative pathway and the membrane attack complex in synovial membrane. RNA was extracted from synovial tissue of patients with rheumatoid arthritis (RA) or osteoarthritis (OA) as well as from normal synovial membrane. Dot blot analysis showed the presence of mRNAs for all the complement components and regulatory proteins (C3, factor B, factor D, C5, C6, C7, C9, factor H, factor I, S-protein, SP-40, 40, DAF, MCP, CR1, CD59), except for properdin, C8 alpha, C8 beta and C8 gamma in all three types of synovial membrane studied. In an attempt to determine which components were synthesised by each cell type, monocytes (mononuclear phagocytes), human umbilical vein endothelial cells (HUVEC), synovial membrane fibroblasts (from normal, OA and RA synovial membrane) and peripheral blood lymphocytes were cultured in vitro and secretion rates of individual components were measured and total cellular RNA analysed by northern blotting. Monocytes secreted properdin, C3, and factor H but not factor B, factor I, C5, C6, C7, C8 or C9. Fibroblasts and endothelial cells secreted factor B, factor H and factor I, but not properdin, C5, C6, C7, C8 or C9. Lymphocytes did not secrete any of these components. mRNAs encoding C3, factor B, factor H, S-protein, SP-40, 40, MCP and DAF were detected in all three other cell types (monocytes, fibroblasts and HU-VEC), but factor I and CD59 mRNAs were not detected in monocytes. C5, C6, C7, C8 alpha, C8 beta, CD8 gamma and C9 mRNAs were not detected in any of the cell types studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthritis, Rheumatoid↗

PubMed1993

Regulation of the synthesis of C1 subcomponents and C1-inhibitor.

We have investigated the synthesis of C1q, C1r, C1s and C1-inhibitor in HepG2 cells, human umbilical vein endothelial cells (HUVEC), fibroblasts (skin and synovial membrane), chondrocytes and monocytes. C1q was only synthesised by monocytes, although the mRNAs for the C1qA and C1qC chains were expressed in HUVEC. C1r, C1s and C1-inhibitor were synthesised by all cell types. The secretion rates of C1r and C1s were approximately equimolar in fibroblasts and chondrocytes whereas the secretion rate for C1s exceeded that for C1r in the other cell types. Molar ratios of C1s to C1r were approximately 2:1 for HepG2 cells, 5:1 for monocytes and 10:1 for HUVEC. Stimulation with interferon-gamma resulted in increased expression of all four proteins. The C1s:C1r ratio did not alter in chondrocytes or fibroblasts, but approached unity in HepG2, monocytes and HUVEC, due to relatively greater stimulation of C1r gene expression.

Carcinoma, Hepatocellular↗

PubMed1992

In vitro biosynthesis of complement factor I by human endothelial cells.

We have studied the secretion of the complement regulatory protein factor I by human umbilical vein endothelial cells (HUVEC). Northern and Western blot analysis and biosynthetic labeling experiments indicate that HUVEC secrete factor I at very low levels in basal conditions and that this secretion is significantly enhanced by interferon-gamma. Analysis of the proteolytic inactivation of C3b by HUVEC supernatants show that factor I is secreted in a functional form and can promote the specific proteolytic inactivation of C3b to iC3b. Together with previous studies establishing the secretion of complement factor H by HUVEC, this work demonstrates that the endothelial cell is able to secrete in its environment two complement regulatory proteins, factor I and factor H, which can mediate the degradation of C3b to iC3b. The secretion of factor I by HUVEC provides a useful in vitro model to analyze the modulation of this secretion and may be relevant to the local deposition of iC3b at the surface of the endothelium during the inflammatory reaction.

Cells, Cultured↗

PubMed1992

Differential modulation by glucocorticoids of alternative complement protein secretion in cells of the monocyte/macrophage lineage.

The effect of the synthetic glucocorticoid dexamethasone (DXM) on the secretion by human monocytes of alternative complement proteins C3, factor B and factor H was investigated. Results indicated that DXM modulates this secretion in a direction which would be consistent with its anti-inflammatory properties. DXM, at therapeutic concentrations, had a suppressive effect on C3 and factor B secretion and a stimulatory effect on factor H secretion by monocytes. This differential modulation on C3, factor B and factor H secretion was similar in mature macrophages. Together with previous studies showing that DXM had a suppressive effect on C3 and factor B secretion and a stimulatory effect on factor H secretion by human endothelial cells, our results indicate that DXM appears to have the general property of regulating local production of complement components so as to control complement activation.

Cell Differentiation↗