RESUMEN
The haptoglobin-hemoglobin receptor CD163 and proTNF-α are transmembrane macrophage proteins subjected to cleavage by the inflammation-responsive protease ADAM17. This leads to release of soluble CD163 (sCD163) and bioactive TNF-α. Sequence comparison of the juxtamembrane region identified similar palindromic sequences in human CD163 ((1044)Arg-Ser-Ser-Arg) and proTNF-α ((78)Arg-Ser-Ser-Ser-Arg). In proTNF-α the Arg-Ser-Ser-Ser-Arg sequence is situated next to the previously established ADAM17 cleavage site. Site-directed mutagenesis revealed that the sequences harbor essential information for efficient cleavage of the two proteins upon ADAM17 stimulation. This was further evidenced by analysis of mouse CD163 that, like CD163 in other non-primates, does not contain the palindromic CD163 sequence in the juxtamembrane region. Mouse CD163 resisted endotoxin- and phorbol ester-induced shedding, and ex vivo analysis of knock-in of the Arg-Ser-Ser-Arg sequence in mouse CD163 revealed a receptor shedding comparable with that of human CD163. In conclusion, we have identified an essential substrate motif for ADAM17-mediated CD163 and proTNF-α cleavage in macrophages. In addition, the present data indicate that CD163, by incorporation of this motif in late evolution, underwent a modification that allows for an instant down-regulation of surface CD163 expression and inhibition of hemoglobin uptake. This regulatory modality seems to have coincided with the evolution of an enhanced hemoglobin-protecting role of the haptoglobin-CD163 system in primates.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Receptores de Superficie Celular/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Sitios de Unión/genética , Células Cultivadas , Endotoxemia/genética , Endotoxemia/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Células HEK293 , Humanos , Immunoblotting , Inflamación/genética , Lipopolisacáridos/farmacología , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Proteolisis/efectos de los fármacos , Interferencia de ARN , Receptores de Superficie Celular/genética , Homología de Secuencia de Aminoácido , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Pseudoxanthoma elasticum (PXE) is a serious genetic disorder with ectopic mineralization affecting the skin, the eye and the cardiovascular system. The disease is predominantly caused by mutations in the transmembrane ABC protein ABCC6, a putative small substrate transporter. Interestingly, ABCC6 seems virtually absent in the affected organs, whereas a high expression is seen in hepatocytes. This and further published experimental evidence indicate that PXE is a systemic, metabolic liver disease where circulatory changes affect the peripheral mineralization process. Owing to the well-characterized transport of organic substrates by related ABC proteins, it has been proposed that PXE is caused by impaired export of an antimineralization compound to the blood. The authors here present an alternative hypothesis that explains ectopic mineralization in PXE as a consequence of hepatic accumulation of ABCC6 substrate(s) that via gene-regulating effects leads to altered hepatic secretion and activation of antimineralization/anticalcification proteins such as fetuin-A and Gla proteins.