RESUMO
Uncontrolled, excessive inflammation contributes to the secondary tissue damage of traumatic spinal cord, and HMGB1 is highlighted for initiation of a vicious self-propagating inflammatory circle by release from necrotic cells or immune cells. Several regenerative-competent vertebrates have evolved to circumvent the second damages during the spontaneous spinal cord regeneration with an unknown HMGB1 regulatory mechanism. By genomic surveys, we have revealed that two paralogs of HMGB1 are broadly retained from fish in the phylogeny. However, their spatial-temporal expression and effects, as shown in lowest amniote gecko, were tightly controlled in order that limited inflammation was produced in spontaneous regeneration. Two paralogs from gecko HMGB1 (gHMGB1) yielded distinct injury and infectious responses, with gHMGB1b significantly up-regulated in the injured cord. The intracellular gHMGB1b induced less release of inflammatory cytokines than gHMGB1a in macrophages, and the effects could be shifted by exchanging one amino acid in the inflammatory domain. Both intracellular proteins were able to mediate neuronal programmed apoptosis, which has been indicated to produce negligible inflammatory responses. In vivo studies demonstrated that the extracellular proteins could not trigger a cascade of the inflammatory cytokines in the injured spinal cord. Signal transduction analysis found that gHMGB1 proteins could not bind with cell surface receptors TLR2 and TLR4 to activate inflammatory signaling pathway. However, they were able to interact with the receptor for advanced glycation end products to potentiate oligodendrocyte migration by activation of both NFκB and Rac1/Cdc42 signaling. Our results reveal that HMGB1 does not mediate the inflammatory response in spontaneous spinal cord regeneration, but it promotes CNS regeneration.
Assuntos
Proteína HMGB1/metabolismo , Inflamação/metabolismo , Regeneração da Medula Espinal/fisiologia , Medula Espinal/metabolismo , Animais , Apoptose/genética , Apoptose/imunologia , Apoptose/fisiologia , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , DNA Complementar/química , DNA Complementar/genética , Células HEK293 , Proteína HMGB1/genética , Proteína HMGB1/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Lagartos , Camundongos , Microglia/imunologia , Microglia/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Neurônios/imunologia , Neurônios/metabolismo , Filogenia , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Coelhos , Regeneração/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Medula Espinal/imunologia , Regeneração da Medula Espinal/genética , Regeneração da Medula Espinal/imunologiaRESUMO
The cDNA encoding stathmin is identified from the brain and spinal cord cDNA library of Gekko japonicus. It contains a 450 bp open-reading-frame, corresponding to a deduced protein of 149 amino acids. At amino acid level. gecko stathmin shares more than 76.4% identities with vertebrate stathmins, and especially, it shares 100% identity with human stathmin, suggesting that the selective pressure must have been extremely high for the conservation of stathmin during the vertebrates including reptile evolution. Reverse transcriptase polymerase chain reaction (RT-PCR) shows that gecko stathmin is ubiquitously expressed in all tissues examined. In situ hybridization reveals that stathmin transcript mainly appear in the gray matter of spinal cord. The change of stathmin expression in spinal cord after tail amputation is examined by semiquantitative RT-PCR. Stathmin expression increases at 1 day and 3 day after amputation and decreases to the control level at I week. However, the expression level increases again at 2 weeks. These suggest that stathmin may be associated with the immune protection of the injury, as well as in the regeneration of spinal cord.
Assuntos
Regulação da Expressão Gênica , Medula Espinal/metabolismo , Estatmina/química , Estatmina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Clonagem Molecular , Evolução Molecular , Humanos , Lagartos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fases de Leitura Aberta , Homologia de Sequência de AminoácidosRESUMO
Transcription factor Pbx4 is recruited to form dimeric or trimeric complexes with Hox and/or Meis homeodomain proteins and participates in patterning the hindbrain and retina during vertebrate CNS development. We characterized a Pbx4 cDNA isolated from a Gekko japonicus brain and spinal cord cDNA library. Northern blot and quantitative real-time PCR revealed that gecko Pbx4 was ubiquitously expressed in several tissues. In the spinal cord after tail amputation, in situ hybridization results showed that Pbx4 mRNA staining was present in the gray matter and ependymal cells of the spinal cord but that additional staining was seen in the white matter in regions close to the amputation stump. Both in situ hybridization and real-time PCR methods detected no obvious changes in Pbx4 expression in segment of the cord farthest from the amputation site, however, Pbx4 mRNA expression increased by 2 fold in segment close to the amputation site after 2 wks. The upregulation of Pbx4 was inhibited by an intraperitoneal injection of retinoic acid (RA) (100 microg/g body weight). These results suggest that gecko Pbx4 is possibly involved in spinal cord regeneration at sites of proximal amputation, and that the expression of Pbx4 in the spinal cord is regulated by retinoic acid in a manner different from that of Pbx1, Pbx2 and Pbx3.
