RESUMO
We have shown previously that repair in the peripheral nervous system is associated with a reversion to an embryonic pattern of alternative splicing of the extracellular matrix molecule fibronectin. One of the consequent changes is a relative increase in the number of fibronectins expressing the binding site for alpha4 integrins. Here we show that alpha4 integrins are expressed on dorsal root ganglion neuron cell bodies and growth cones in the sciatic nerve during regeneration and that the interaction of alpha4 integrin with alternatively spliced isoforms of recombinant fibronectins containing the alpha4 binding site enhances neurite outgrowth in dorsal root ganglion neurons. The pheochromocytoma (PC12) neuronal cell line, which normally extends neurites poorly on fibronectin, does so efficiently when alpha4 is expressed in the cells. Experiments using chimeric integrins expressed in PC12 cells show that the alpha4 cytoplasmic domain is necessary and sufficient for this enhanced neurite outgrowth. In both dorsal root ganglion neurons and PC12 cells the alpha4 cytoplasmic domain is tightly linked to the intracellular adapter protein paxillin. These experiments suggest an important role for alpha4 integrin and paxillin in peripheral nerve regeneration and show how alternative splicing of fibronectin may provide a mechanism to enhance repair after injury.
Assuntos
Antígenos CD/biossíntese , Regeneração Nervosa/fisiologia , Neuritos/metabolismo , Nervos Periféricos/metabolismo , Processamento Alternativo , Animais , Antígenos CD/farmacologia , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Fibronectinas/biossíntese , Fibronectinas/genética , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Cones de Crescimento/metabolismo , Integrina alfa4 , Camundongos , Compressão Nervosa , Neuritos/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Paxilina , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Traumatismos dos Nervos Periféricos , Fosfoproteínas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Nervo Isquiático/lesões , Nervo Isquiático/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Wallerian degeneration following peripheral nerve injury is associated with increased production of fibronectin and other extracellular matrix molecules that are thought to enhance repair. We have shown previously that alternative splicing of the mRNA for fibronectin also changes following sciatic nerve lesions so as to reexpress forms of mRNA seen during embryogenesis. In the present study, we have examined the role of the regenerating axons in the regulation of this splicing. We have compared the patterns of fibronectin mRNA splicing seen in sciatic nerve development with that seen in cut nerves (that do not regenerate), crushed nerves (that regenerate successfully), and Schwann cells cultured in forskolin so as to mimic axonal signals. By using a reverse transcriptase polymerase chain reaction assay to examine all three regions of fibronectin mRNA splicing in a quantitative manner, we found that embryonic patterns of fibronectin mRNA splicing appear rapidly following injury and are not then altered by reestablishment of axons in the nerve. In addition, we found that forskolin has no effect on fibronectin mRNA splicing in cultured cells. We conclude that axonal signals do not regulate the pattern of fibronectin alternative splicing in peripheral nerve repair.
