RESUMEN
Osteoporosis and sarcopenia are common comorbid diseases, yet their shared mechanisms are largely unknown. We found that genetic variation near FAM210A was associated, through large genome-wide association studies, with fracture, bone mineral density (BMD), and appendicular and whole body lean mass, in humans. In mice, Fam210a was expressed in muscle mitochondria and cytoplasm, as well as in heart and brain, but not in bone. Grip strength and limb lean mass were reduced in tamoxifen-inducible Fam210a homozygous global knockout mice (TFam210a-/- ), and in tamoxifen-inducible Fam210 skeletal muscle cell-specific knockout mice (TFam210aMus-/- ). Decreased BMD, bone biomechanical strength, and bone formation, and elevated osteoclast activity with microarchitectural deterioration of trabecular and cortical bones, were observed in TFam210a-/- mice. BMD of male TFam210aMus-/- mice was also reduced, and osteoclast numbers and surface in TFam210aMus-/- mice increased. Microarray analysis of muscle cells from TFam210aMus-/- mice identified candidate musculoskeletal modulators. FAM210A, a novel gene, therefore has a crucial role in regulating bone structure and function, and may impact osteoporosis through a biological pathway involving muscle as well as through other mechanisms.
Asunto(s)
Peso Corporal/genética , Densidad Ósea/genética , Mitocondrias Musculares/metabolismo , Proteínas Mitocondriales/genética , Osteoporosis/metabolismo , Sarcopenia/metabolismo , Adulto , Animales , Células Cultivadas , Niño , Femenino , Perfilación de la Expresión Génica , Genes Letales , Genes Reporteros , Fuerza de la Mano , Humanos , Masculino , Ratones , Ratones Noqueados , Fuerza Muscular/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mioblastos/metabolismo , Especificidad de Órganos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoporosis/genética , Fenotipo , Polimorfismo de Nucleótido Simple , Sarcopenia/genética , Soporte de PesoRESUMEN
Circulating levels of fibroblast growth factor 23 (FGF23) increase during the early stages of kidney disease, but the underlying mechanism remains incompletely characterized. We investigated the role of vitamin D metabolites in regulating intact FGF23 production in genetically modified mice without and with adenine-induced uremia. Exogenous calcitriol (1,25-dihydroxyvitamin D) and high circulating levels of calcidiol (25-hydroxyvitamin D) each increased serum FGF23 levels in wild-type mice and in mice with global deficiency of the Cyp27b1 gene encoding 25-hydroxyvitamin D 1-α-hydroxylase, which produces 1,25-hydroxyvitamin D. Compared with wild-type mice, normal, or uremic mice lacking Cyp27b1 had lower levels of serum FGF23, despite having high concentrations of parathyroid hormone, but administration of exogenous 1,25-dihydroxyvitamin D increased FGF23 levels. Furthermore, raising serum calcium levels in Cyp27b1-depleted mice directly increased FGF23 levels and indirectly enhanced the action of ambient vitamin D metabolites via the vitamin D receptor. In chromatin immunoprecipitation assays, 25-hydroxyvitamin D promoted binding of the vitamin D receptor and retinoid X receptor to the promoters of osteoblastic target genes. Conditional osteoblastic deletion of Cyp27b1 caused lower serum FGF23 levels, despite normal circulating levels of vitamin D metabolites. In adenine-induced uremia, only a modest increase in serum FGF23 levels occurred in mice with osteoblastic deletion of Cyp27b1 (12-fold) compared with a large increase (58-fold) in wild-type mice. Therefore, in addition to the direct effect of high circulating concentrations of 25-hydroxyvitamin D, local osteoblastic conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D appears to be an important positive regulator of FGF23 production, particularly in uremia.
Asunto(s)
Factores de Crecimiento de Fibroblastos/fisiología , Osteoblastos/metabolismo , Vitamina D/análogos & derivados , Animales , Factor-23 de Crecimiento de Fibroblastos , Enfermedades Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Vitamina D/biosíntesis , Vitamina D/fisiologíaRESUMEN
Vitamin D signaling regulates cell proliferation and differentiation, and epidemiological data suggest that it functions as a cancer chemopreventive agent, although the underlying mechanisms are poorly understood. Vitamin D signaling can suppress expression of genes regulated by c-MYC, a transcription factor that controls epidermal differentiation and cell proliferation and whose activity is frequently elevated in cancer. We show through cell- and animal-based studies and mathematical modeling that hormonal 1,25-dihydroxyvitamin D (1,25D) and the vitamin D receptor (VDR) profoundly alter, through multiple mechanisms, the balance in function of c-MYC and its antagonist the transcriptional repressor MAD1/MXD1. 1,25D inhibited transcription of c-MYC-regulated genes in vitro, and topical 1,25D suppressed expression of c-MYC and its target setd8 in mouse skin, whereas MXD1 levels increased. 1,25D inhibited MYC gene expression and accelerated its protein turnover. In contrast, it enhanced MXD1 expression and stability, dramatically altering ratios of DNA-bound c-MYC and MXD1. Remarkably, F-box protein FBW7, an E3-ubiquitin ligase, controlled stability of both arms of the c-MYC/MXD1 push-pull network, and FBW7 ablation attenuated 1,25D regulation of c-MYC and MXD1 turnover. Additionally, c-MYC expression increased upon VDR knockdown, an effect abrogated by ablation of MYC regulator ß-catenin. c-MYC levels were widely elevated in vdr(-/-) mice, including in intestinal epithelium, where hyperproliferation has been reported, and in skin epithelia, where phenotypes of VDR-deficient mice and those overexpressing epidermal c-MYC are similar. Thus, 1,25D and the VDR regulate the c-MYC/MXD1 network to suppress c-MYC function, providing a molecular basis for cancer preventive actions of vitamin D.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Calcitriol/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Receptores de Calcitriol/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Calcitriol/farmacología , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD , Regulación de la Expresión Génica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Noqueados , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/prevención & control , Estabilidad Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Receptores de Calcitriol/genética , Proteínas Represoras/genética , Transducción de Señal/efectos de los fármacos , Piel/metabolismo , Transcripción Genética/efectos de los fármacos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Increased risk of bone fractures is observed in patients with chronic inflammatory conditions, such as inflammatory bowel disease and rheumatoid arthritis. Members of the Interferon Response Factor family of transcriptional regulators, IRF1 and IRF8, have been identified as genetic risk factors for several chronic inflammatory and autoimmune diseases. We have investigated a potential role for the Irf1 gene in bone metabolism. Here, we report that Irf1(-/-) mutant mice show altered bone morphology in association with altered trabecular bone architecture and increased cortical thickness and cellularity. Ex vivo studies on cells derived from bone marrow stimulated with Rank ligand revealed an increase in size and resorptive activity of tartrate-resistant acid-positive cells from Irf1(-/-) mutant mice compared with wild-type control mice. Irf1 deficiency was also associated with decreased proliferation of bone marrow-derived osteoblast precursors ex vivo, concomitant with increased mineralization activity compared with control cells. We show that Irf1 plays a role in bone metabolism and suggest that Irf1 regulates the maturation and activity of osteoclasts and osteoblasts. The altered bone phenotype of Irf1(-/-) mutants is strikingly similar to that of Stat1(-/-) mice, suggesting that the two interacting proteins play a critical enabling role in the common regulation of these two cell lineages.
Asunto(s)
Resorción Ósea/metabolismo , Huesos/metabolismo , Diferenciación Celular , Factor 1 Regulador del Interferón/fisiología , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Animales , Western Blotting , Resorción Ósea/patología , Huesos/citología , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/citología , Osteoclastos/citología , Fenotipo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The differential effects between cinacalcet and active vitamin D compounds on parathyroid function, mineral metabolism, and skeletal function are incompletely understood. Here, we studied cinacalcet and active vitamin D compounds in mice expressing the null mutation for Cyp27b1, which encodes 25-hydroxyvitamin D-1α-hydroxylase, thereby lacking endogenous 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Vehicle-treated mice given high dietary calcium had hypocalcemia, hypophosphatemia, and marked secondary hyperparathyroidism. Doxercalciferol and 1,25(OH)(2)D(3) each normalized these parameters and corrected both the abnormal growth plate architecture and the diminished longitudinal bone growth observed in these mice. In contrast, cinacalcet suppressed serum parathyroid hormone (PTH) cyclically and did not correct the skeletal abnormalities and hypocalcemia persisted. Vehicle-treated mice given a "rescue diet" (high calcium and phosphorus, 20% lactose) had normal serum calcium and PTH levels; cinacalcet induced transient hypocalcemia and mild hypercalciuria. The active vitamin D compounds and cinacalcet normalized the increased osteoblast activity observed in mice with secondary hyperparathyroidism; cinacalcet, however, increased the number and activity of osteoclasts. In conclusion, cinacalcet reduces PTH in a cyclical manner, does not eliminate hypocalcemia, and does not correct abnormalities of the growth plate. Doxercalciferol and 1,25(OH)(2)D(3) reduce PTH in a sustained manner, normalize serum calcium, and improve skeletal abnormalities.
Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Huesos/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Naftalenos/farmacología , Glándulas Paratiroides/efectos de los fármacos , Vitamina D/farmacología , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/deficiencia , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Animales , Densidad Ósea/efectos de los fármacos , Conservadores de la Densidad Ósea/uso terapéutico , Cinacalcet , Homeostasis/efectos de los fármacos , Ratones , Mutación , Naftalenos/uso terapéutico , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Glándulas Paratiroides/patología , Vitamina D/uso terapéutico , Deficiencia de Vitamina D/sangre , Deficiencia de Vitamina D/tratamiento farmacológico , Deficiencia de Vitamina D/patologíaRESUMEN
We induced chronic kidney disease (CKD) with adenine in WT mice, mice with osteocyte-specific deletion of Cyp27b1, encoding the 25-hydroxyvitamin D 1(OH)ase [Oct-1(OH)ase-/-], and mice with global deletion of Cyp27b1 [global-1α(OH)ase-/-]; we then compared extraskeletal calcification. After adenine treatment, mice displayed increased blood urea nitrogen, decreased serum 1,25(OH)2D, and severe hyperparathyroidism. Skeletal expression of Cyp27b1 and of sclerostin and serum sclerostin all increased in WT mice but not in Oct-1α(OH)ase-/- mice or global-1α(OH)ase-/- mice. In contrast, skeletal expression of BMP2 and serum BMP2 rose in the Oct-1α(OH)ase-/- mice and in the global-1α(OH)ase-/- mice. Extraskeletal calcification occurred in muscle and blood vessels of mice with CKD and was highest in Oct-1α(OH)ase-/-mice. In vitro, recombinant sclerostin (100 ng/mL) significantly suppressed BMP2-induced osteoblastic transdifferentiation of vascular smooth muscle A7r5 cells and diminished BMP2-induced mineralization. Our study provides evidence that local osteocytic production of 1,25(OH)2D stimulates sclerostin and inhibits BMP2 production in murine CKD, thus mitigating osteoblastic transdifferentiation and mineralization of soft tissues. Increased osteocytic 1,25(OH)2D production, triggered by renal malfunction, may represent a "primary defensive response" to protect the organism from ectopic calcification by increasing sclerostin and suppressing BMP2 production.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Calcinosis/patología , Calcitriol/metabolismo , Insuficiencia Renal Crónica/complicaciones , Uremia/complicaciones , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Proteínas Adaptadoras Transductoras de Señales/sangre , Adenina/toxicidad , Animales , Nitrógeno de la Urea Sanguínea , Proteína Morfogenética Ósea 2/sangre , Huesos/citología , Huesos/metabolismo , Calcinosis/sangre , Calcinosis/etiología , Calcitriol/sangre , Transdiferenciación Celular , Modelos Animales de Enfermedad , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Masculino , Ratones , Osteocitos/metabolismo , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/inducido químicamente , Uremia/sangre , Uremia/inducido químicamenteRESUMEN
Links between solar UV exposure and immunity date back to the ancient Greeks with the development of heliotherapy. Skin contains several UV-sensitive chromophores and exposure to sunlight can produce molecules, such as vitamin D3, that act in an endocrine manner. We investigated the role of the aryl hydrocarbon receptor (AHR), an environmental sensor and ligand-regulated transcription factor activated by numerous planar compounds of endogenous, dietary or environmental origin. 15- to 30-minute exposure of cells to a minimal erythemal dose of UVB irradiation in vitro induced translocation of the AHR to the nucleus, rapidly inducing site-specific DNA binding and target gene regulation. Importantly, ex vivo studies with Ahr wild-type or null fibroblasts showed that serum from mice whose skin was exposed to a 15 min UVB dose, but not control serum, contained agonist activity within 30 min of UV irradiation, inducing AHR-dependent gene expression. Moreover, a 15-min cutaneous UVB exposure induced AHR site-specific DNA binding and target gene regulation in vivo within 3-6 hr post-irradiation in blood and in peripheral tissues, including intestine. These results show that cutaneous exposure of mice to a single minimal erythemic dose of UVB induces rapid AHR signaling in multiple peripheral organs, providing compelling evidence that moderate sun exposure can exert endocrine control of immunity through the AHR.
Asunto(s)
Sistema Endocrino/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal , Piel/metabolismo , Piel/efectos de la radiación , Rayos Ultravioleta , Animales , Línea Celular , Femenino , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Masculino , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genéticaRESUMEN
In the version of this article initially published, in Fig. 5a, the data in the right column of 'DAAM2 gRNA1' were incorrectly plotted as circles indicating 'untreated' rather than as squares indicating 'treated'. The error has been corrected in the HTML and PDF versions of the article.
RESUMEN
Osteoporosis is a common aging-related disease diagnosed primarily using bone mineral density (BMD). We assessed genetic determinants of BMD as estimated by heel quantitative ultrasound in 426,824 individuals, identifying 518 genome-wide significant loci (301 novel), explaining 20% of its variance. We identified 13 bone fracture loci, all associated with estimated BMD (eBMD), in ~1.2 million individuals. We then identified target genes enriched for genes known to influence bone density and strength (maximum odds ratio (OR) = 58, P = 1 × 10-75) from cell-specific features, including chromatin conformation and accessible chromatin sites. We next performed rapid-throughput skeletal phenotyping of 126 knockout mice with disruptions in predicted target genes and found an increased abnormal skeletal phenotype frequency compared to 526 unselected lines (P < 0.0001). In-depth analysis of one gene, DAAM2, showed a disproportionate decrease in bone strength relative to mineralization. This genetic atlas provides evidence linking associated SNPs to causal genes, offers new insight into osteoporosis pathophysiology, and highlights opportunities for drug development.
Asunto(s)
Densidad Ósea/genética , Predisposición Genética a la Enfermedad/genética , Osteoporosis/genética , Adulto , Anciano , Animales , Femenino , Fracturas Óseas/genética , Estudio de Asociación del Genoma Completo/métodos , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Fenotipo , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
We examined the role of bone remodeling in the regulation of circulating concentrations of FGF23 using mouse models manifesting differing degrees of coupled and uncoupled bone turnover. Administration of the antiresorptive agent osteoprotegerin produced a profound reduction in bone resorption and formation in male and oophorectomized female mice, accompanied by an increase in serum levels of fibroblast growth factor 23 (FGF23) and a reduction in circulating 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. In contrast, exogenous PTH(1-34) administration increased bone turnover and reduced circulating FGF23. In 1,25(OH)(2)D-deficient, 25-hydroxyvitamin D 1alpha-hydroxylase null mice on a high-calcium diet, endogenous PTH was elevated, bone formation but not resorption was increased, and serum FGF23 was virtually undetectable; on a rescue diet, serum calcium was normalized, PTH levels were reduced, bone formation was reduced, and serum FGF23 levels increased. After PTH treatment of wild-type mice, gene expression of dentin matrix protein 1 (DMP1) in bone was increased, whereas gene expression of FGF23 was reduced. In vitro studies in the osteoblastic cell line UMR-106 showed that externally added DMP1 could inhibit FGF23 gene expression and production stimulated by 1,25(OH)(2)D(3). The results show that osteoblastic bone formation is a potent modulator of FGF23 production and release into the circulation, suggest that the biological consequences on mineral homeostasis of circulating FGF23 may also be dependent on the prevailing rate of bone turnover, and provide evidence that DMP1 may be a direct negative regulator of FGF23 production in osteoblastic cells.
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Factores de Crecimiento de Fibroblastos/sangre , Osteogénesis , Animales , Huesos/metabolismo , Línea Celular , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/metabolismo , Vitamina D/análogos & derivados , Vitamina D/sangreRESUMEN
The retinoid X receptor alpha (RXRalpha) is a member of the nuclear receptor superfamily that regulates transcription of target genes through heterodimerization with several partners, including peroxisome proliferator-activated receptor, retinoic acid receptor, thyroid receptor, and vitamin D receptor (VDR). We have shown previously that signaling through VDR.RXRalpha heterodimers was attenuated in ras-transformed keratinocytes due to phosphorylation of serine 260 of the RXRalpha via the activated Ras-Raf-MAPK cascade in these cells. In this study we demonstrate that phosphorylation at serine 260, a site located in the omega loop-AF-2 interacting domain of RXRalpha, inhibits signaling through several heterodimeric partners of the RXRalpha. The inhibition of signaling results in reduced transactivational response to ligand presentation and the reduced physiological response of growth inhibition not only of 1,25-dihydroxyvitamin D3 but also of retinoic acid receptor alpha ligands and LG1069 (an RXRalpha ligand). This partial resistance to ligands could be reversed by inhibition of MAPK activity or by overexpression of a non-phosphorylable RXRalpha mutant at serine 260 (RXRalpha Ser-260-->Ala). Importantly, phosphorylation of RXRalpha at serine 260 impaired the recruitment of DRIP205 and other coactivators to the VDR.RXRalpha complex. Chromatin immunoprecipitation and pulldown assays further demonstrated that coactivator recruitment to the VDR.RXR complex could be restored by treatment with a MAPK inhibitor. Our data suggest that phosphorylation at serine 260 plays a critical role in inducing hormone resistance of RXRalpha-mediated signaling likely through structural changes in the H1-H3 omega loop-AF2 coactivator(s) interacting domain.
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Complejos Multiproteicos/metabolismo , Receptores de Calcitriol/metabolismo , Receptor alfa X Retinoide/metabolismo , Serina/metabolismo , Factores de Transcripción/metabolismo , Calcitriol/metabolismo , Calcitriol/farmacología , Línea Celular Transformada , Resistencia a Medicamentos/fisiología , Quinasas MAP Reguladas por Señal Extracelular , Hormonas/genética , Hormonas/metabolismo , Humanos , Ligandos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Complejo Mediador , Complejos Multiproteicos/genética , Proteína Oncogénica p21(ras)/genética , Proteína Oncogénica p21(ras)/metabolismo , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , Receptores de Calcitriol/genética , Receptor alfa X Retinoide/genética , Serina/genética , Factores de Transcripción/genética , Vitaminas/metabolismo , Vitaminas/farmacología , Quinasas raf/genética , Quinasas raf/metabolismoRESUMEN
BACKGROUND: Non-(1-84) parathyroid hormone (PTH) fragments are large circulating carboxyl-terminal (C) fragments with a partially preserved amino-terminal (N) structure. hPTH (7-84), a synthetic surrogate, has been demonstrated to exert biologic effects in vivo and in vitro which are opposite to those of hPTH (1-34) on the PTH/PTHrP type I receptor through a C-PTH receptor. We wanted to determine the N structure of non-(1-84) PTH fragments. METHODS: Parathyroid cells isolated from glands obtained at surgery from three patients with primary hyperparathyroidism and three patients with secondary hyperparathyroidism were incubated with 35S-methionine to internally label their secretion products. Incubations were performed for 8 hours at the patient-ionized calcium concentration and in the presence of various protease inhibitors. The supernatant was fractionated by high-performance liquid chromatography (HPLC) and fractions were analyzed with PTH assays having (1 to 4) and (12 to 23) epitopes, respectively. The serum of each patient was similarly analyzed. Peaks of immunoreactivity identified were submitted to sequence analysis to recover the 35S-methionine residues in positions 8 and 18. RESULTS: Three regions of interest were identified with PTH assays. They corresponded to non-(1-84) PTH fragments (further divided in regions 3 and 4), a peak of N-PTH migrating in front of hPTH (1-84) (region 2) and a peak of immunoreactivity corresponding to the elution position of hPTH (1-84) (region 1). The last corresponded to a single sequence starting at position 1. Region 2 gave similar results in all cases (a major signal starting at position 1) but also sometimes minor sequences starting at position 4 or 7. Regions 3 and 4 always identified a major sequence starting at positions 7 and minor sequences starting at positions 8, 10, and 15. Surprisingly, a major signal starting at position 1 was also present in region 3. The HPLC profile obtained from a given patient's parathyroid cells was qualitatively similar to the one obtained with his/her serum in each case. CONCLUSION: These results indicate that non-(1-84) PTH fragments are composed of a family of fragments which may be generated by specific or progressive cleavage at the N region. The longest fragment starts at position 4 and the shortest at position 15. A peptide starting at position 7 appears as the major component of non-(1-84) PTH fragments. The generation process is similar to the one described for smaller C-PTH fragments a number of years ago, suggesting a similar production mechanism and source for all C-PTH fragments.