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1.
Bone Res ; 12(1): 3, 2024 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-38253615

RESUMEN

Disc degeneration primarily contributes to chronic low back and neck pain. Consequently, there is an urgent need to understand the spectrum of disc degeneration phenotypes such as fibrosis, ectopic calcification, herniation, or mixed phenotypes. Amongst these phenotypes, disc calcification is the least studied. Ectopic calcification, by definition, is the pathological mineralization of soft tissues, widely studied in the context of conditions that afflict vasculature, skin, and cartilage. Clinically, disc calcification is associated with poor surgical outcomes and back pain refractory to conservative treatment. It is frequently seen as a consequence of disc aging and progressive degeneration but exhibits unique molecular and morphological characteristics: hypertrophic chondrocyte-like cell differentiation; TNAP, ENPP1, and ANK upregulation; cell death; altered Pi and PPi homeostasis; and local inflammation. Recent studies in mouse models have provided a better understanding of the mechanisms underlying this phenotype. It is essential to recognize that the presentation and nature of mineralization differ between AF, NP, and EP compartments. Moreover, the combination of anatomic location, genetics, and environmental stressors, such as aging or trauma, govern the predisposition to calcification. Lastly, the systemic regulation of calcium and Pi metabolism is less important than the local activity of PPi modulated by the ANK-ENPP1 axis, along with disc cell death and differentiation status. While there is limited understanding of this phenotype, understanding the molecular pathways governing local intervertebral disc calcification may lead to developing disease-modifying drugs and better clinical management of degeneration-related pathologies.


Asunto(s)
Calcinosis , Condrocalcinosis , Degeneración del Disco Intervertebral , Disco Intervertebral , Animales , Ratones , Degeneración del Disco Intervertebral/genética , Calcinosis/genética , Inflamación
2.
Cell Death Dis ; 14(7): 447, 2023 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-37468461

RESUMEN

Pathological mineralization of intervertebral disc is debilitating and painful and linked to disc degeneration in a subset of human patients. An adenosine triphosphate efflux transporter, progressive ankylosis (ANK) is a regulator of extracellular inorganic pyrophosphate levels and plays an important role in tissue mineralization. However, the function of ANK in intervertebral disc has not been fully explored. Herein we analyzed the spinal phenotype of Ank mutant mice (ank/ank) with attenuated ANK function. Micro-computed tomography and histological analysis showed that loss of ANK function results in the aberrant annulus fibrosus mineralization and peripheral disc fusions with cranial to caudal progression in the spine. Vertebrae in ank mice exhibit elevated cortical bone mass and increased tissue non-specific alkaline phosphatase-positive endplate chondrocytes with decreased subchondral endplate porosity. The acellular dystrophic mineral inclusions in the annulus fibrosus were localized adjacent to apoptotic cells and cells that acquired osteoblast-like phenotype. Fourier transform infrared spectral imaging showed that the apatite mineral in the outer annulus fibrosus had similar chemical composition to that of vertebral bone. Transcriptomic analysis of annulus fibrosus and nucleus pulposus tissues showed changes in several biological themes with a prominent dysregulation of BMAL1/CLOCK circadian regulation. The present study provides new insights into the role of ANK in the disc tissue compartments and highlights the importance of local inorganic pyrophosphate metabolism in inhibiting the mineralization of this important connective tissue.


Asunto(s)
Calcinosis , Degeneración del Disco Intervertebral , Disco Intervertebral , Animales , Humanos , Ratones , Calcinosis/patología , Difosfatos/metabolismo , Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/patología , Mutación con Pérdida de Función , Fenotipo , Microtomografía por Rayos X
3.
Anal Bioanal Chem ; 415(3): 481-492, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36400967

RESUMEN

Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe a PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (< 3.5%) and accuracy (93-106%) of PPi spiked into human plasma samples. We found that of several anticoagulants tested only EDTA effectively blocked conversion of ATP into PPi in plasma after blood collection. Moreover, filtration over a 300,000-Da molecular weight cut-off membrane reduced variability of plasma PPi and removed ATP present in a membrane-enclosed compartment, possibly platelets. Applied to plasma samples of wild-type and Abcc6-/- rats, an animal model with established low circulating levels of PPi, the new assay showed lower variability than the assay that was previously in routine use in our laboratory. In conclusion, we here report a new and robust assay to determine PPi concentrations in plasma, which outperforms currently available assays because of its high sensitivity, precision, and accuracy.


Asunto(s)
Calcinosis , Difosfatos , Humanos , Ratas , Animales , Luciferasas de Luciérnaga , Adenosina Trifosfato
4.
Hum Mutat ; 43(12): 1872-1881, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36317459

RESUMEN

ABCC6 promotes ATP efflux from hepatocytes to bloodstream. ATP is metabolized to pyrophosphate, an inhibitor of ectopic calcification. Pathogenic variants of ABCC6 cause pseudoxanthoma elasticum, a highly variable recessive ectopic calcification disorder. Incomplete penetrance may initiate disease heterogeneity, hence symptoms may not, or differently manifest in carriers. Here, we investigated whether incomplete penetrance is a source of heterogeneity in pseudoxanthoma elasticum. By integrating clinical and genetic data of 589 patients, we created the largest European cohort. Based on allele frequency alterations, we identified two incomplete penetrant pathogenic variants, c.2359G>A (p.Val787Ile) and c.1171A>G (p.Arg391Gly), with 6.5% and 2% penetrance, respectively. However, when penetrant, the c.1171A>G (p.Arg391Gly) manifested a clinically unaltered severity. After applying in silico and in vitro characterization, we suggest that incomplete penetrant variants are only deleterious if a yet unknown interacting partner of ABCC6 is mutated simultaneously. The low penetrance of these variants should be contemplated in genetic counseling.


Asunto(s)
Seudoxantoma Elástico , Humanos , Mutación , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/metabolismo , Seudoxantoma Elástico/patología , Penetrancia , Adenosina Trifosfato , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética
5.
Nature ; 606(7915): 785-790, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35705806

RESUMEN

Exercise confers protection against obesity, type 2 diabetes and other cardiometabolic diseases1-5. However, the molecular and cellular mechanisms that mediate the metabolic benefits of physical activity remain unclear6. Here we show that exercise stimulates the production of N-lactoyl-phenylalanine (Lac-Phe), a blood-borne signalling metabolite that suppresses feeding and obesity. The biosynthesis of Lac-Phe from lactate and phenylalanine occurs in CNDP2+ cells, including macrophages, monocytes and other immune and epithelial cells localized to diverse organs. In diet-induced obese mice, pharmacological-mediated increases in Lac-Phe reduces food intake without affecting movement or energy expenditure. Chronic administration of Lac-Phe decreases adiposity and body weight and improves glucose homeostasis. Conversely, genetic ablation of Lac-Phe biosynthesis in mice increases food intake and obesity following exercise training. Last, large activity-inducible increases in circulating Lac-Phe are also observed in humans and racehorses, establishing this metabolite as a molecular effector associated with physical activity across multiple activity modalities and mammalian species. These data define a conserved exercise-inducible metabolite that controls food intake and influences systemic energy balance.


Asunto(s)
Ingestión de Alimentos , Conducta Alimentaria , Obesidad , Fenilalanina , Condicionamiento Físico Animal , Adiposidad/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Diabetes Mellitus Tipo 2 , Modelos Animales de Enfermedad , Ingestión de Alimentos/fisiología , Metabolismo Energético , Conducta Alimentaria/fisiología , Glucosa/metabolismo , Ácido Láctico/metabolismo , Ratones , Obesidad/metabolismo , Obesidad/prevención & control , Fenilalanina/administración & dosificación , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Fenilalanina/farmacología , Condicionamiento Físico Animal/fisiología
6.
Front Cell Dev Biol ; 10: 823249, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35186933

RESUMEN

Chronic low back pain is a highly prevalent health condition intricately linked to intervertebral disc degeneration. One of the prominent features of disc degeneration that is commonly observed with aging is dystrophic calcification. ATP-binding cassette sub-family C member 6 (ABCC6), a presumed ATP efflux transporter, is a key regulator of systemic levels of the mineralization inhibitor pyrophosphate (PPi). Mutations in ABCC6 result in pseudoxanthoma elasticum (PXE), a progressive human metabolic disorder characterized by mineralization of the skin and elastic tissues. The implications of ABCC6 loss-of-function on pathological mineralization of structures in the spine, however, are unknown. Using the Abcc6 -/- mouse model of PXE, we investigated age-dependent changes in the vertebral bone and intervertebral disc. Abcc6 -/- mice exhibited diminished trabecular bone quality parameters at 7 months, which remained significantly lower than the wild-type mice at 18 months of age. Abcc6 -/- vertebrae showed increased TRAP staining along with decreased TNAP staining, suggesting an enhanced bone resorption as well as decreased bone formation. Surprisingly, however, loss of ABCC6 resulted only in a mild, aging disc phenotype without evidence of dystrophic mineralization. Finally, we tested the utility of oral K3Citrate to treat the vertebral phenotype since it is shown to regulate hydroxyapatite mechanical behavior. The treatment resulted in inhibition of the osteoclastic response and an early improvement in mechanical properties of the bone underscoring the promise of potassium citrate as a therapeutic agent. Our data suggest that although ectopic mineralization is tightly regulated in the disc, loss of ABCC6 compromises vertebral bone quality and dysregulates osteoblast-osteoclast coupling.

7.
Am J Pathol ; 192(5): 762-770, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35182493

RESUMEN

Pathologic soft tissue calcification can occur in both genetic and acquired clinical conditions, causing significant morbidity and mortality. Although the pathomechanisms of pathologic calcification are poorly understood, major progress has been made in recent years in defining the underlying genetic defects in Mendelian disorders of ectopic calcification. This review presents an overview of the pathophysiology of five monogenic disorders of pathologic calcification: pseudoxanthoma elasticum, generalized arterial calcification of infancy, arterial calcification due to deficiency of CD73, ankylosis, and progeria. These hereditary disorders, caused by mutations in genes encoding ATP binding cassette subfamily C member 6, ectonucleotide pyrophosphatase/phosphodiesterase 1, CD73, progressive ankylosis protein, and lamin A/C proteins, respectively, are inorganic pyrophosphate (PPi) deficiency syndromes with reduced circulating levels of PPi, the principal physiologic inhibitor of calcium hydroxyapatite deposition in soft connective tissues. In addition to genetic diseases, PPi deficiency has been encountered in acquired clinical conditions accompanied by pathologic calcification. Because specific and effective treatments are lacking for pathologic calcification, the unifying finding of PPi deficiency suggests that PPi-targeted therapies may be beneficial to counteract pathologic soft tissue calcification in both genetic and acquired diseases.


Asunto(s)
Anquilosis , Calcinosis , Coristoma , Seudoxantoma Elástico , Calcificación Vascular , Anquilosis/tratamiento farmacológico , Calcinosis/genética , Calcinosis/terapia , Difosfatos/metabolismo , Humanos , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/metabolismo , Seudoxantoma Elástico/terapia , Síndrome , Calcificación Vascular/tratamiento farmacológico , Calcificación Vascular/terapia
8.
J Bone Miner Res ; 37(5): 1024-1031, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35147247

RESUMEN

The plasma membrane protein ankylosis homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints. We recently showed that when overproduced in HEK293 cells, ANKH mediates release of large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. ATP is converted extracellularly into PPi and AMP by the ectoenzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANKH. We now addressed the question of whether PPi leaves cells via ANKH using HEK293 cells that completely lack ENPP1. Introduction of ANKH in these ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi found in ENPP1-proficient cells. Ank activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1-/- mice contained <2.5% of the PPi found in bones of wild-type mice, showing that Enpp1 activity is also a prerequisite for Ank-dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1-mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60% to 70% of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification therefore do so by extruding NTPs rather than PPi itself. © 2022 American Society for Bone and Mineral Research (ASBMR).


Asunto(s)
Adenosina Trifosfato , Calcinosis , Difosfatos , Proteínas de Transporte de Fosfato , Adenosina Trifosfato/metabolismo , Animales , Huesos/metabolismo , Huesos/patología , Calcificación Fisiológica , Calcinosis/metabolismo , Calcinosis/patología , Difosfatos/metabolismo , Células HEK293 , Humanos , Ratones , Proteínas de Transporte de Fosfato/metabolismo
9.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-34199119

RESUMEN

Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC4), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC4 binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC4 binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC4-binding site.


Asunto(s)
Sitios de Unión , Modelos Moleculares , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Mutación , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Ligandos , Conformación Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutagénesis , Unión Proteica , Transporte de Proteínas , Ratas , Relación Estructura-Actividad , Especificidad por Sustrato
10.
FEBS Lett ; 595(6): 799-810, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33058196

RESUMEN

ABCC6 mediates release of ATP from hepatocytes into the blood. Extracellularly, ATP is converted into the mineralization inhibitor pyrophosphate. Consequently, inactivating mutations in ABCC6 give low plasma pyrophosphate and underlie the ectopic mineralization disorder pseudoxanthoma elasticum. How ABCC6 mediates cellular ATP release is still unknown. Fluorescent ABCC6 fusion proteins would allow mechanistic studies, but fluorophores attached to the ABCC6 N- or C-terminus result in intracellular retention and degradation. Here we describe that intramolecular introduction of fluorophores yields fully functional ABCC6 fusion proteins. A corresponding ABCC6 variant in which the catalytic glutamate of the second nucleotide binding domain was mutated, correctly routed to the plasma membrane but was inactive. Finally, N-terminal His10 or FLAG tags did not affect activity of the fusion proteins, allowing their purification for biochemical characterization.


Asunto(s)
Adenosina Trifosfato/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Recombinantes de Fusión/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Células HEK293 , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
11.
Front Cell Dev Biol ; 8: 573727, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33363139

RESUMEN

Epidemiological studies indicate that elevated alkaline phosphatase activity is associated with increased cardiovascular disease risk. Other epidemiological data demonstrate that mothers giving multiple childbirths (multipara) are also at increased risk of developing late-onset cardiovascular disease. We hypothesized that these two associations stem from a common cause, the insufficient plasma level of the ectopic mineralization inhibitor inorganic pyrophosphate, which is a substrate of alkaline phosphatase. As alkaline phosphatase activity is elevated in pregnancy, we hypothesized that pyrophosphate concentrations decrease gestationally, potentially leading to increased maternal vascular calcification and cardiovascular disease risk in multipara. We investigated plasma pyrophosphate kinetics pre- and postpartum in sheep and at term in humans and demonstrated its shortage in pregnancy, mirroring alkaline phosphatase activity. Next, we tested whether multiparity is associated with increased vascular calcification in pseudoxanthoma elasticum patients, characterized by low intrinsic plasma pyrophosphate levels. We demonstrated that these patients had increased vascular calcification when they give birth multiple times. We propose that transient shortages of pyrophosphate during repeated pregnancies might contribute to vascular calcification and multiparity-associated cardiovascular disease risk threatening hundreds of millions of healthy women worldwide. Future trials are needed to assess if gestational pyrophosphate supplementation might be a suitable prophylactic treatment to mitigate maternal cardiovascular disease risk in multiparous women.

12.
PLoS Genet ; 16(7): e1008884, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32639996

RESUMEN

The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality.


Asunto(s)
Huesos/metabolismo , Calcinosis/genética , Ácido Cítrico/metabolismo , Proteínas de Transporte de Fosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Desarrollo Óseo/genética , Calcinosis/metabolismo , Calcinosis/patología , Diferenciación Celular , Células Cultivadas , Difosfatos/metabolismo , Humanos , Fenómenos Mecánicos , Ratones , Mutación/genética , Proteínas de Transporte de Fosfato/metabolismo
13.
Aging Cell ; 19(5): e13148, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32319726

RESUMEN

Intervertebral disc degeneration presents a wide spectrum of clinically degenerative disc phenotypes; however, the contribution of genetic background to the degenerative outcomes has not been established. We characterized the spinal phenotype of 3 mouse strains with varying cartilage-regenerative potential at 6 and 23 months: C57BL/6, LG/J and SM/J. All strains showed different aging phenotypes. Importantly, LG/J mice showed an increased prevalence of dystrophic disc calcification in caudal discs with aging. Quantitative-histological analyses of LG/J and SM/J caudal discs evidenced accelerated degeneration compared to BL6, with cellular disorganization and cell loss together with fibrosis of the NP, respectively. Along with the higher grades of disc degeneration, SM/J, at 6M, also differed the most in terms of NP gene expression compared to other strains. Moreover, although we found common DEGs between BL6 and LG/J aging, most of them were divergent between the strains. Noteworthy, the common DEGs altered in both LG/J and BL6 aging were associated with inflammatory processes, response to stress, cell differentiation, cell metabolism and cell division. Results suggested that disc calcification in LG/J resulted from a dystrophic calcification process likely aggravated by cell death, matrix remodelling, changes in calcium/phosphate homeostasis and cell transformation. Lastly, we report 7 distinct phenotypes of human disc degeneration based on transcriptomic profiles, that presented similar pathways and DEGs found in aging mouse strains. Together, our results suggest that disc aging and degeneration depends on the genetic background and involves changes in various molecular pathways, which might help to explain the diverse phenotypes seen during disc disease.


Asunto(s)
Senescencia Celular , Disco Intervertebral/patología , Animales , Disco Intervertebral/metabolismo , Ratones , Ratones Endogámicos , Fenotipo
14.
Trends Biochem Sci ; 44(2): 125-140, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30446375

RESUMEN

Ever since Garrod deduced the existence of inborn errors in 1901, a vast array of metabolic diseases has been identified and characterized in molecular terms. In 2018 it is difficult to imagine that there is any uncharted backyard left in the metabolic disease landscape. Nevertheless, it took until 2013 to identify the cause of a relatively frequent inborn error, pseudoxanthoma elasticum (PXE), a disorder resulting in aberrant calcification. The mechanism found was not only biochemically interesting but also points to possible new treatments for PXE, a disease that has remained untreatable. In this review we sketch the tortuous road that led to the biochemical understanding of PXE and to new ideas for treatment. We also discuss some of the controversies still haunting the field.


Asunto(s)
Errores Innatos del Metabolismo/genética , Seudoxantoma Elástico/genética , Humanos , Errores Innatos del Metabolismo/metabolismo , Errores Innatos del Metabolismo/terapia , Seudoxantoma Elástico/metabolismo , Seudoxantoma Elástico/terapia
15.
Am J Pathol ; 189(2): 216-225, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30414410

RESUMEN

Ectopic mineralization is a global problem and leading cause of morbidity and mortality. The pathomechanisms of ectopic mineralization are poorly understood. Recent studies on heritable ectopic mineralization disorders with defined gene defects have been helpful in elucidation of the mechanisms of ectopic mineralization in general. The prototype of such disorders is pseudoxanthoma elasticum (PXE), a late-onset, slowly progressing disorder with multisystem clinical manifestations. Other conditions include generalized arterial calcification of infancy (GACI), characterized by severe, early-onset mineralization of the cardiovascular system, often with early postnatal demise. In addition, arterial calcification due to CD73 deficiency (ACDC) occurs late in life, mostly affecting arteries in the lower extremities in elderly individuals. These three conditions, PXE, GACI, and ACDC, caused by mutations in ABCC6, ENPP1, and NT5E, respectively, are characterized by reduced levels of inorganic pyrophosphate (PPi) in plasma. Because PPi is a powerful antimineralization factor, it has been postulated that reduced PPi is a major determinant for ectopic mineralization in these conditions. These and related observations on complementary mechanisms of ectopic mineralization have resulted in development of potential treatment modalities for PXE, including administration of bisphosphonates, stable PPi analogs with antimineralization activity. It is conceivable that efficient treatments may soon become available for heritable ectopic mineralization disorders with application to common calcification disorders.


Asunto(s)
5'-Nucleotidasa/deficiencia , Difosfonatos/uso terapéutico , Seudoxantoma Elástico , Calcificación Vascular , Difosfatos/sangre , Proteínas Ligadas a GPI/deficiencia , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Seudoxantoma Elástico/sangre , Seudoxantoma Elástico/tratamiento farmacológico , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/patología , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Calcificación Vascular/sangre , Calcificación Vascular/tratamiento farmacológico , Calcificación Vascular/genética , Calcificación Vascular/patología
16.
Cancer Res ; 78(19): 5668-5679, 2018 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-30115694

RESUMEN

In human cancers, FGFR signaling is frequently hyperactivated by deregulation of FGF ligands or by activating mutations in the FGFR receptors such as gene amplifications, point mutations, and gene fusions. As such, FGFR inhibitors are considered an attractive therapeutic strategy for patients with mutations in FGFR family members. We previously identified Fgfr2 as a key driver of invasive lobular carcinoma (ILC) in an in vivo insertional mutagenesis screen using the Sleeping Beauty transposon system. Here we explore whether these FGFR-driven ILCs are sensitive to the FGFR inhibitor AZD4547 and use transposon mutagenesis in these tumors to identify potential mechanisms of resistance to therapy. Combined with RNA sequencing-based analyses of AZD4547-resistant tumors, our in vivo approach identified several known and novel potential resistance mechanisms to FGFR inhibition, most of which converged on reactivation of the canonical MAPK-ERK signaling cascade. Observed resistance mechanisms included mutations in the tyrosine kinase domain of FGFR2, overexpression of MET, inactivation of RASA1, and activation of the drug-efflux transporter ABCG2. ABCG2 and RASA1 were identified only from de novo transposon insertions acquired during AZD4547 treatment, demonstrating that insertional mutagenesis in mice is an effective tool for identifying potential mechanisms of resistance to targeted cancer therapies.Significance: These findings demonstrate that a combined approach of transcriptomics and insertional mutagenesis in vivo is an effective method for identifying potential targets to overcome resistance to therapy in the clinic. Cancer Res; 78(19); 5668-79. ©2018 AACR.


Asunto(s)
Benzamidas/química , Elementos Transponibles de ADN , Resistencia a Antineoplásicos , Mutagénesis , Piperazinas/química , Pirazoles/química , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Carcinoma Lobular/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Amplificación de Genes , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Mutación , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Análisis de Secuencia de ARN , Transcriptoma , Proteína Activadora de GTPasa p120/metabolismo
17.
Elife ; 62017 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-28849762

RESUMEN

The urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein that promotes tissue remodeling, tumor cell adhesion, migration and invasion. uPAR mediates degradation of the extracellular matrix through protease recruitment and enhances cell adhesion, migration and signaling through vitronectin binding and interactions with integrins. Full-length uPAR is released from the cell surface, but the mechanism and significance of uPAR shedding remain obscure. Here we identify transmembrane glycerophosphodiesterase GDE3 as a GPI-specific phospholipase C that cleaves and releases uPAR with consequent loss of function, whereas its homologue GDE2 fails to attack uPAR. GDE3 overexpression depletes uPAR from distinct basolateral membrane domains in breast cancer cells, resulting in a less transformed phenotype, it slows tumor growth in a xenograft model and correlates with prolonged survival in patients. Our results establish GDE3 as a negative regulator of the uPAR signaling network and, furthermore, highlight GPI-anchor hydrolysis as a cell-intrinsic mechanism to alter cell behavior.


Asunto(s)
Neoplasias de la Mama/genética , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Hidrolasas Diéster Fosfóricas/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Femenino , Técnicas de Inactivación de Genes/métodos , Células HEK293 , Humanos , Hidrólisis , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Desnudos , Modelos Moleculares , Trasplante de Neoplasias , Hidrolasas Diéster Fosfóricas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Transducción de Señal , Carga Tumoral , Vitronectina/genética , Vitronectina/metabolismo
18.
EMBO Mol Med ; 9(11): 1463-1470, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28701330

RESUMEN

Various disorders including pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI), which are caused by inactivating mutations in ABCC6 and ENPP1, respectively, present with extensive tissue calcification due to reduced plasma pyrophosphate (PPi). However, it has always been assumed that the bioavailability of orally administered PPi is negligible. Here, we demonstrate increased PPi concentration in the circulation of humans after oral PPi administration. Furthermore, in mouse models of PXE and GACI, oral PPi provided via drinking water attenuated their ectopic calcification phenotype. Noticeably, provision of drinking water with 0.3 mM PPi to mice heterozygous for inactivating mutations in Enpp1 during pregnancy robustly inhibited ectopic calcification in their Enpp1-/- offspring. Our work shows that orally administered PPi is readily absorbed in humans and mice and inhibits connective tissue calcification in mouse models of PXE and GACI PPi, which is recognized as safe by the FDA, therefore not only has great potential as an effective and extremely low-cost treatment for these currently intractable genetic disorders, but also in other conditions involving connective tissue calcification.


Asunto(s)
Difosfatos/uso terapéutico , Seudoxantoma Elástico/tratamiento farmacológico , Calcificación Vascular/tratamiento farmacológico , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Administración Oral , Adulto , Anciano , Animales , Calcio/análisis , Tejido Conectivo/metabolismo , Difosfatos/sangre , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Miocardio/química , Miocardio/metabolismo , Hidrolasas Diéster Fosfóricas/deficiencia , Hidrolasas Diéster Fosfóricas/genética , Embarazo , Seudoxantoma Elástico/patología , Pirofosfatasas/deficiencia , Pirofosfatasas/genética , Calcificación Vascular/patología , Adulto Joven
19.
Am J Pathol ; 187(6): 1258-1272, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28416300

RESUMEN

Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6-/- mice fully inhibited the acute dystrophic cardiac calcification phenotype, whereas alendronate had no significant effect. We also found that daily injection of PPi to Abcc6-/- mice over several months prevented the development of pseudoxanthoma elasticum-like spontaneous calcification, but failed to reverse already established lesions. Furthermore, we found that the expression of low amounts of the human ABCC6 in liver of transgenic Abcc6-/- mice, resulting in only a 27% increase in plasma PPi levels, led to a major reduction in acute and chronic calcification phenotypes. This proof-of-concept study shows that the development of both acute and chronic calcification associated with ABCC6 deficiency can be prevented by compensating PPi deficits, even partially. Our work indicates that PPi substitution represents a promising strategy to treat ABCC6-dependent calcification disorders.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Calcinosis/prevención & control , Difosfatos/uso terapéutico , Seudoxantoma Elástico/prevención & control , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Enfermedad Aguda , Animales , Calcinosis/metabolismo , Calcinosis/patología , Enfermedad Crónica , Difosfatos/administración & dosificación , Difosfatos/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Ácido Etidrónico/uso terapéutico , Femenino , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Fenotipo , Seudoxantoma Elástico/metabolismo , Seudoxantoma Elástico/patología , Transgenes
20.
J Invest Dermatol ; 137(4): 790-795, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28340679

RESUMEN

Pseudoxanthoma elasticum is a prototype of heritable ectopic mineralization disorders, with phenotypic overlap with generalized arterial calcification of infancy and arterial calcification due to CD73 deficiency. Recent observations have suggested that the reduced inorganic pyrophosphate/phosphate ratio is the cause of soft connective tissue mineralization in these disorders. PXE International, a patient advocacy organization, supports research in part by sponsoring biennial research symposia on these disorders; the latest meeting was held in September 2016 at Thomas Jefferson University, Philadelphia. This report summarizes the progress in pseudoxanthoma elasticum and other ectopic mineralization disorders, as presented in the symposium, with focus on translational aspects of precision medicine toward improved diagnostics and treatment development for these currently intractable disorders.


Asunto(s)
Difosfatos/metabolismo , Ácido Etidrónico/farmacología , Predisposición Genética a la Enfermedad , Seudoxantoma Elástico/genética , Calcificación Vascular/genética , 5'-Nucleotidasa/efectos de los fármacos , 5'-Nucleotidasa/genética , Fosfatasa Alcalina/efectos de los fármacos , Fosfatasa Alcalina/genética , Animales , Biopsia con Aguja , Ensayos Clínicos como Asunto , Congresos como Asunto , Modelos Animales de Enfermedad , Ácido Etidrónico/uso terapéutico , Proteínas Ligadas a GPI/efectos de los fármacos , Proteínas Ligadas a GPI/genética , Humanos , Inmunohistoquímica , Internacionalidad , Ratones , Mutación , Hidrolasas Diéster Fosfóricas/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/genética , Seudoxantoma Elástico/patología , Pirofosfatasas/efectos de los fármacos , Pirofosfatasas/genética , Enfermedades Raras , Calcificación Vascular/fisiopatología
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