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R St-Arnaud

Publications and source records attributed to R St-Arnaud.

At least 37 records · Page 2Linked to original sources
PubMed1996

New mechanisms of regulation of the genomic actions of vitamin D in bone cells: interaction of the vitamin D receptor with non-classical response elements and with the multifunctional protein, calreticulin.

Vitamin D exerts its genomic effects following binding to a specific receptor which is a member of the steroid hormone receptor superfamily. The vitamin D receptor (VDR) forms heterodimers with retinoid X receptors (RXRs) and the dimer then interacts with its cognate binding site, termed vitamin D response element (VDRE), to affect the transcription of target genes. Recent studies have identified novel sequence motifs for VDREs as well as novel protein-protein interactions involving the VDR. These will be reviewed with particular emphasis on the complex VDRE from the c-fos proto-oncogene promoter region and the inhibition of the vitamin D signal transduction pathway by the multifunctional protein, calreticulin. Thus research on the control of gene transcription by vitamin D reveals examples of molecular interplay between transcriptional regulatory pathways and provides new insight into the molecular mechanism of action of vitamin D.

Base Sequence↗
PubMed1996

Differential splicing-in of a proline-rich exon converts alphaNAC into a muscle-specific transcription factor.

NAC (nascent polypeptide-associated complex) was recently purified as an alpha/beta heterodimeric complex binding the newly synthesized polypeptide chains as they emerge from the ribosome. We have identified, cloned, and characterized a muscle-specific isoform of alphaNAC. The 7.0-kb mRNA arises from differential splicing-in of a 6.0 kb-exon giving rise to a proline-rich isoform that we termed skNAC. The skNAC protein was specifically expressed in differentiated myotubes but not in myoblasts. We have identified a specific DNA binding site for skNAC and shown that it can activate transcription through that element. The murine myoglobin promoter contains three putative skNAC-binding sites. skNAC was shown to activate transcription from the myoglobin promoter, and site-specific mutation of the skNAC response elements abrogated skNAC-dependent activation. We also examined the role of the NAC isoforms in the myogenic program. Whereas overexpression of alphaNAC prevented C2C12 differentiation and myotube fusion, the overexpression of skNAC in C2C12 myoblasts led to early fusion of the cells into gigantic myosacs, suggesting that skNAC may be involved in normal differentiation along the myogenic lineage and in the regulation of myoblast fusion. Our data demonstrate that differential splicing converts alphaNAC into a tissue-specific DNA-binding activator and suggest that this regulation may be an important event in the proper control of gene expression during myogenic differentiation.

Amino Acid Sequence↗
PubMed1996

A composite element binding the vitamin D receptor, retinoid X receptor alpha, and a member of the CTF/NF-1 family of transcription factors mediates the vitamin D responsiveness of the c-fos promoter.

The hormonal form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 [1,25- (OH)2D3], transiently stimulates the transcription of the c-fos proto-oncogene in osteoblastic cells. We have identified and characterized a vitamin D response element (VDRE) in the promoter of c-fos. The 1,25-(OH)2D3-responsive region was delineated between residues -178 and -144 upstream of the c-fos transcription start site. A mutation that inhibited binding to the sequence concomitantly abolished 1,25-(OH)2D3-induced transcriptional responsiveness; similarly, cloning to the site upstream of a heterologous promoter conferred copy-number-dependent vitamin D responsiveness to a reporter gene, demonstrating that we have identified a functional response element. The structure of the c-fos VDRE was found to be unusual. Mutational analysis revealed that the c-fos VDRE does not conform to the direct repeat configuration in which hexameric core-binding sites are spaced by a few nucleotide residues. In contrast, the entire 36-bp sequence was essential for binding. We identified the vitamin D receptor and the retinoid X receptor alpha as components of the complex that bound the c-fos VDRE. However, our results also show that a putative CCAAT-binding transcription factor/nuclear factor 1 (CTF/NF-1) family member bound the response element in conjunction with the nuclear hormone receptors. The expression of this CTF/NF-1 family member appeared restricted to bone cells. These data hint at new molecular mechanisms of action for vitamin D.

Base Sequence↗
PubMed1995

Increased expression of the c-fos proto-oncogene in bone from patients with fibrous dysplasia.

BACKGROUND: Fibrous dysplasia is characterized by intense marrow fibrosis and increased rates of bone turnover. The lesions of fibrous dysplasia resemble those described in the long bones of transgenic mice overexpressing the c-fos proto-oncogene. Activating mutations in the alpha subunit of the stimulatory guanine-nucleotide-binding protein (GS alpha) linked to adenylate cyclase have recently been described in bone cells from patients with the McCune-Albright syndrome and fibrous dysplasia. METHODS: We used in situ hybridization to determine the level of expression of c-fos in bone-biopsy specimens from two normal subjects, eight patients with fibrous dysplasia, and six patients with other bone disorders characterized by high rates of bone turnover. The probe used corresponded to the fourth exon of the c-fos gene. RESULTS: High levels of c-fos expression were detected in the bone lesions from all eight patients with fibrous dysplasia. No expression of c-fos was detected in bone specimens from the normal subjects or from specimens of normal bone obtained from patients with fibrous dysplasia. The cells that expressed c-fos in the dysplastic lesions were fibroblastic and populated the marrow space. A very low level of c-fos expression was detected in the biopsy specimens from the patients with other bone diseases. One patient with polyostotic fibrous dysplasia and one patient with the McCune-Albright syndrome were tested for the previously described GS alpha gene mutations and were found to express these mutations in bone. CONCLUSIONS: Increased expression of the c-fos proto-oncogene, presumably a consequence of increased adenylate cyclase activity, may be important in the pathogenesis of the bone lesions in patients with fibrous dysplasia.

Adolescent↗
PubMed1995

Constitutive expression of calreticulin in osteoblasts inhibits mineralization.

Recent studies have shown that the multifunctional protein calreticulin can localize to the cell nucleus and regulate gene transcription via its ability to bind a protein motif in the DNA-binding domain of nuclear hormone receptors. A number of known modulators of bone cell function, including vitamin D, act through this receptor family, suggesting that calreticulin may regulate their action in bone cells. We have used a gain-of-function strategy to examine this putative role of calreticulin in MC3T3-E1 osteoblastic cells. Purified calreticulin inhibited the binding of the vitamin D receptor to characterized vitamin D response elements in gel retardation assays. This inhibition was due to direct protein-protein interactions between the vitamin D receptor and calreticulin. Expression of calreticulin transcripts declined during MC3T3-E1 osteoblastic differentiation. MC3T3-E1 cells were transfected with calreticulin expression vectors; stably transfected cell lines overexpressing recombinant calreticulin were established and assayed for vitamin D-induced gene expression and the capacity to mineralize. Constitutive calreticulin expression inhibited basal and vitamin D-induced expression of the osteocalcin gene, whereas osteopontin gene expression was unaffected. This pattern mimicked the gene expression pattern observed in parental cells before down-regulation of endogenous calreticulin expression. In long-term cultures of parental or vector-transfected cells, 1 alpha,25-dihydroxyvitamin D3 (1,25[OH]2D3) induced a two- to threefold stimulation of 45Ca accumulation into the matrix layer. Constitutive expression of calreticulin inhibited the 1,25(OH)2D3-induced 45Ca accumulation. This result correlated with the complete absence of mineralization nodules in long-term cultures of calreticulin-transfected cells. These data suggest that calreticulin can regulate bone cell function by interacting with specific nuclear hormone receptor-mediated pathways.

Amino Acid Sequence↗
PubMed1995

An upstream activator sequence regulates the murine Pgk-1 promoter and binds multiple nuclear proteins.

The murine Pgk-1 gene is driven by a strong promoter that is regulated by a 304 bp upstream activator sequence (UAS). The activity of the UAS is high in undifferentiated embryonal carcinoma cells but declines when these cells are induced to differentiate with retinoic acid. The effect of the UAS on promoter activity is particularly striking when the activity of the Pgk-1 promoter is assayed following its integration into the genome, suggesting that it may function by regulating chromatin structure in the region of the core promoter. Three sites on the UAS bind nuclear proteins. Two of these sites bind factors present in both embryonal carcinoma cells and their differentiated derivatives whereas one site binds factors present only in differentiated cells. There appears to be both cooperation and antagonism in the binding of proteins to different sites in the UAS, suggesting that the activity of the Pgk-1 promoter is determined by the constellation of proteins assembled upstream of its transcription start site.

Animals↗
PubMed1993

Cloning and functional expression analysis of the alpha subunit of mouse ATP synthase.

The alpha subunit of the mitochondrial ATP synthase is part of the F1 enzymatic complex known to bind ADP, phosphate and ATP and is at the heart of the mitochondrial energy-producing mechanism. The mouse embryonal carcinoma variant of the alpha subunit cDNA was cloned and the complete nucleotide sequences of two different lengths of clones were determined. Two distinct polyadenylation sites in the cDNA sequence were detected and two sizes of mRNAs were confirmed by Northern blot hybridization. Two putative ATP-binding motifs - A and B, have been hypothesized for this enzyme based on previous NMR work on another ATP-binding enzyme, adenylate kinase. We have constructed four deletion mutants of the alpha subunit of the mouse F1-ATP synthase to examine the putative role of these domains. The resulting recombinant proteins were expressed and purified. Functional studies with the immobilized mutants proved the significance of both sites for ATP binding.

Adenosine Triphosphate↗
PubMed1993

Wnt-1-inducing factor-1: a novel G/C box-binding transcription factor regulating the expression of Wnt-1 during neuroectodermal differentiation.

The Wnt-1 proto-oncogene is essential for proper development of the midbrain and is expressed in a spatially and temporally restricted manner during central nervous system development in mice. In vitro, the gene is specifically transcribed during the retinoic acid (RA)-induced neuroectodermal differentiation of the P19 line of embryonal carcinoma cells. The P19 cells differentiate into neurons, astrocytes, and fibroblast-like cells when treated with RA. Treatment of the cells with dimethyl sulfoxide leads to differentiation along mesodermal lineages, including skeletal and cardiac muscle. We have used the P19 cell line to study the Wnt-1 promoter and identify and characterize the transcription factor(s) that regulates the differentiation-specific transcription of Wnt-1 in RA-treated P19 cultures. Transient-transfection assays have revealed that a 230-bp region comprising positions -278 to -47 of the 5' upstream Wnt-1 sequence was sufficient to direct RA-specific transcription. This promoter fragment was shown to contain a binding site for a nuclear factor that was not detected in undifferentiated P19 stem cells or their dimethyl sulfoxide-treated derivatives but was induced in differentiating RA-treated cells. This factor was termed Wnt-1-inducing factor-1 (WiF-1). DNase I footprinting analysis has identified the G/C-rich WiF-1 binding site, and UV cross-linking studies have shown that WiF-1 is a protein with an M(r) of 65,000. WiF-1 binding activity was also detected in postpubertal mouse testis, the only tissue that expresses Wnt-1 in adults. Site-directed mutations that inhibited WiF-1 binding to the Wnt-1 promoter concomitantly abolished the activity of the promoter in RA-treated P19 cells. The active WiF-1 protein was purified by DNA affinity chromatography. Our data suggest that WiF-1 is a novel G/C box-binding transcription factor and support a physiological role for WiF-1 in the developmentally regulated expression of Wnt-1.

Animals↗
PubMed1991

Differential stimulation of fos and jun family members by calcitriol in osteoblastic cells.

We have looked at the effects of calcitriol (1 alpha,25-dihydroxyvitamin D3) on the expression of the members of the fos and jun families of protooncogenes in an osteoblastic cell line and in primary cultures of osteoblasts. Calcitriol treatment of starved, confluent cultures of MC3T3-E1 cells induced a rapid and transient stimulation of the expression of c-fos, fos-B, c-jun, and jun-B with varying kinetics. The expression of fra-1 and jun-D was not affected by calcitriol in those cells. The selective stimulation of fos and jun family members by calcitriol was also observed in primary cultures of osteoblasts isolated from newborn mouse calvaria, suggesting that this modulation is a physiological response of the bone cells and not an artefact of the established cell line. The calcitriol effect was specific and dose-dependent. The expression of the c-Fos protein correlated with the expression of the mRNA in calcitriol-treated cells. The calcitriol-induced stimulation of c-fos expression was modulated, at least in part, at the level of the initiation and elongation of transcription, whereas its effects on c-jun and jun-B expression was controlled at the posttranscriptional level by a mechanism that does not implicate stabilization of their respective mRNAs. The differential stimulation of the expression of certain members of the fos and jun families by calcitriol support a role for these oncoproteins in bone cell physiology.

Animals↗
PubMed1989

The int-1 proto-oncogene is transcriptionally activated during neuroectodermal differentiation of P19 mouse embryonal carcinoma cells.

We have used the P19 line of mouse embryonal carcinoma (EC) cells to initiate studies on the putative role of the int-1 proto-oncogene during neuronal differentiation. P19 cells are induced to differentiate into neurons, astrocytes and fibroblast-like cells following exposure to retinoic acid (RA). Treatment of the same P19 cells with dimethyl sulfoxide (DMSO) leads to differentiation into mesodermal derivatives, including skeletal and cardiac muscle. Northern blot analysis showed that int-1 RNA was not present in the EC cells but appeared 48 h after RA exposure and could be detected for at least the next 8 days. No int-1 RNA was detected in P19 cells induced to differentiate with DMSO. Nuclear run-on transcription assays showed that int-1 expression in RA-treated P19 cells was induced at the transcriptional level. Immunofluorescent staining with an antibody directed against an int-1 peptide identified immunoreactive material in cytoplasmic granules of fibroblast-like cells in RA-treated P19 cultures. Thus the P19 cell line is a suitable experimental system to study int-1 gene expression and function during neuroectodermal development.

Animals↗
PubMed1988

The rise in testicular androgens during the first days of treatment with an LHRH agonist in the dog can be blocked by aminoglutethimide or ketoconazole.

Up to day 6 of treatment of adult dogs, daily subcutaneous administration of 50 micrograms of the LHRH agonist [D-Trp6, des-Gly-NH2-10]LHRH ethylamide causes up to a 3-fold increase in serum testosterone (T) concentration which is followed by a progressive decrease to castration levels (less than or equal to 0.2 ng/ml) at later time intervals (up to 21 days, the last time interval studied). Both aminoglutethimide and ketoconazole, two inhibitors of steroid biosynthesis, cause a 30-40% rise in serum T when administered alone. However, either drug administered in combination with the LHRH agonist completely blocks the transient rise in serum T observed when the LHRH agonist is administered alone. On the other hand, the LHRH agonist prevents the secondary rise in steroid secretion observed when either of the two inhibitors of steroid secretion is used alone. Administration of the pure antiandrogen Flutamide alone or in combination with LHRH-A and an inhibitor of steroid biosynthesis does not influence serum T levels. When the serum levels of pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH-progesterone, dehydroepiandrosterone (DHEA), androstenedione (delta 4-dione), androst-5-ene-3 beta, 17 beta-diol (delta 5-diol), T, dihydrotestosterone (DHT), androstane-3 alpha, 17 beta-diol, androstane-3 beta. 17 beta-diol and 17 beta-estradiol (E2) are analyzed in detail, it can be seen that both aminoglutethimide and ketoconazole not only prevent the rise in serum steroids observed during the first 8 days of treatment with the LHRH agonist but that both compounds enhance the inhibitory effect of the LHRH agonist at later time intervals. A predominant inhibitory effect of ketoconazole is exerted on 17,20-desmolase activity. Aminoglutethimide has little influence on the loss of serum LH bioactivity induced by the LHRH agonist while ketoconazole stimulates the concentration of serum bioactive LH in the absence or presence of simultaneous treatment with the LHRH agonist. The present data clearly demonstrate that aminoglutethimide or ketoconazole can prevent the rise in serum androgens accompanying the first days of treatment with an LHRH agonist in the dog. Moreover, after 3 weeks of treatment, the inhibitory effect of the LHRH agonist on serum androgen levels is enhanced by addition of aminoglutethimide or ketoconazole. Moreover, Flutamide does not interfere with the inhibitory action of the LHRH agonist, aminoglutethimide or ketoconazole, thus suggesting that maximal inhibition of androgen action is likely to be achieved by a combination of these drugs.

Aldehyde-Lyases↗
PubMed1988

Androgens modulate epidermal growth factor receptor levels in the rat ventral prostate.

In order to further understand the factors which influence the normal or pathologic growth of the prostate, we have characterized the receptor for epidermal growth factor (EGF) in the rat ventral prostate and have studied the hormonal regulation in this receptor. EGF binds to a single class of saturable, high affinity binding sites in total prostatic homogenate. Scatchard analysis of the binding data reveals an apparent dissociation constant (KD) of 0.93 +/- 0.08 nM and a number of sites of 4.01 +/- 0.24 fmol per mg protein. Among the peptides tested, only native EGF can displace bound [125I]EGF. Castration stimulates the concentration of prostatic EGF receptors from 25.5 +/- 3.0 to 43.4 +/- 5.4 fmol/100 mg tissue in intact and castrated animals, respectively (P less than 0.01). Treatment of castrated rats with dihydrotestosterone (DHT) inhibits the rise in prostatic EGF receptor concentration induced by orchiectomy, while estradiol, progesterone or the dopaminergic agonist CB-154, have no effect. Combined administration of DHT with the other above-mentioned steroids or CB-154 does not modify the inhibition of prostatic EGF receptor concentration induced by the androgen in castrated animals. When the data are expressed as changes in EGF receptor number in the total prostate, DHT treatment reverses the inhibitory effect induced by castration and yields an EGF binding capacity comparable to that measured in intact animals. Chronic treatment with a pure antiandrogen or a potent LHRH agonist (LHRH-A) alone has no significant effect on EGF receptor concentration in prostatic tissue, although, secondary to a reduction in prostatic weight, total prostatic EGF binding capacity is reduced following antiandrogen or LHRH-A treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed1988

A pure antiandrogen does not interfere with the LHRH agonist-induced blockade of testicular androgen secretion in the dog.

Daily subcutaneous administration of 50 micrograms of the luteinizing hormone-releasing hormone (LHRH) agonist [D-Trp6]LHRH ethylamide in adult dogs causes a transient increase in the serum testosterone (T) concentration which reaches a maximum at 200% above control on days 2-4 of treatment and progressively decreases to 7% of the pretreatment value on day 21, the last time interval studied. After a transient increase, the concentration of serum bioactive luteinizing hormone (LH) was progressively decreased on days 11 and 19, thus suggesting that in analogy with human findings, the loss of LH bioactivity is responsible for the inhibition of testicular steroidogenesis induced in the dog by LHRH agonists. Of major significance is the finding that the changes in serum T levels observed during the first 3 weeks of treatment, as well as the complete inhibition of the intratesticular concentration of sex steroids observed at the end of this period of treatment with the LHRH agonist were not affected by simultaneous administration of flutamide (125 mg per os every 8 h). Such findings indicate that at the dose used, the LHRH agonist is in full control of gonadotropin secretion, thus completely overcoming feedback influences. Since the administration of the antiandrogen flutamide does not decrease the efficacy of the LHRH agonist as blocker of testicular androgen biosynthesis, the present data support the use of a pure antiandrogen in order to neutralize the effect of the transient rise in testicular androgen secretion which always accompanies the first days of treatment with LHRH agonists in patients with advanced prostate cancer.

Anilides↗
PubMed1988

Two transient increases in c-myc gene expression during neuroectodermal differentiation of mouse embryonal carcinoma cells.

Mouse embryonal carcinoma (EC) cells of the P19 line can be induced to differentiate into neurons, astrocytes and fibroblasts by exposure to retinoic acid (RA), whereas treatment of the EC cells with dimethyl sulfoxide (DMSO) leads to differentiation into mesodermal tissues, including cardiac and skeletal muscle. In RA- but not DMSO-treated cultures, the level of c-myc mRNA underwent two transient increases. The concentration of c-myc mRNA in RA-treated cultures increased 3-fold after 3 h in the presence of the drug, returned to normal by 9 h, increased again 5-fold from 48 to 96 h, and finally decreased below pre-treatment values by 144 h. Increased levels of c-myc protein were observed at the times of elevated c-myc mRNA. Nuclear run-on assays of the c-myc gene and measurements of c-myc mRNA stability indicated that both transcriptional and post-transcriptional mechanisms contribute to the modulated expression of the c-myc gene. The two transient increases in c-myc expression suggest a role for the c-myc protein in the differentiation of cells along neuroectodermal lineages.

Animals↗
PubMed1987

Characteristics of radioimmunoassays for the alpha- and beta-subunits of human luteinizing hormone.

The binding characteristics and specificities of the National Hormone and Pituitary Program (NHPP) kits for the radioimmunoassay of the alpha- and beta-subunits of human luteinizing hormone (hLH-alpha and hLH-beta) were studied, as well as the specificities of the anti-hLH and anti-human follicle stimulating hormone (anti-hFSH) antisera distributed by the same organization. The affinity constants of the anti-hLH-alpha and anti-hLH-beta antisera were calculated at 157 +/- 8.4 nM-1 and 109 +/- 7.4 nM-1, respectively. Both antisera were highly specific with regard to the other subunit. However, in the homologous hLH-alpha RIA, native hLH cross-reacted at 21.9%, hFSH at 17.5% and hTSH at 7.9%. The alpha-subunit of the human chorionic gonadotropin, hCG-alpha, was equipotent with the hLH-alpha standard in this assay. In the homologous hLH-beta RIA, hLH showed a cross-reactivity of 14.7% while the cross-reactivities of hCG-beta, hFSH and hTSH were 3.5%, 1.2% and 0.6%, respectively. The anti-hFSH antiserum was highly specific, while the anti-hLH antiserum showed non parallel competition curves. With this knowledge of the specificity of each antiserum, corrections can be properly made for the assays of hLH, hLH-alpha and hLH-beta while the hFSH RIA can be used without correction for the presence of the three other components.

Antibody Specificity↗