Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 46
Filter
Add more filters










Publication year range
1.
Oncogene ; 37(4): 512-521, 2018 01 25.
Article in English | MEDLINE | ID: mdl-28991225

ABSTRACT

Metabolic changes are linked to epigenetic reprogramming and play important roles in several tumor types. PGC-1α is a transcriptional coactivator controlling mitochondrial biogenesis and is linked to oxidative phosphorylation. We provide evidence that melanoma models with elevated PGC-1α levels are characteristic of the proliferative phenotype and are sensitive to bromodomain and extra-terminal domain (BET) inhibitor treatment. A super-enhancer region highly occupied by the BET family member BRD4 was identified for the PGC-1α gene. BET inhibitor treatment prevented this interaction, leading to a dramatic reduction of PGC-1α expression. Accordingly, BET inhibition diminished respiration and mitochondrial function in cells. In vivo, melanoma models with high PGC-1α expression strongly responded to BET inhibition by reduction of PGC-1α and impaired tumor growth. Altogether, our findings identify epigenetic regulatory elements that define a subset of melanomas with high sensitivity to BET inhibition, which opens up the opportunity to define melanoma patients most likely to respond to this treatment, depending on their tumor characteristics.


Subject(s)
Antineoplastic Agents/pharmacology , Enhancer Elements, Genetic/genetics , Melanoma/genetics , Nuclear Proteins/antagonists & inhibitors , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics , Transcription Factors/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Azepines/pharmacology , Azepines/therapeutic use , Benzodiazepines/pharmacology , Benzodiazepines/therapeutic use , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Drug Resistance, Neoplasm/genetics , Epigenesis, Genetic , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Melanoma/drug therapy , Melanoma/pathology , Mice , Mice, Inbred C57BL , Mice, Nude , Mitochondria/drug effects , Mitochondria/metabolism , Nuclear Proteins/metabolism , Patient Selection , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Protein Binding/genetics , SOXE Transcription Factors/genetics , SOXE Transcription Factors/metabolism , Skin Neoplasms/drug therapy , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Transcription Factors/metabolism , Treatment Outcome , Triazoles/pharmacology , Triazoles/therapeutic use , Xenograft Model Antitumor Assays
2.
Handb Exp Pharmacol ; (214): 543-87, 2012.
Article in English | MEDLINE | ID: mdl-23027466

ABSTRACT

Sex steroid receptors are ligand-triggered transcription factors. Oestrogen, progesterone and androgen receptors form, together with the glucocorticoid and mineralocorticoid receptors, a subgroup of the superfamily of nuclear receptors. They share a common mode of action, namely translating a hormone-i.e. a small-molecule signal-from outside to changes in gene expression and cell fate, and thereby represent "natural" pharmacological targets.For pharmacological therapy, these receptors have originally been addressed by hormones and synthetic hormone analogues in order to overcome pathologies related to deficiencies in the natural ligands. Another major use for female sex hormone receptor modulators is oral contraception, i.e. birth control.On the other side, blocking the activity of sex steroid receptors has become an established way to treat hormone-dependent malignancies, such as breast and prostate cancer.In this review, we will discuss how the experience gained from the classical pharmacology of these receptors and their molecular similarities led to new options for the treatment of gender-specific diseases and highlight recent progress in medicinal chemistry of sex hormone-modulating drugs.


Subject(s)
Hormone Antagonists/therapeutic use , Receptors, Androgen/drug effects , Receptors, Estrogen/drug effects , Receptors, Progesterone/drug effects , Breast Neoplasms/drug therapy , Female , Humans , Male , Prostatic Neoplasms/drug therapy , Receptors, Androgen/physiology , Receptors, Estrogen/physiology , Receptors, Progesterone/physiology , Selective Estrogen Receptor Modulators/therapeutic use
3.
Mol Cell Endocrinol ; 333(1): 37-46, 2011 Feb 10.
Article in English | MEDLINE | ID: mdl-21138753

ABSTRACT

The ovarian steroid hormone progesterone is essential for normal mammary gland physiology but may also play a role in breast cancer. Highly potent and selective antiprogestins may therefore represent a new treatment option for this disease. Here we studied the effects of the new antiprogestin Lonaprisan on the T47D breast cancer cell line. Strong inhibition of cell proliferation and arrest in the G0/G1 phase were observed, as well as induction of a senescence-like phenotype. This was accompanied by p21 induction through direct binding of Lonaprisan-bound progesterone receptor (PR) to the promoter. Reduction of p21 levels blunted the antiproliferative effects of Lonaprisan. Mutation analysis showed that intact PR DNA-binding properties were needed for p21 induction. Phosphorylation of PR Ser345 was stimulated by Lonaprisan, but this post-translational modification was not required for p21 promoter activation, nor was the interaction with c-Src needed. These results support the rationale for using antiprogestins in breast cancer treatment and warrant further studies to better understand their mode of action.


Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21/genetics , Estrenes/pharmacology , Progestins/antagonists & inhibitors , Antineoplastic Agents/metabolism , Apoptosis , Breast Neoplasms/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cellular Senescence/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Estrenes/metabolism , Female , Humans , Ligands , Mutation , Phosphorylation , Progesterone/antagonists & inhibitors , Promoter Regions, Genetic , RNA Interference , Receptors, Progesterone/genetics , Receptors, Progesterone/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sp1 Transcription Factor/metabolism
4.
Biomed Pharmacother ; 60(9): 520-8, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16949786

ABSTRACT

The multiple physiological functions of steroid hormones have been known for many years. The cloning of the steroid receptors in the mid-1980s led to the concept of ligand-activated transcription factors and to the identification of specific DNA response elements in the regulatory regions of target genes. The next main development was the identification of cofactors with activating or repressing functions, of which several act by modifying histones and locally affecting the chromatin structure. Work from several groups shows that the steroid receptors themselves can also be modified at various positions. Besides the long-known phosphorylation at tyrosines and serine/threonine residues, other covalent additions such as acetylation, ubiquitylation and sumoylation have been evidenced for steroid receptors in recent years. These modifications affect receptor stability and activity, and provide potential mechanisms for cell- or gene-specific regulation. A better understanding of the impact of these post-translational modifications (PTMs) on steroid receptor function should help in the identification of novel ligands with improved clinical profiles.


Subject(s)
Protein Processing, Post-Translational , Receptors, Steroid/metabolism , Acetylation , Animals , Humans , Phosphorylation , Receptors, Androgen/metabolism , Receptors, Estrogen/metabolism , Receptors, Glucocorticoid/metabolism , Receptors, Progesterone/metabolism , SUMO-1 Protein/metabolism , Ubiquitin/metabolism
7.
Biomed Pharmacother ; 56(2): 78-83, 2002 Mar.
Article in English | MEDLINE | ID: mdl-12000138

ABSTRACT

Androgens are essential for normal prostate growth and function but are also intimately associated with prostate cancer, an important cause of mortality in the ageing male population. The effects of androgens are mediated via a specific androgen receptor (AR) belonging to the nuclear receptor family and acting as a ligand-dependent transcription factor. The AR is built in a modular fashion and composed of a long N-terminal region with transactivation functions, a central DNA-binding domain, an intermediate hinge region and a C-terminal ligand-binding domain with additional transactivation functions. In its inactive form, the AR is complexed to heat-shock proteins, and mainly cytoplasmic. Following activation, the AR enters the nucleus, binds to its cognate DNA response elements as a homodimer and stimulates gene transcription. Various cofactors directly interact with the AR to modulate gene transcription. In addition, cross-talk between the AR and other signalling pathways has been proven for several prostate-expressed genes. Understanding the intricate networks underlying androgen-selective gene regulation represents a formidable challenge but might also offer the chance to identify new drug targets for the treatment of prostate carcinoma.


Subject(s)
Androgens/metabolism , Gene Expression Regulation , Prostate/metabolism , Humans , Male , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Protein Binding , Receptor Cross-Talk , Receptors, Androgen/metabolism , Response Elements , Signal Transduction
8.
Mol Endocrinol ; 15(10): 1803-16, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11579212

ABSTRACT

The Pem homeobox transcription factor is expressed under androgen control in the testis and epididymis. It is also transcribed in the ovary, muscle, and placenta. The mouse Pem gene promoter was cloned and sequenced. It was analyzed in transactivation tests using CV-1 and PC-3 cells expressing the AR and found to be strongly stimulated by androgens. EMSAs and mutational analysis of the Pem promoter allowed the identification of two functional androgen response elements named ARE-1 and ARE-2. They both differed from the consensus semipalindromic steroid response element and exhibited characteristics of direct repeats of the TGTTCT half-site. Unlike the steroid response element, both Pem androgen response elements were selectively responsive to androgen stimulation. Specific mutations in the left half-site of Pem ARE-1 and ARE-2, but not of the steroid response element, were still compatible with AR binding in the EMSA. In addition, Pem ARE-1, but not ARE-2 or the steroid response element, showed some flexibility with regard to spacing between half-sites. These results strongly suggest that the AR interacts differently with direct repeats than with inverted repeats, potentially leading to cis element-driven selective properties. Thus, the existence of several classes of DNA response elements might be an essential feature of differential androgen regulation.


Subject(s)
Androgens/pharmacology , Homeodomain Proteins/genetics , Promoter Regions, Genetic , Response Elements , Transcription Factors/genetics , Transcriptional Activation , Animals , Base Sequence , Cloning, Molecular , Conserved Sequence , DNA/chemistry , DNA/metabolism , Electrophoretic Mobility Shift Assay , Glucocorticoids/pharmacology , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Progestins/pharmacology , Rats , Receptors, Androgen/metabolism , Sequence Alignment , Structure-Activity Relationship , Transfection
9.
Biol Reprod ; 64(4): 1115-21, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11259257

ABSTRACT

Based on strong epididymal expression of the mouse glutathione peroxidase 5 (GPX5) and cysteine-rich secretory protein-1 (CRISP-1) genes, we evaluated whether the 5.0-kilobase (kb)-long GPX5 and 3.8-kb-long CRISP-1 gene 5'-flanking regions could be used to target expression of genes of interest into the epididymis in transgenic mice. Of the two candidate promoters investigated, the CRISP-1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene was highly expressed in the tubular compartment of the testis in all stages of the seminiferous epithelial cycle between pachytene spermatocytes at stage VII to elongated spermatids at step 16. In contrast to CRISP-1, the 5.0-kb 5' region of the mouse GPX5 gene directed EGFP expression to the epididymis. In the various GPX5-EGFP mouse lines, strongest expression of EGFP mRNA was found in the epididymis, but low levels of reporter gene mRNA were detected in several other tissues. Strong EGFP fluorescence was found in the principal cells of the distal caput region of epididymis, and few fluorescent cells were also detected in the cauda region. No EGFP fluorescence was detected in the corpus region or in the other tissues analyzed. Hence, it is evident that the 5.0-kb 5'-flanking region of GPX5 promoter is suitable for directing the expression of structural genes of interest into the caput epididymidis in transgenic mice.


Subject(s)
Epididymis/metabolism , Gene Expression , Glutathione Peroxidase/genetics , Membrane Glycoproteins , Salivary Proteins and Peptides/genetics , Testicular Hormones , Animals , Blotting, Northern , Embryo Transfer , Epididymis/chemistry , Green Fluorescent Proteins , Luminescent Proteins/genetics , Male , Mice , Mice, Transgenic , Promoter Regions, Genetic , RNA, Messenger/analysis , Recombinant Fusion Proteins , Reverse Transcriptase Polymerase Chain Reaction , Seminiferous Epithelium/metabolism , Seminiferous Tubules/metabolism , Spermatogenesis , Tissue Distribution , Transfection
10.
Mol Cell Endocrinol ; 173(1-2): 63-73, 2001 Feb 28.
Article in English | MEDLINE | ID: mdl-11223178

ABSTRACT

Androgen receptor (AR) signalling was analysed using as models the cysteine-rich secretory protein-1 (CRISP-1) and CRISP-3 gene promoters, which are differentially regulated by androgen in vivo and contain multiple potential androgen response elements. Using electrophoretic mobility shift assay, we identified several elements with differing affinities for the AR at positions -3706, -1270, -1253 and -350 of the CRISP-1 promoter and at positions -369 and -349 of the CRISP-3 promoter. The strongest binding was observed for the -1253 element of CRISP-1. In transactivation assays using a PC-3 cell line stably transfected with the AR (PC-3/AR), the -1253 element placed as two or four copies upstream of the TK minimal promoter yielded a strong induction of luciferase reporter gene activity in the presence of the androgen methyltrienolone (R1881). In the context of the CRISP promoters a 2-fold induction by R1881 was measured for the CRISP-3 upstream region whereas only limited effects were noted for the CRISP-1 upstream region. The androgenic stimulation of the p(-1253 ARE)(4x)-TK-luciferase reporter construct was dose-dependently inhibited by geldanamycin and radicicol, two compounds that selectively interact with the chaperone protein, heat-shock protein 90. Cotransfection with an expression vector for the 14-3-3eta protein markedly enhanced the androgen-dependent stimulation. These results emphasize the influence of promoter context on androgen regulation and the importance of AR-associated proteins.


Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Membrane Glycoproteins , Receptors, Androgen/metabolism , Response Elements/genetics , Seminal Plasma Proteins , Tyrosine 3-Monooxygenase/metabolism , 14-3-3 Proteins , Benzoquinones , Cell Line , Enzyme Inhibitors/pharmacology , Genes, Reporter/genetics , Humans , Lactams, Macrocyclic , Lactones/pharmacology , Macrolides , Male , Metribolone/pharmacology , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Prostate/drug effects , Prostate/metabolism , Quinones/pharmacology , Salivary Proteins and Peptides/genetics , Signal Transduction/drug effects , Testosterone Congeners/pharmacology , Transcriptional Activation/drug effects , Tyrosine 3-Monooxygenase/genetics
11.
Protein Sci ; 9(12): 2386-93, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11206060

ABSTRACT

The immunosuppressant drug cyclosporin A (CsA) inhibits T-cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high-affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural differences in the calcineurin-binding region. To delineate the calcineurin-binding region of CyPB, we mutated several amino acids, located in two loops corresponding to CyPA regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild-type CyPB, the G77H, D155R, and D158R mutants had intact isomerase and CsA-binding activities, indicating that no major conformational changes had taken place. When complexed to CsA, they all displayed only reduced affinity for calcineurin and much decreased inhibition of calcineurin phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of CyPB/CsA with calcineurin, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in CyPA and might therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.


Subject(s)
Calcineurin/metabolism , Cyclophilins/chemistry , Amino Acid Sequence , Binding Sites/genetics , Cyclophilins/genetics , Cyclophilins/metabolism , Cyclosporine/metabolism , Cyclosporine/pharmacology , Humans , Models, Molecular , Mutagenesis, Site-Directed , Peptidylprolyl Isomerase , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Point Mutation , Protein Binding/genetics
12.
J Biol Chem ; 274(16): 10990-8, 1999 Apr 16.
Article in English | MEDLINE | ID: mdl-10196180

ABSTRACT

Cyclophilin B is a cyclosporin A-binding protein exhibiting peptidyl-prolyl cis/trans isomerase activity. We have previously shown that it interacts with two types of binding sites on T lymphocytes. The type I sites correspond to specific functional receptors and the type II sites to sulfated glycosaminoglycans. The interactions of cyclophilin B with type I and type II sites are reduced in the presence of cyclosporin A and of a synthetic peptide mimicking the N-terminal part of cyclophilin B, respectively, suggesting that the protein possesses two distinct binding regions. In this study, we intended to characterize the areas of cyclophilin B involved in the interactions with binding sites present on Jurkat cells. The use of cyclophilin B mutants modified in the N-terminal region demonstrated that the 3Lys-Lys-Lys5 and 14Tyr-Phe-Asp16 clusters are probably solely required for the interactions with the type II sites. We further engineered mutants of the conserved central core of cyclophilin B, which bears the catalytic and the cyclosporin A binding sites as an approach to localize the binding regions for the type I sites. The enzymatic activity of cyclophilin B was dramatically reduced after substitution of the Arg62 and Phe67 residues, whereas the cyclosporin A binding activity was destroyed by mutation of the Trp128 residue and strongly decreased after modification of the Phe67 residue. Only the substitution of the Trp128 residue reduced the binding of the resulting cyclophilin B mutant to type I binding sites. The catalytic site of cyclophilin B therefore did not seem to be essential for cellular binding and the cyclosporin A binding site appeared to be partially involved in the binding to type I sites.


Subject(s)
Cyclophilins , Glycosaminoglycans/metabolism , Immunophilins/metabolism , T-Lymphocytes/metabolism , Amino Acid Sequence , Binding Sites , Humans , Immunophilins/chemistry , Immunophilins/genetics , Jurkat Cells , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptidylprolyl Isomerase , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid
13.
J Cell Physiol ; 178(3): 371-8, 1999 Mar.
Article in English | MEDLINE | ID: mdl-9989783

ABSTRACT

Cysteine-rich secretory proteins (CRISPs) represent a family of evolutionarily conserved proteins which may play a role in the innate immune system and are transcriptionally regulated by androgens in several tissues. Transcripts for all three members of the CRISP family have now been identified in the murine lacrimal gland. RT-PCR using primers able to discriminate between the related CRISP forms allowed the amplification of fragments with the expected length. DNA sequencing revealed a complete identity with the hitherto characterized epididymal CRISP-1, testicular CRISP-2, and salivary gland CRISP-3. An analysis of several mouse strains indicated that all expressed the three CRISP forms, but in differing amounts. RT-PCR analysis of RNA isolated from acinar cells of lacrimal glands revealed that they expressed CRISP-1 and CRISP-2. Semiquantitative and quantitative analyses furthermore showed higher CRISP-1 and CRISP-3 mRNA levels in the lacrimal glands of male BALB/c and NOD mice when compared to females. Testosterone treatment of C3H/HeJ female mice was followed by an upregulation of the steady-state CRISP-1 but not CRISP-2 transcript levels. A comparable stimulation was observed for the mRNAs coding for parotid secretory protein (PSP), a factor previously shown to exhibit sexual dimorphism in the murine lacrimal gland. The expression of CRISP transcripts in the lacrimal gland is consistent with a function in the innate immune system.


Subject(s)
Glycoproteins/genetics , Lacrimal Apparatus/metabolism , Membrane Glycoproteins , Salivary Proteins and Peptides/genetics , Seminal Plasma Proteins , Testosterone/pharmacology , Transcription, Genetic , Animals , Cell Adhesion Molecules , DNA Primers , Drug Implants , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Exocrine Glands/metabolism , Female , Genitalia, Male/metabolism , Male , Membrane Proteins , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred NOD , RNA, Messenger/genetics , Receptors, Androgen/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sex Characteristics , Transcription, Genetic/drug effects
14.
Biochem J ; 321 ( Pt 2): 325-32, 1997 Jan 15.
Article in English | MEDLINE | ID: mdl-9020862

ABSTRACT

In mice, cysteine-rich secretory protein-1 (CRISP-1) is mainly found in the epididymis and also, to a lesser extent, in the salivary gland of males, where androgens control its expression. We have now isolated and characterized overlapping phage clones covering the entire length of the CRISP-1 gene. DNA sequencing revealed that the gene is organized into eight exons, ranging between 55 and 748 bp in size, and seven introns. All exon-intron junctions conformed to the GT/AG rule established for eukaryotic genes. The intron length, as determined by PCR, varied between 1.05 and 4.0 kb so that the CRISP-1 gene spans over 20 kb of the mouse genome. The transcription-initiation site was determined by primer extension and localized at the expected distance downstream of a consensus TATA box. Approximately 3.7 kb of the CRISP-1 promoter region were isolated and sequenced, and several stretches fitting the androgen-responsive element consensus were found. Those that most resembled the consensus were analysed by electrophoretic mobility-shift assay and found to form specific complexes with the liganded androgen receptor in vitro, but with different affinities. Putative binding elements for the transcription factors Oct, GATA, PEA3, CF1. AP-1 and AP-3 were also found in the promoter region.


Subject(s)
Androgens/physiology , Cysteine , Epididymis/chemistry , Genes , Membrane Glycoproteins , Salivary Proteins and Peptides/genetics , Salivary Proteins and Peptides/metabolism , Animals , Bacteriophage lambda , Base Sequence , Cloning, Molecular , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Promoter Regions, Genetic , Receptors, Androgen/chemistry , Receptors, Androgen/genetics , Salivary Proteins and Peptides/isolation & purification , Sequence Analysis, DNA , Transcription, Genetic
15.
Eur J Biochem ; 250(2): 440-6, 1997 Dec 01.
Article in English | MEDLINE | ID: mdl-9428696

ABSTRACT

The androgen dependency of the genes coding for the cysteine-rich secretory proteins (CRISP) was analysed in their main sites of expression. Male mice were treated with the gonadotropin-releasing hormone antagonist Ac-DNapAla-DClPhAla-DPyrAla-Ser-Tyr-DCtl-Leu-Lys (Mor)-Pro-DAla-NH2 [DNapAla, D-2-naphthyl-Ala; DClPhAla, D-4-chlorphenyl-Ala; DPyrAla, D-pyridyn-3-yl-Ala; DCtl, D-citrulline; Lys(Mor), L-2-amino-6-(morpholin-4-yl)-hexanoic acid], and CRISP RNA levels were assessed by northern blot and competitive reverse transcriptase-mediated (RT)-PCR. In the salivary gland, CRISP-1 and to a lesser extent CRISP-3 expression was markedly reduced, in spite of an up-regulation of androgen receptor transcript levels. A down-regulation of CRISP-1 expression was also observed in the epididymis. Conversely, the levels of the testicular CRISP-2 transcripts were hardly affected at all. Female mice were ovariectomised and treated with testosterone propionate, and their salivary gland RNAs analysed. CRISP-1 and CRISP-3 RNA levels were significantly increased, and these effects were prevented by a concomitant treatment with the antiandrogen flutamide. Androgen receptor transcript levels were not affected by androgen administration but increased following antiandrogen treatment. CRISP expression during postnatal development was monitored by northern blot analysis. CRISP-1 and CRISP-2 transcripts were detected as early as 22 days after birth in the epididymis and testis, respectively, whereas CRISP-3 mRNA was visible only from day 30 in the salivary gland. A sharp increase of all CRISP levels was noted on day 40, coincident with the onset of sexual maturity. Altogether these results indicate that despite their high similarity, the CRISP genes are differentially regulated by androgens.


Subject(s)
Androgens/physiology , Gene Expression Regulation , Membrane Glycoproteins , Salivary Proteins and Peptides/genetics , Seminal Plasma Proteins , Animals , Blotting, Northern , Epididymis/metabolism , Female , Male , Mice , Organ Specificity , Polymerase Chain Reaction , RNA, Messenger/analysis , Receptors, Androgen/genetics , Salivary Glands/metabolism
16.
Mol Cell Biol ; 16(11): 6160-8, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8887646

ABSTRACT

The Oct2 transcription factor is expressed throughout the B-lymphoid lineage and plays an essential role during the terminal phase of B-cell differentiation. Several genes specifically expressed in B lymphocytes have been identified that contain a functional octamer motif in their regulatory elements. However, expression of only a single gene, the murine CD36 gene, has been shown to date to be dependent on Oct2. Here, we present the identification and characterization of a further gene, coding for cysteine-rich secreted protein 3 (CRISP-3), whose expression in B cells is regulated by Oct2. We show that CRISP-3 is expressed in the B-lymphoid lineage specifically at the pre-B-cell stage. By using different experimental strategies, including nuclear run-on experiments, we demonstrate that this gene is transcriptionally activated by Oct2. Furthermore, analysis of CRISP-3 expression in primary B cells derived from either wild-type or Oct2-deficient mice demonstrates the dependence on Oct2. Two variant octamer motifs were identified in the upstream promoter region of the crisp-3 gene, and Oct2 interacts with both of them in vitro. Cotransfection experiments with expression vectors for Oct1 and Oct2 together with a reporter driven by the crisp-3 promoter showed that transcriptional activation of this promoter can only be achieved with Oct2. The C-terminal transactivation domain of Oct2 is required for this activation. Finally, introducing specific mutations in the two variant octamer motifs revealed that both of them are important for full transcriptional activation by Oct2.


Subject(s)
B-Lymphocytes/metabolism , Salivary Proteins and Peptides/biosynthesis , Seminal Plasma Proteins , Transcription Factors/metabolism , Transcription, Genetic/drug effects , 3T3 Cells , Animals , Blood Proteins/chemistry , Cell Line , Cysteine , DNA Primers , DNA-Binding Proteins/metabolism , Defensins , Estradiol/pharmacology , Female , Lymphoid Tissue/metabolism , Male , Mice , Octamer Transcription Factor-2 , Organ Specificity , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Salivary Proteins and Peptides/chemistry , Sequence Homology, Amino Acid , Sex Characteristics , Simplexvirus/enzymology , Simplexvirus/genetics , T-Lymphocytes/metabolism , Transfection , Tumor Cells, Cultured
17.
Biochem J ; 317 ( Pt 2): 571-6, 1996 Jul 15.
Article in English | MEDLINE | ID: mdl-8713087

ABSTRACT

Cyclophilin B (CyPB) is secreted in biological fluids such as blood or milk and binds to a specific receptor present on the human lymphoblastic cell line Jurkat and on human peripheral blood lymphocytes. This study was intended to specify the areas of CyPB that are involved in the interaction with the receptor. A synthetic peptide corresponding to the first 24 N-terminal amino acid residues of CyPB was shown to specifically recognize the receptor. Moreover, modification of Arg18 of CyPB by p-hydroxyphenlglyoxal led to a dramatic loss of affinity for the receptor. However, when this residue was replaced by an alanine residue using site-directed mutagenesis, no modification of the binding properties was found, suggesting that Arg18 is not directly involved but is sufficiently close to the interaction site to interfere with the binding when modified. Competitive binding experiments using a chimaeric protein made up of the 24 N-terminal amino acid residues of CyPB fused to the cyclophilin A core sequence confirmed the involvement of this region of CyPB in receptor binding.


Subject(s)
Amino Acid Isomerases/metabolism , Carrier Proteins/metabolism , Cyclophilins , Peptide Fragments/metabolism , Receptors, Peptide/metabolism , T-Lymphocytes/metabolism , Amino Acid Isomerases/antagonists & inhibitors , Amino Acid Isomerases/genetics , Amino Acid Sequence , Binding Sites , Binding, Competitive , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Cyclophilin C , Enzyme Inhibitors/pharmacology , Humans , Isoenzymes/metabolism , Molecular Sequence Data , Peptidylprolyl Isomerase , Phenylglyoxal/analogs & derivatives , Phenylglyoxal/pharmacology , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Spectrophotometry, Ultraviolet
18.
Eur J Biochem ; 236(3): 827-36, 1996 Mar 15.
Article in English | MEDLINE | ID: mdl-8665901

ABSTRACT

We report the isolation and characterisation of cDNAs encoding three different, human members of the cysteine-rich secretory protein (CRISP) family. The novel CRISP-1 exists in five cDNA subtypes differing by the presence or absence of a stretch coding for a C-terminal cysteine-rich domain so far found in all members of the family, and by the length of their 3'-untranslated region. CRISP-2 cDNA corresponds to the previously described TPX1 form, with so far unreported 5'-untranslated sequence heterogeneities while CRISP-3 cDNA codes for a new, unique protein. Northern blot analysis of various human organs indicates that CRISP-1 transcripts are epididymis-specific whereas CRISP-2/TPX1 transcripts are detected mainly in the testis and also in the epididymis. CRISP-3 transcripts are more widely distributed and found predominantly in the salivary gland, pancreas and prostate, and in less abundance in the epididymis, ovary, thymus and colon. A protein reacting with an anti-mouse CRISP-1 antibody was isolated from human epididymal extracts and N-terminal sequencing revealed that it corresponded to the CRISP-1 cDNA we have isolated. In contrast to findings on its rat counterpart epididymal protein DE/acidic epididymal glycoprotein (AEG), no significant association of CRISP-1 with human spermatozoa was observed.


Subject(s)
Epididymis/metabolism , Glycoproteins/biosynthesis , Glycoproteins/chemistry , Membrane Glycoproteins , Salivary Proteins and Peptides/biosynthesis , Salivary Proteins and Peptides/chemistry , Seminal Plasma Proteins , Testis/metabolism , Aged , Amino Acid Sequence , Animals , Base Sequence , Cell Adhesion Molecules , DNA, Complementary , Enzyme-Linked Immunosorbent Assay , Gene Library , Glycoproteins/analysis , Humans , Male , Membrane Proteins , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides , Organ Specificity , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Rats , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Salivary Proteins and Peptides/analysis , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Spermatozoa/metabolism , Transcription, Genetic
19.
Blood Coagul Fibrinolysis ; 7(2): 183-6, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8735814

ABSTRACT

Pallidipin is a platelet aggregation inhibitor protein originating from the saliva of the haematophageous triatomine bug Triatoma pallidipennis. Its inhibitory effects are specific for collagen-induced platelet aggregation. The recombinant form of the protein was expressed in the periplasmic space of transformed Escherichia coli using a vector based on the alkaline phosphatase gene promoter and leader peptide. Recombinant pallidipin was purified in three chromatographic steps including cation exchange, anion exchange and size exclusion gel chromatography. SDS/PAGE and N-terminal amino acid sequencing showed that recombinant pallidipin had a molecular weight similar to that of the salivary protein (approximately 19 kDa) and had been correctly processed. The yield was 864 micrograms of pure protein from one litre of bacterial culture. The biological activity of recombinant pallidipin was assessed in a platelet aggregation assay using collagen at a concentration of 2 micrograms/ml as inducer, and the IC50 found to be 33 nM, similar to that determined for the native protein. When the collagen concentration used for induction was increased, higher pallidipin concentrations were also needed to achieve a comparable inhibition of platelet aggregation.


Subject(s)
Platelet Aggregation Inhibitors/metabolism , Salivary Proteins and Peptides/metabolism , Animals , Collagen/administration & dosage , Collagen/pharmacology , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Humans , Molecular Weight , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/chemistry , Platelet Aggregation Inhibitors/isolation & purification , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Salivary Proteins and Peptides/chemistry , Salivary Proteins and Peptides/isolation & purification , Triatoma
20.
J Biol Chem ; 270(48): 28629-34, 1995 Dec 01.
Article in English | MEDLINE | ID: mdl-7499380

ABSTRACT

Triabin, a new thrombin inhibitor, has been purified from the saliva of Triatoma pallidipennis, a blood-sucking triatomine bug. It forms a noncovalent complex with thrombin at a molar ratio of 1:1, inhibits thrombin-induced platelet aggregation, and prolongs thrombin clotting time and activated partial thromboplastin time. However, it only minimally suppresses the amidolytic activity of thrombin, as measured by a chromogenic peptide substrate assay. It completely blocks trypsin-catalyzed cleavage of thrombin, probably via protection of the anion-binding exosite and inhibits the effect of thrombomodulin on thrombin in a dose-dependent fashion. These results indicate that the inhibitor is directed toward the anion-binding exosite of thrombin. The protein was partially sequenced and the information used to isolate cDNA clones from a T. pallidipennis salivary gland library. Four slightly polymorphic variants coding for mature proteins of 142 amino acids preceded by a putative leader sequence were obtained. The recombinant protein expressed in the periplasmic space of Escherichia coli has a biological activity similar to that of salivary triabin, as tested in a thrombin-induced platelet aggregation assay. In addition, recombinant triabin inhibits thrombin-catalyzed hydrolysis of fibrinogen with a Ki of about 3 pM.


Subject(s)
Platelet Aggregation Inhibitors/pharmacology , Salivary Proteins and Peptides/pharmacology , Serine Proteinase Inhibitors/pharmacology , Thrombin/antagonists & inhibitors , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , DNA Primers/chemistry , DNA, Complementary/chemistry , Humans , Hydrolysis , Insect Proteins , Molecular Sequence Data , Platelet Aggregation Inhibitors/chemistry , Platelet Aggregation Inhibitors/metabolism , Saliva/chemistry , Salivary Proteins and Peptides/genetics , Salivary Proteins and Peptides/metabolism , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/metabolism , Thrombin/metabolism , Thrombomodulin/metabolism , Triatoma , Trypsin/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...