RESUMO
BACKGROUND: The lifespan of red blood cells is terminated when macrophages remove senescent red blood cells by erythrophagocytosis. This puts macrophages at the center of systemic iron recycling in addition to their functions in tissue remodeling and innate immunity. Thus far, erythrophagocytosis has been studied by evaluating phagocytosis of erythrocytes that were damaged to mimic senescence. These studies have demonstrated that acquisition of some specific individual senescence markers can trigger erythrophagocytosis by macrophages, but we hypothesized that the mechanism of erythrophagocytosis of such damaged erythrocytes might differ from erythrophagocytosis of physiologically aged erythrocytes. DESIGN AND METHODS: To test this hypothesis we generated an erythrocyte population highly enriched in senescent erythrocytes by a hypertransfusion procedure in mice. Various erythrocyte-aging signals were analyzed and erythrophagocytosis was evaluated in vivo and in vitro. RESULTS: The large cohort of senescent erythrocytes from hypertransfused mice carried numerous aging signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from hypertransfused mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. However, neither erythrocytes from hypertransfused mice, nor those from control mice were phagocytosed in vitro by primary macrophage cultures, even though these cultures were able to phagocytose oxidatively damaged erythrocytes. CONCLUSIONS: The large senescent erythrocyte population found in hypertransfused mice mimics physiologically aged erythrocytes. For effective erythrophagocytosis of these senescent erythrocytes, macrophages depend on some features of the intact phagocytosing tissue for support.
Assuntos
Envelhecimento Eritrocítico/fisiologia , Eritrócitos/fisiologia , Macrófagos/fisiologia , Fagocitose/fisiologia , Animais , Biomarcadores/análise , Biotinilação , Transfusão de Eritrócitos , Eritrócitos/citologia , Eritropoese/fisiologia , Feminino , Citometria de Fluxo , Humanos , Ferro/metabolismo , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismoRESUMO
Normophosphatemic familial tumoral calcinosis (NFTC) is an autosomal recessive disorder characterized by calcium deposition in skin and mucosae and associated with unremitting pain and life-threatening skin infections. A homozygous missense mutation (p.K1495E), resulting in SAMD9 protein degradation, was recently shown to cause NFTC in five families of Jewish-Yemenite origin. In this study, we evaluated another Jewish-Yemenite NFTC kindred. All patients were compound heterozygous for two mutations in SAMD9: K1495E and a previously unreported nonsense mutation, R344X, predicted to result in a markedly truncated molecule. Screening of unaffected population-matched controls revealed heterozygosity for K1495E and R344X only in individuals of Jewish-Yemenite ancestry, but not in more than 700 control samples of other origins, including 93 non-Jewish Yemenite. These data may be suggestive of positive selection, considering the rarity of NFTC and the small size of the Jewish-Yemenite population; alternatively, they may reflect genetic drift or the effect of a population-specific modifier trait. Calcifications in NFTC generally develop over areas subjected to repeated trauma and are associated with marked inflammatory manifestations, indicating that SAMD9 may play a role in the inflammatory response to tissue injury. We therefore assessed the effect of cellular stress and tumor necrosis factor-alpha (TNF-alpha), a potent pro-inflammatory cytokine, on SAMD9 gene expression. Whereas exogenous hydrogen peroxide and heat shock did not affect SAMD9 transcription, osmotic shock was found to markedly upregulate SAMD9 expression. In addition, incubation of endothelial cells with TNF-alpha caused a dose-related, p38-dependant increase in SAMD9 expression. These data link NFTC and SAMD9 to the TNF-alpha signaling pathway, suggesting a role for this system in the regulation of extra-osseous calcification.
Assuntos
Calcinose/genética , Regulação da Expressão Gênica , Mutação , Proteínas/genética , Fator de Necrose Tumoral alfa/genética , Calcinose/etnologia , Linhagem Celular Transformada , Células Cultivadas , Análise Mutacional de DNA , Haplótipos , Heterozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Judeus , Repetições de Microssatélites , Transdução de Sinais , Veias Umbilicais/citologiaRESUMO
Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, which results in painful ulcerative lesions and severe skin and bone infections. Two major types of FTC have been recognized: hyperphosphatemic FTC (HFTC) and normophosphatemic FTC (NFTC). HFTC was recently shown to result from mutations in two different genes: GALNT3, which codes for a glycosyltransferase, and FGF23, which codes for a potent phosphaturic protein. To determine the molecular cause of NFTC, we performed homozygosity mapping in five affected families of Jewish Yemenite origin and mapped NFTC to 7q21-7q21.3. Mutation analysis revealed a homozygous mutation in the SAMD9 gene (K1495E), which was found to segregate with the disease in all families and to interfere with the protein expression. Our data suggest that SAMD9 is involved in the regulation of extraosseous calcification, a process of considerable importance in a wide range of diseases as common as atherosclerosis and autoimmune disorders.
Assuntos
Calcinose/genética , Proteínas/genética , Neoplasias Cutâneas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Calcinose/patologia , Linhagem Celular , Conjuntivite/genética , Família , Feminino , Fator de Crescimento de Fibroblastos 23 , Gengivite/genética , Gengivite/patologia , Proteínas de Fluorescência Verde , Haplótipos , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular , Escore Lod , Masculino , Linhagem , Proteínas/química , Alinhamento de Sequência , Dermatopatias/genética , Dermatopatias/patologia , Neoplasias Cutâneas/patologia , Úlcera Cutânea/genética , Úlcera Cutânea/patologia , TransfecçãoAssuntos
Calcinose/genética , Calcinose/metabolismo , N-Acetilgalactosaminiltransferases/genética , Proteínas de Neoplasias/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/ultraestrutura , Predisposição Genética para Doença , Humanos , Mutação/genética , Neoplasias Cutâneas/genética , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Hyperphosphatemic Familial Tumoral Calcinosis (HFTC; MIM211900) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, associated with elevated circulating levels of phosphate. The disease was initially found to result from mutations in GALNT3 encoding a glycosyltransferase. However, more recently, the S71G missense mutation in FGF23, encoding a potent phosphaturic protein, was identified in two families. In the present report, we describe a second mutation in FGF23 underlying a severe case displaying calcifications of cutaneous and numerous extracutaneous tissues. The mutation (M96T) was found to affect a highly conserved methionine residue at position 96 of the protein. These observations illustrate the extent of genetic and phenotypic heterogeneity in HFTC.
Assuntos
Substituição de Aminoácidos , Calcinose/genética , Fatores de Crescimento de Fibroblastos/genética , Mutação de Sentido Incorreto , Neoplasias Cutâneas/genética , Calcinose/patologia , Feminino , Fator de Crescimento de Fibroblastos 23 , Humanos , Masculino , N-Acetilgalactosaminiltransferases/genética , Proteínas de Neoplasias/genética , Neoplasias Cutâneas/patologia , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Neurocutaneous syndromes represent a vast, largely heterogeneous group of disorders characterized by neurological and dermatological manifestations, reflecting the common embryonic origin of epidermal and neural tissues. In the present report, we describe a novel neurocutaneous syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK syndrome). Using homozygosity mapping in two large families, we localized the disease gene to 22q11.2 and identified, in all patients, a 1-bp deletion in SNAP29, which codes for a SNARE protein involved in vesicle fusion. SNAP29 expression was decreased in the skin of the patients, resulting in abnormal maturation of lamellar granules and, as a consequence, in mislocation of epidermal lipids and proteases. These data underscore the importance of vesicle trafficking regulatory mechanisms for proper neuroectodermal differentiation.
Assuntos
Encefalopatias/genética , Encéfalo/anormalidades , Ictiose/genética , Ceratodermia Palmar e Plantar/genética , Mutação , Malformações do Sistema Nervoso/genética , Proteínas de Transporte Vesicular/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Biópsia , Western Blotting , Diferenciação Celular , Proliferação de Células , Mapeamento Cromossômico , Análise Mutacional de DNA , Epiderme/metabolismo , Saúde da Família , Feminino , Fibroblastos/citologia , Genótipo , Haplótipos , Homozigoto , Humanos , Imuno-Histoquímica , Masculino , Repetições de Microssatélites , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Modelos Genéticos , Oligonucleotídeos/genética , Transporte Proteico , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , SíndromeRESUMO
Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare autosomal recessive disorder characterized by progressive, tumor-like calcifications in the dermis and subcutaneous tissues. The disease is associated with primary hyperphosphatemia due to increased renal tubular reabsorption of phosphate. We recently identified mutations in GALNT3 as the proximal cause of this metabolic disorder. GALNT3 encodes the glycosyltransferase UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 3 (ppGalNAc-T3), which initiates mucin-type O-glycosylation and thus takes part in posttranslational modification and formation of mucin-type glycoproteins. A number of studies have previously described the histopathological and ultrastructural features of lesional skin in HFTC, but little is currently known about the morphology of the normal-appearing non-lesional skin. We obtained biopsies of uninvolved skin from two HFTC patients carrying a known splice site mutation in GALNT3. Light and electron microscopic examination of a biopsy of one of the two patients did not reveal abnormal findings in the epidermis or dermis. However, immunohistochemical studies of frozen skin sections of biopsies of the two patients using monoclonal antibodies directed against three ppGalNac isoforms revealed the complete absence of immunostaining for ppGalNAc-T3 while the staining pattern for ppGalNAc-T2 and -T6 was identical in skin biopsies obtained from HFTC patients and healthy control individuals. Our data provide for the first time evidence for ppGalNAc-T3 deficiency in the skin of HFTC patients and suggest that immunostaining of skin biopsy samples for ppGal-Nac-T3 might be a useful tool for the diagnosis of HFTC.
Assuntos
Calcinose/enzimologia , N-Acetilgalactosaminiltransferases/biossíntese , Dermatopatias/enzimologia , Pele/ultraestrutura , Calcinose/diagnóstico , Humanos , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Mutação , N-Acetilgalactosaminiltransferases/genética , Proteínas de Neoplasias/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/enzimologia , Pele/patologia , Dermatopatias/patologia , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Hyperphosphatemia-hyperostosis syndrome (HHS) is a rare autosomal recessive metabolic disorder characterized by elevated serum phosphate levels and repeated attacks of acute, painful swellings of the long bones with radiological evidence of periosteal reaction and cortical hyperostosis. HHS shares several clinical and metabolic features with hyperphosphatemic familial tumoral calcinosis (HFTC), which is caused by mutations in GALNT3 encoding a glycosyltransferase responsible for initiating O-glycosylation. To determine whether GALNT3 is involved in the pathogenesis of HHS we screened two unrelated Arab-Israeli HHS families for pathogenic mutations in this gene. All affected individuals harbored a homozygous splice site mutation (1524+1G-->A) in GALNT3. This mutation was previously described in a large Druze HFTC kindred and has been shown to alter GALNT3 expression and result in ppGalNAc-T3 deficiency. Genotype analysis of six microsatellite markers across the GALNT3 region on 2q24-q31 revealed that the HHS and HFTC families share a common haplotype spanning approximately 0.14 Mb. Our results demonstrate that HHS and HFTC are allelic disorders despite their phenotypic differences and suggest a common origin of the 1524+1G-->A mutation in the Middle East (founder effect). The heterogeneous phenotypic expression of the identified splice site mutation implies the existence of inherited or epigenetic modifying factors of importance in the regulation of ppGalNAc-T3 activity.
Assuntos
Calcinose/genética , Cálcio/metabolismo , Hiperostose/genética , N-Acetilgalactosaminiltransferases/genética , Proteínas de Neoplasias/genética , Fosfatos/sangue , Calcinose/metabolismo , Criança , Colecalciferol/metabolismo , Feminino , Haplótipos , Humanos , Hiperostose/metabolismo , Masculino , N-Acetilgalactosaminiltransferases/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Osteomalacia/metabolismo , Hormônio Paratireóideo/metabolismo , Linhagem , Análise de Sequência de DNA , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Familial tumoral calcinosis (FTC; OMIM 211900) is a severe autosomal recessive metabolic disorder that manifests with hyperphosphatemia and massive calcium deposits in the skin and subcutaneous tissues. Using linkage analysis, we mapped the gene underlying FTC to 2q24-q31. This region includes the gene GALNT3, which encodes a glycosyltransferase responsible for initiating mucin-type O-glycosylation. Sequence analysis of GALNT3 identified biallelic deleterious mutations in all individuals with FTC, suggesting that defective post-translational modification underlies the disease.