RESUMO
Infantile hypercalcemia type 1 (HCINF1), formerly known as Lightwood syndrome, is a subtype of hypercalcemia caused by loss-of-function biallelic mutations in the vitamin D catabolic enzyme, CYP24A1, which 24-hydroxylates the hormone 1,25-(OH)2D3. This short review focuses on the main features of the HCINF1 disease; emerging knowledge of the structure and function of the cytochrome P450, CYP24A1 and the location of inactivating mutations; the development of a rapid LC-MS/MS-based laboratory test for defective 24-hydroxylation; and future implications for bioanalytical assay and treatment of all types of vitamin D-related hypercalcemic conditions.
RESUMO
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.
Assuntos
Calcificação Fisiológica/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Osteoblastos/fisiologia , Ribonucleoproteínas/fisiologia , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/genética , Calreticulina , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica , Dados de Sequência Molecular , Osteocalcina/genética , Osteocalcina/metabolismo , Osteopontina , Ligação Proteica , Receptores de Calcitriol/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Receptores X de Retinoides , Ribonucleoproteínas/genética , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteína de Ligação a Vitamina D/metabolismoRESUMO
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.
Assuntos
Proteínas de Homeodomínio , Músculo Esquelético/citologia , Proteínas Repressoras/farmacologia , Fatores de Transcrição/farmacologia , Transporte Ativo do Núcleo Celular , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proteínas Correpressoras , Humanos , Camundongos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Proteínas Musculares/efeitos dos fármacos , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/crescimento & desenvolvimento , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas Repressoras/fisiologia , Fatores de Transcrição HES-1 , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Técnicas do Sistema de Duplo-Híbrido , LevedurasRESUMO
BACKGROUND: Integrins are transmembrane cell surface receptors that mediate cell-cell and cell-matrix contacts. Integrin-linked kinase (ILK) is the binding partner of beta1 and beta3 integrins, and has been ascribed essential roles in development, angiogenesis and tumourigenesis. However, in vivo evidence for the latter is currently lacking. AIM: The hypothesis that epithelial cell-specific deletion of ILK would impact on murine tumourigenesis was tested using a colitis-associated cancer model. METHODS: To create intestinal epithelial cell ILK knockout animals, Fabp/Cre mice (Cre recombinase expressed under the control of a modified Fabp promoter) were used, and they were mated with mice carrying a loxP-flanked (floxed) ILK gene (ILK(flox/flox)). RESULTS: ILK intestinal knockout mice exhibited a reduction in the size of the caecum, and reduced crypt height in the colon. Immunohistochemical analysis confirmed that there was diminished ILK expression, and bromodeoxyuridine (BrdU) staining was significantly reduced in the knockout animals as compared with the wild-type animals in both the caecum and colon (p<0.001 for both). Following azoxymethane and dextran sodium sulfate (DSS) treatment, fewer total tumours were observed in the ILK knockout animals, which were mosaic with respect to ILK expression. Cyclin D1, Snail, fibronectin and matrix metalloproteinase 9 (MMP9) were all reduced, and active caspase 3 increased, in tumours from ILK knockout mice, as compared with wild-type mice, on immunohistochemical analysis. Using small interfering RNA (siRNA) to knock down ILK in colonic cancer cell lines, it was confirmed that it is capable of regulating cyclin D1, Snail, MMP9 and fibronectin transcription. CONCLUSIONS: From these findings, it is concluded that ILK plays an important role in intestinal epithelial cell proliferation, and that it influences the development of colitis-associated cancer, through modulation of cyclin D1, the extracellular matrix and MMP9.
Assuntos
Transformação Celular Neoplásica/metabolismo , Colite/complicações , Neoplasias do Colo/etiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Apoptose , Ceco/crescimento & desenvolvimento , Proliferação de Células , Transformação Celular Neoplásica/patologia , Colite/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ciclina D , Ciclinas/metabolismo , Fibronectinas/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Mosaicismo , Reação em Cadeia da Polimerase/métodos , Proteínas Serina-Treonina Quinases/deficiência , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismoRESUMO
It is now well established that chronic treatment with GnRH agonists offers an advantageous alternative to orchiectomy and estrogens for the treatment of prostate cancer. Castration levels of androgens can thus be easily achieved without side effects other than those related to castration levels of serum androgens. However, man is unique among species in having a high secretion rate of precursor adrenal steroids which are converted into active androgens in the normal prostate and prostatic cancer. All the enzymes required for the transformation of dehydroepiandrosterone sulfate, dehydroepiandrosterone, androstenedione, and androst-5-ene-3 beta, 17 beta-diol are present in prostatic tissue. Moreover, as shown in many systems, castration levels of serum testosterone (T) at 0.2-0.4 ng/ml exert significant androgenic activity in target tissues. In order to inhibit the action of androgens of both testicular and adrenal origin, GnRH agonists have been administered in association with the pure antiandrogen Flutamide in patients having clinical stage D2 (bone metastases) prostate cancer. A positive objective response assessed according to the criteria of the United States National Prostatic Cancer Project (USNPCP) has been observed in 84 of the 88 patients who had received no previous treatment (95.4%). After 2 yr of treatment, the probability of continuing response is 70% compared to 0-10% by previous approaches. In addition, the death rate at 2 yr is at 10.9% as compared to approximately 50% after standard hormonal therapy. When the same treatment was applied to patients who had received previous hormonal therapy (orchiectomy, estrogens or GnRH agonists alone) before showing a relapse, the response rate decreased to 62.9% and the death rate at 2 yr was 52%.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hormônios Liberadores de Hormônios Hipofisários/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Antagonistas de Androgênios/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Di-Hidrotestosterona/análise , Estrogênios/uso terapêutico , Flutamida/administração & dosagem , Humanos , Hormônio Luteinizante/sangue , Masculino , Orquiectomia , Hormônios Liberadores de Hormônios Hipofisários/administração & dosagem , Radioimunoensaio , Testosterona/sangueRESUMO
BACKGROUND: Vitamin D compounds are effective in managing elevated PTH levels in secondary hyperparathyroidism (SHPT) of renal failure. However, undesired increases in serum calcium and phosphorus associated with compounds such as calcitriol [1,25(OH)2D3] has prompted a search for compounds with improved safety profiles. 1alpha,24(S)(OH)2D2 (1,24(OH)2D2) is a vitamin D2 metabolite with low calcium-mo bilizing activity in vivo. We studied the efficacy of 1,24(OH)2D2 in mice lacking the CYP27B1 enzyme [25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase)], a novel vitamin D deficiency model with SHPT. MATERIALS AND METHODS: 1alpha-OHase-deficient (-/-) mice and normal (+/-) heterozygous littermates re ceived 1,24(OH)2D2 (100, 300, 1000, and 3000 pg/g/day) or 1,25(OH)2D3 (30, 300, and 500 pg/g/day) for 5 weeks via daily sc injection. Control groups received vehicle. RESULTS: Vehicle-treated 1alpha-OHase-deficient mice were hypocalcemic and had greatly elevated serum PTH. 1,24(OH)2D2 at doses above 300 pg/g/day normalized serum calcium, serum PTH, bone growth plate morphology, and other bone parameters. No hy percalcemia was observed at any dose of 1,24(OH)2D2 in normal or 1alpha-OHase-deficient animals. In contrast, 1,25(OH)2D3 at only 30 pg/g/day normalized calcemia, serum PTH, and bone parameters, but at higher doses completely suppressed PTH and caused hypercalcemia in both 1alpha-OHase-deficient and normal mice. Treatment with 500 pg/g/day of 1,25(OH)2D3 also induced osteomalacia in normal animals. CONCLUSION: 1,25(OH)2D3 was maximally active at 10-fold lower doses than 1,24(OH)2D2, but induced hypercalcemia and osteomalacia at high doses. 1,24(OH)2D2 normalized serum calcium, serum PTH, and bone histomorphometry without hypercalcemia in 1alpha-OHase-deficient mice with SHPT.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Osso e Ossos/efeitos dos fármacos , Ergocalciferóis/uso terapêutico , Hipercalcemia/prevenção & controle , Hormônio Paratireóideo/sangue , Deficiência de Vitamina D/tratamento farmacológico , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/deficiência , Animais , Osso e Ossos/anatomia & histologia , Calcinose/induzido quimicamente , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ergocalciferóis/efeitos adversos , Ergocalciferóis/farmacologia , Fêmur/efeitos dos fármacos , Fêmur/patologia , Hipercalcemia/genética , Hiperparatireoidismo Secundário/sangue , Hiperparatireoidismo Secundário/etiologia , Hiperparatireoidismo Secundário/genética , Hiperparatireoidismo Secundário/patologia , Camundongos , Camundongos Transgênicos , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/genética , Deficiência de Vitamina D/patologiaRESUMO
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.
Assuntos
Camundongos/genética , Sistema Nervoso/embriologia , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/biossíntese , Fatores de Transcrição/isolamento & purificação , Transcrição Gênica , Proteínas de Peixe-Zebra , Animais , Sequência de Bases , Diferenciação Celular/genética , Masculino , Camundongos/embriologia , Dados de Sequência Molecular , Mutagênese , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Deleção de Sequência , Testículo/embriologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/efeitos da radiação , Transcrição Gênica/efeitos dos fármacos , Tretinoína/farmacologia , Raios Ultravioleta , Proteínas Wnt , Proteína Wnt1RESUMO
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.
Assuntos
Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA , Proteína Vmw65 do Vírus do Herpes Simples/genética , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Camundongos , Chaperonas Moleculares , Osteoblastos/citologia , Osteoblastos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fase de Repouso do Ciclo Celular , Transativadores/genética , Fator de Transcrição TFIID , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição TFII/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Osso e Ossos/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT , Calcitriol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Osteoblastos/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/genética , Sequência de Bases , Sítios de Ligação , Osso e Ossos/citologia , Células Cultivadas , Análise Mutacional de DNA , Dados de Sequência Molecular , Fatores de Transcrição NFI , Osteoblastos/citologia , Ligação Proteica , Receptores de Calcitriol/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Transdução de Sinais , Distribuição Tecidual , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Animais , Sítios de Ligação , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Linhagem Celular , Dimerização , Expressão Gênica , Camundongos , Chaperonas Moleculares , Proteínas Proto-Oncogênicas c-jun/genética , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/biossíntese , Transativadores/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
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.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Células Tumorais Cultivadas/citologia , Animais , Astrócitos/citologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Dactinomicina/farmacologia , Dimetil Sulfóxido/farmacologia , Íntrons , Camundongos , Neurônios/citologia , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-myc , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Teratoma , Transcrição Gênica , Tretinoína/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
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.
Assuntos
Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Transcrição Gênica , Proteínas de Peixe-Zebra , Animais , Diferenciação Celular , Células-Tronco de Carcinoma Embrionário , Imunofluorescência , Camundongos , Proteínas Proto-Oncogênicas/análise , RNA Mensageiro/análise , Proteínas Wnt , Proteína Wnt1RESUMO
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.
Assuntos
Calcitriol/farmacologia , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/genética , Animais , Northern Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Camundongos , Osteoblastos/química , Osteoblastos/citologia , Proteínas Proto-Oncogênicas c-fos/análise , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/análise , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
Proteínas Nucleares/metabolismo , Receptores de Calcitriol/metabolismo , Transdução de Sinais , Ativação Transcricional , Vitamina D/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Células COS , Ligantes , Dados de Sequência Molecular , Mutação , Proteína 1 de Interação com Receptor Nuclear , Dobramento de Proteína , Receptores de Calcitriol/química , Receptores de Calcitriol/genéticaRESUMO
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.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Sistema Enzimático do Citocromo P-450/genética , Raquitismo/genética , Esteroide Hidroxilases/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/química , Sequência de Aminoácidos , Animais , Calcitriol/química , Calcitriol/uso terapêutico , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/química , DNA Complementar/química , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Dados de Sequência Molecular , Peso Molecular , Hormônio Paratireóideo/uso terapêutico , Ratos , Ratos Sprague-Dawley , Mapeamento por Restrição , Raquitismo/enzimologia , Raquitismo/etiologia , Homologia de Sequência de Aminoácidos , Esteroide Hidroxilases/química , Transcrição Gênica/genética , Transfecção , Deficiência de Vitamina D/enzimologia , Deficiência de Vitamina D/genética , Vitamina D3 24-HidroxilaseRESUMO
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.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Inativação Gênica/fisiologia , Marcação de Genes , Raquitismo/etiologia , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/genética , Animais , Modelos Animais de Doenças , Fêmur/patologia , Expressão Gênica/fisiologia , Hiperparatireoidismo/etiologia , Hipocalcemia/etiologia , Hipofosfatemia/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Raquitismo/patologia , Vitamina D/fisiologia , Deficiência de Vitamina D/sangueRESUMO
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.
Assuntos
24,25-Di-Hidroxivitamina D 3/deficiência , Densidade Óssea , Calcitriol/metabolismo , Sistema Enzimático do Citocromo P-450/deficiência , Receptores de Calcitriol/deficiência , Esteroide Hidroxilases/deficiência , Alelos , Animais , Calcitriol/sangue , Calcitriol/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Feminino , Hibridização Genética , Rim/efeitos dos fármacos , Rim/patologia , Camundongos , Camundongos Knockout/genética , Mutação/fisiologia , Fenótipo , Ratos , Receptores de Calcitriol/genética , Esteroide Hidroxilases/genética , Vitamina D3 24-HidroxilaseRESUMO
We recently reported almost complete disappearance of serum LH biological activity in previously untreated patients with advanced prostatic cancer receiving combined therapy with a LHRH agonist and a pure antiandrogen. This decrease in LH bioactivity was most likely responsible for the fall of circulating testosterone to castration levels during such treatment. Since patients previously treated with high doses of estrogens or orchiectomy before receiving combined therapy had a less favorable response to the new treatment, we measured serum LH levels by RIA and the mouse interstitial cell bioassay in these 2 groups of patients. Serum samples were obtained from 14 men with advanced prostatic cancer treated from 9-41 months (24 +/- 9 months) with diethylstilbestrol before receiving 500 micrograms/day LHRH agonist ([D-Trp6]LH/RH ethylamide) in combination with 3 daily oral doses of 250 mg pure antiandrogen flutamide and from 21 men castrated for at least 9 months (32 +/- 26 months) before receiving the antiandrogen alone. In previously castrated patients, both bio- and immunoactive LH serum levels were elevated and did not change during at least 3 months of antiandrogen treatment. In estrogen-pretreated men, however, bioactive LH concentrations declined from 1.2 +/- 0.5 (+/- SEM) to 0.04 +/- 0.01 ng/ml after 1 month of combined treatment and remained low thereafter, while serum LH levels, measured by RIA, did not significantly decline (1.4 +/- 0.5 vs. 0.9 +/- 0.1 ng/ml on days--2 and 30, respectively). This decrease in LH biopotency caused the biological to immunological activity ratio to fall from 0.5 +/- 0.2 before the onset of the combined therapy to 0.05 +/- 0.01 after 3 months. Thus, estrogen pretreatment did not prevent the ability of the LHRH agonist-antiandrogen combination to decrease serum LH biological activity. Moreover, the absence of an effect in castrated patients receiving antiandrogen alone indicates that the LHRH agonist, and not flutamide, was responsible for the effects of the combined therapy.
Assuntos
Anilidas/uso terapêutico , Dietilestilbestrol/uso terapêutico , Flutamida/uso terapêutico , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Luteinizante/sangue , Orquiectomia , Neoplasias da Próstata/tratamento farmacológico , Pamoato de Triptorrelina/análogos & derivados , Idoso , Bioensaio , Terapia Combinada , Quimioterapia Combinada , Hormônio Liberador de Gonadotropina/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/sangue , Neoplasias da Próstata/cirurgia , Radioimunoensaio , Fatores de TempoRESUMO
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.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica , Mesoderma/citologia , Transativadores/metabolismo , Transcrição Gênica , Animais , Humanos , Splicing de RNARESUMO
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.