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R St-Arnaud

Publications and source records attributed to R St-Arnaud.

At least 19 recordsLinked to original sources
PubMed2003

Correction of the abnormal mineral ion homeostasis with a high-calcium, high-phosphorus, high-lactose diet rescues the PDDR phenotype of mice deficient for the 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1).

Mutations in the 25-hydroxyvitamin D-1alpha-hydroxylase gene (CYP27B1; 1alpha-OHase) cause pseudo vitamin D deficiency rickets (PDDR), while mutations in the vitamin D receptor (VDR) cause hereditary vitamin D resistance rickets. Animal models of both diseases have been engineered. The bone phenotype of VDR-ablated mice can be completely rescued by feeding the animals with a high-calcium, high-phosphorus, high-lactose diet. We have attempted to rescue the PDDR phenotype of mice deficient for the 1alpha-OHase gene by feeding them with the high-calcium diet. The rescue regimen consisted of feeding a diet containing 2% calcium, 1.25% phosphorus, 20% lactose (rescue diet) from 3 weeks of age until sacrifice at 8.5 weeks of age. Blood biochemistry analysis revealed that the rescue diet corrected the hypocalcemia and secondary hyperparathyroidism. Despite the restoration of normocalcemia, 1alpha-OHase(-/-) (and 1alpha-OHase(+/-)) animals fed the rescue diet initially gained weight less rapidly than control mice fed normal mouse chow. Although 1alpha-OHase(-/-) mice fed the rescue diet eventually reached the same weight as control animals, the treatment did not entirely correct bone growth, as femur size remained significantly smaller than that of control. Bone histology and histomorphometry confirmed that the rickets and osteomalacia were cured. The rescue diet also restored the biomechanical properties of the bone tissue within normal parameters. These results demonstrate that correction of the abnormal mineral ion homeostasis by feeding with a high-calcium rescue diet is effective to rescue the PDDR phenotype of 1alpha-OHase mutant mice. This treatment, however, does not appear as effective as 1,25(OH)(2)D(3) replacement therapy since bone growth remained impaired.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2001

HES6 acts as a transcriptional repressor in myoblasts and can induce the myogenic differentiation program.

HES6 is a novel member of the family of basic helix-loop-helix mammalian homologues of Drosophila Hairy and Enhancer of split. We have analyzed the biochemical and functional roles of HES6 in myoblasts. HES6 interacted with the corepressor transducin-like Enhancer of split 1 in yeast and mammalian cells through its WRPW COOH-terminal motif. HES6 repressed transcription from an N box-containing template and also when tethered to DNA through the GAL4 DNA binding domain. On N box-containing promoters, HES6 cooperated with HES1 to achieve maximal repression. An HES6-VP16 activation domain fusion protein activated the N box-containing reporter, confirming that HES6 bound the N box in muscle cells. The expression of HES6 was induced when myoblasts fused to become differentiated myotubes. Constitutive expression of HES6 in myoblasts inhibited expression of MyoR, a repressor of myogenesis, and induced differentiation, as evidenced by fusion into myotubes and expression of the muscle marker myosin heavy chain. Reciprocally, blocking endogenous HES6 function by using a WRPW-deleted dominant negative HES6 mutant led to increased expression of MyoR and completely blocked the muscle development program. Our results show that HES6 is an important regulator of myogenesis and suggest that MyoR is a target for HES6-dependent transcriptional repression.

Active Transport, Cell Nucleus↗
PubMed2001

Targeted inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets.

Pseudovitamin D-deficiency rickets is caused by mutations in the cytochrome P450 enzyme, 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-OHase). Patients with the disease exhibit growth retardation, rickets, and osteomalacia. Serum biochemistry is characterized by hypocalcemia, secondary hyperparathyroidism, and undetectable levels of 1alpha,25-dihydroxyvitamin D(3). We have inactivated the 1alpha-OHase gene in mice after homologous recombination in embryonic stem cells. Serum analysis of homozygous mutant animals confirmed that they were hypocalcemic, hypophosphatemic, hyperparathyroidic, and that they had undetectable 1alpha,25-dihydroxyvitamin D(3). Histological analysis of the bones from 3-week-old mutant animals confirmed the evidence of rickets. At the age of 8 weeks, femurs from 1alpha-OHase-ablated mice present a severe disorganization in the architecture of the growth plate and marked osteomalacia. These results show that we have successfully inactivated the 1alpha-OHase gene in mice and established a valid animal model of pseudovitamin D-deficiency rickets.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2000

Heart, brain, and body wall defects in mice lacking calreticulin.

Calreticulin is a ubiquitously expressed protein, which has been implicated in a large number of cellular functions, including calcium storage and signaling, protein folding, and cell attachment. To examine the role of calreticulin during in vivo development, mice deficient in calreticulin were generated by targeted inactivation of the calreticulin gene. Calreticulin-deficient mutants die in utero, mostly in late gestation. Half of these embryos had decreased cardiac cell mass, associated with increased apoptosis of cardiac myocytes. In vitro differentiation cultures of calreticulin-deficient embryonic stem cells resulted in fewer embryoid bodies with contractile activity than cultures derived from calreticulin +/- stem cells (P < 0.001). Sixteen percent of the mutants exhibited exencephaly secondary to a defect in neural tube closure. Embryos surviving until Embryonic Day 16.5 had omphalocele. Lack of calreticulin did not influence survival of embryonic fibroblasts under various endoplasmic reticulum stress conditions. However, calreticulin did influence cell migration in a calcium- and substrate-dependent manner. We conclude that calreticulin is not essential during the early stages of embryonic development, but is important for the development of heart and brain and for ventral body wall closure. The observed abnormalities are compatible with a role of calreticulin in the modulation of cellular calcium signaling.

Abnormalities, Multiple↗
PubMed2000

Deficient mineralization of intramembranous bone in vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D.

The 25-hydroxyvitamin D-24-hydroxylase enzyme (24-OHase) is responsible for the catabolic breakdown of 1,25-dihydroxyvitamin D [1,25(OH)2D], the active form of vitamin D. The 24-OHase enzyme can also act on the 25-hydroxyvitamin D substrate to generate 24,25-dihydroxyvitamin D, a metabolite whose physiological importance remains unclear. We report that mice with a targeted inactivating mutation of the 24-OHase gene had impaired 1,25(OH)2D catabolism. Surprisingly, complete absence of 24-OHase activity during development leads to impaired intramembranous bone mineralization. This phenotype was rescued by crossing the 24-OHase mutant mice to mice harboring a targeted mutation in the vitamin D receptor gene, confirming that the elevated 1,25(OH)2D levels, acting through the vitamin D receptor, were responsible for the observed accumulation of osteoid. Our results confirm the physiological importance of the 24-OHase enzyme for maintaining vitamin D homeostasis, and they reveal that 24,25-dihydroxyvitamin D is a dispensable metabolite during bone development.

24,25-Dihydroxyvitamin D 3↗
PubMed1999

Targeted inactivation of vitamin D hydroxylases in mice.

Vitamin D undergoes a first hydroxylation in the liver to generate 25-hydroxyvitamin D, then this metabolite is further hydroxylated in the kidney to yield either 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D], or 24R,25-dihydroxyvitamin D[24,25(OH)2D]. The production of 1alpha,25(OH)2D is catalyzed by the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase), while the synthesis of 24,25(OH)2D is catalyzed by the enzyme 25-hydroxyvitamin D-24-hydroxylase (24-OHase). To determine the role of each of these enzymes in vivo and their putative role during development, we have inactivated each gene by homologous recombination in embryonic stem cells. The targeting vector for the 1alpha-OHase gene was constructed to allow tissue-specific gene inactivation in order to study the hypothesized paracrine/autocrine roles of the 1alpha-OHase enzyme in particular target tissues such as skin, brain, or macrophages. The targeting vector for the 24-OHase gene utilized standard methodology, and analysis of the phenotype of 24-OHase-deficient mice confirmed the role of the 24-OHase enzyme in the catabolism of 1alpha,25(OH)2D. The phenotype of the second generation 24-OHase-null mice also suggests a key role for 24,25(OH)2D in intramembranous bone formation during development.

Animals↗
PubMed1999

Novel findings about 24,25-dihydroxyvitamin D: an active metabolite?

The physiological role of 24,25-dihydroxyvitamin D remains controversial. Recent results suggest that 24,25-dihydroxyvitamin D is essential for fracture healing, and binding sites for 24,25-dihydroxyvitamin D have been identified in fracture callus tissue. Mice deficient in the 25-hydroxyvitamin D-24-hydroxylase enzyme provide novel genetic tools in which to study the role of 24,25-dihydroxyvitamin D in bone development and fracture repair.

24,25-Dihydroxyvitamin D 3↗
PubMed1999

Dual functions for transcriptional regulators: myth or reality?

Several transcriptional regulatory molecules have been described that appear to possess dual function in separate cellular compartments. It remains unclear whether the proteins really exert dual functions, or which of the transcriptional regulatory role or the cytoplasm-associated function is the physiologically relevant action of the protein. This review will briefly describe the cases at hand and attempt to sort the true bifunctional proteins from the aritfactual trespassers. J. Cell. Biochem. Suppls. 32/33:32-40, 1999.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

Transcriptional coactivators potentiating AP-1 function in bone.

The AP-1 proteins are formed by the heterodimerization of Fos family members and Jun family members through a structural motif called the leucine zipper. The heterodimer can then bind DNA at a consensus site termed the AP-1 site and act as a transcription factor to modulate the expression of AP-1-responsive genes. All the Jun family members can also homodimerize to exert the same function. Genetic studies including gain-of-function and loss-of-function mutations have shown that AP-1 components, particularly the c-Fos protein, are essential for proper bone development. Both Fos and Jun family members interact with coactivator molecules to activate transcription. To date, the coactivator proteins CBP (CREB-binding protein), JAB1 (Jun-activation domain-binding protein 1), and alpha-NAC (Nascent polypeptide associated complex And Coactivator alpha) have been shown to potentiate the AP-1 transcriptional activating function. We have shown that all three proteins are expressed in bone during mouse development. These findings raise the intriguing possibility that multiple coactivators may be involved in mediating AP-1-dependent transcription and increase the specificity of target gene activation by AP-1 proteins in differentiating bone cells.

Animals↗
PubMed1998

The alpha chain of the nascent polypeptide-associated complex functions as a transcriptional coactivator.

We report the characterization of clone 1.9.2, a gene expressed in mineralizing osteoblasts. Remarkably, clone 1.9.2 is the murine homolog of the alpha chain of the nascent polypeptide-associated complex (alpha-NAC). Based on sequence similarities between alpha-NAC/1.9.2 and transcriptional regulatory proteins and the fact that the heterodimerization partner of alpha-NAC was identified as the transcription factor BTF3b (B. Wiedmann, H. Sakai, T. A. Davis, and M. Wiedmann, Nature 370:434-440, 1994), we investigated a putative role for alpha-NAC/ 1.9.2 in transcriptional control. The alpha-NAC/1.9.2 protein potentiated by 10-fold the activity of the chimeric activator GAL4/VP-16 in vivo. The potentiation was shown to be mediated at the level of gene transcription, because alpha-NAC/1.9.2 increased GAL4/VP-16-mediated mRNA synthesis without affecting the half-life of the GAL4/VP-16 fusion protein. Moreover, the interaction of alpha-NAC/1.9.2 with a transcriptionally defective mutant of GAL4/VP-16 was severely compromised. Specific protein-protein interactions between alpha-NAC/1.9.2 and GAL4/VP-16 were demonstrated by gel retardation, affinity chromatography, and protein blotting assays, while interactions with TATA box-binding protein (TBP) were detected by immunoprecipitation, affinity chromatography, and protein blotting assays. Based on these interactions that define the coactivator class of proteins, we conclude that the alapha-NAC/1.9.2 gene product functions as a transcriptional coactivator.

Animals↗
PubMed1998

Bone-specific expression of the alpha chain of the nascent polypeptide-associated complex, a coactivator potentiating c-Jun-mediated transcription.

The alpha chain of the nascent polypeptide-associated complex (alpha-NAC) coactivator was shown to potentiate the activity of the homodimeric c-Jun activator, while transcription mediated by the c-Fos/c-Jun heterodimer was unaffected. The use of deletion mutants in pull-down assays revealed that alpha-NAC interacted with amino acids 1 to 89 of the c-Jun protein and that the coactivator could interact with both the unphosphorylated and the serine 73-phosphorylated form of c-Jun. N-terminal-deleted c-Jun protein failed to interact with alpha-NAC in mammalian two-hybrid assays, while mutant c-Jun proteins lacking the leucine zipper or the basic domain retained interaction with alpha-NAC in vivo. Kinetics studies with purified c-Jun homodimer and recombinant alpha-NAC proteins allowed determination of the mechanism of coactivation by alpha-NAC: the coactivator stabilized the AP-1 complex formed by the c-Jun homodimer on its DNA recognition sequence through an eightfold reduction in the dissociation constant (kd) of the complex. This effect of alpha-NAC was specific, because alpha-NAC could not stabilize the interactions of JunB or Sp1 with their cognate binding sites. Interestingly, the expression of alpha-NAC was first detected at 14.5 to 15 days postconception, concomitantly with the onset of ossification during embryogenesis. The alpha-NAC protein was specifically expressed in differentiated osteoblasts at the centers of ossification. Thus, the alpha-NAC gene product exhibits the properties of a developmentally regulated, bone-specific transcriptional coactivator.

Animals↗
PubMed1998

Transcriptional regulation during mesenchymal cell differentiation: the role of coactivators.

We have begun to understand the molecular mechanisms involved in the differentiation of pluripotent mesenchymal stem cells through the identification and characterization of the sequence-specific DNA-binding transcriptional activators that control differentiation along specific lineages. However, recent progress in the study of the mechanisms of gene transcription has identified an additional class of proteins essential for activated gene transcription, namely, the coactivators (sometimes also referred to as adaptors, mediators, or integrator molecules). This review focuses on the identified coactivators that could play a regulatory role in the differentiation of mesenchymal precursors along the myogenic and osteoblastic programs. Interestingly, one such coactivator specifically expressed in bone cells during development, alpha NAC (Nascent-polypeptide-associated complex And Coactivator alpha), is converted into a DNA-binding activator by differential splicing in differentiated myotubes. This suggests that NAC isoforms may be involved in multiple steps along the lineage-making decisions facing pluripotent mesenchymal precursors.

Animals↗
PubMed1998

Molecular cloning of (25-OH D)-1 alpha-hydroxylase: an approach to the understanding of vitamin D pseudo-deficiency.

Pseudovitamin D-deficiency rickets (PDDR) is the first identified inborn error of vitamin D metabolism. Its clinical course is similar to that of nutritional rickets due to simple vitamin D deficiency. The treatment of choice is replacement therapy with calcitriol [1,25(OH)2D3]. PDDR is inherited as a simple autosomal recessive trait. The PDDR locus has been mapped to chromosome 12q13-q14. The molecular defect underlying the 25-hydroxyvitamin D-1 alpha-hydroxylase enzyme dysfunction has remained elusive due to the lack of sequence information for the gene encoding the cytochrome P450 moiety of the enzyme. We have used a probe derived from the rat 25-hydroxyvitamin D-24-hydroxylase sequence to identify and clone the 1 alpha-OHase cDNA. The candidate gene was transiently expressed in P19 embryonal carcinoma cells. Only those cells that were transfected with the candidate cDNA in the sense orientation were able to produce a compound that co-eluted with the 1 alpha, 25 vitamin D3 standard. Mass spectrometry analysis confirmed the identity of the produced metabolite. A human genomic clone was isolated from a chromosome 12 cosmid library and subsequently mapped to human chromosome 12q13.1-q13.3. To address the putative biological function of 24,25-dihydroxyvitamin I) 24,25(OH)2D, we also engineered a null mutation in the 24-OHase gene in embryonic stem cells (ES). Animals heterozygous for the engineered mutation are normal and fertile. One half of the homozygous animals die before weaning. Breeding of surviving females gives an F2 generation in which bone development is abnormal at sites of intramembranous ossification. Growthplate maturation and endochondral ossification appeared to proceed normally. The results show that a complete absence of vitamin D metabolites hydroxylated in position 24 during embryogenesis leads to abnormal bone structure and suggests a key role for 24,25(OH)2D in the developmental regulation of intramembranous ossification.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1997

Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion.

Integrins are important mediators of cell adhesion to extracellular ligands and can transduce biochemical signals both into and out of cells. The cytoplasmic domains of integrins interact with several structural and signalling proteins and consequently participate in the regulation of cell shape, motility, growth and differentiation. It has been shown that calreticulin associates with the cytoplasmic domains of integrin alpha-subunits and that this interaction can influence integrin-mediated cell adhesion to extracellular matrix. We have now developed calreticulin-deficient embryonic stem (ES) cells and isolated embryonic fibroblasts from calreticulin mutant mice. We find that in both cell types integrin-mediated adhesion is severely impaired, although integrin expression is unaltered. Expression of recombinant calreticulin in double knockout ES cells by complementary DNA transfection rescued integrin-mediated adhesion. In wild-type cells, engagement of surface integrins induced a transient elevation in cytosolic calcium concentration owing to influx of extracellular calcium. This calcium transient was absent in calreticulin-deficient cells. In contrast, the amount of calcium in endomembrane stores, which is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin, was indistinguishable in the two cell types. Our results indicate that calreticulin is an essential modulator both of integrin adhesive functions and integrin-initiated signalling, but that it may not play a significant role in the storage of luminal calcium.

Animals↗
PubMed1997

Evidence for ligand-dependent intramolecular folding of the AF-2 domain in vitamin D receptor-activated transcription and coactivator interaction.

A ligand-dependent transcriptional activation domain (AF-2) exists in region E of the nuclear receptors. This highly conserved domain may contact several coactivators that are putatively involved in nuclear receptor-mediated transcription. In this study, a panel of vitamin D receptor (VDR) AF-2 mutants was created to examine the importance of several conserved residues in VDR-activated transcription. Two AF-2 mutants (L417S and E420Q) exhibited normal ligand binding, heterodimerization with retinoid X receptor, and vitamin D-responsive element interaction, but they were transcriptionally inactive in a VDR-responsive reporter gene assay. All AF-2 mutations that abolished VDR-mediated transactivation also eliminated interactions between VDR and several putative coactivator proteins including suppressor of gal1 (SUG1), steroid hormone receptor coactivator-1 (SRC-1), or receptor interacting protein (RIP140), suggesting that coactivator interaction is important for AF-2-mediated transcription. In support of this concept, the minimal AF-2 domain [VDR(408-427)] fused to the gal4 DNA binding domain was sufficient to mediate transactivation as well as interaction with putative coactivators. Introducing the L417S and E420Q mutations into the minimal AF-2 domain abolished this autonomous transactivation and coactivator interactions. Finally, we demonstrate that the minimal AF-2 domain interacted with an AF-2 deletion mutant of the VDR in a 1,25-(OH)2D3-dependent manner, suggesting a ligand-induced intramolecular folding of the VDR AF-2 domain. The L417S mutant of this domain disrupted the interaction with VDR ligand-binding domain, while the E420Q mutant did not affect this interaction. These studies suggest that the conserved AF-2 motif may mediate transactivation through ligand-dependent intermolecular interaction with coactivators and through ligand-induced intramolecular contacts with the VDR ligand-binding domain itself.

Adaptor Proteins, Signal Transducing↗
PubMed1997

The 25-hydroxyvitamin D 1-alpha-hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus.

Pseudovitamin D-deficiency rickets (PDDR) is an autosomal recessive disorder that may be due to impaired activity of 25-hydroxyvitamin D-1alpha-hydroxylase, a renal cytochrome P450 enzyme (P450[1alpha]) of the vitamin D pathway. The disease locus for PDDR has been mapped by linkage analysis to 12q13-q14, but the molecular defect underlying the enzyme dysfunction has remained elusive due to the lack of sequence information for the P450(1alpha) gene (hereafter referred to as 1alpha-OHase). We have used a probe derived from the rat 25-hydroxyvitamin D-24-hydroxylase (CYP24; 24-OHase) sequence to identify and clone the 1alpha-OHase cDNA. The full-length 1alpha-OHase clone of 2.4 kb codes for a protein of predicted Mr 55 kDa. Functional activity of the cloned sequence was assessed using transient transfection, and the production of authentic 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] was confirmed using high performance liquid chromatography fractionation and time-of-flight mass spectrometry. The expression of the gene was analyzed in vitamin D-replete animals; treatment with 1alpha,25(OH)2D3 reduced 1alpha-OHase transcript levels by 70%, while administration of parathyroid hormone led to a 2-fold increase in the expression of the gene, thus confirming the hormonal regulation previously described using biochemical methods. The rat cDNA was used to obtain a human genomic clone. Interestingly, the human 1alpha-OHase gene mapped to 12q13.1-q13.3, providing strong evidence that a mutation in the 1alpha-OHase gene is responsible for the PDDR phenotype. The availability of a cloned sequence for 1alpha-OHase generates novel tools for the study of the molecular etiology of PDDR, and will allow the investigation of other disturbances of vitamin D metabolism.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗