ABSTRACT
Regulation of gene expression is critical to the proper development of neuronal cells. The methyl-CpG binding protein 2 (MeCP2) operates as a transcriptional repressor by facilitating histone deacetylation and DNA methylation-dependent transcriptional silencing. This study examined the importance of MeCP2 in the regulation of neurite formation in PC12 cells. Expression of MeCP2 increased in a time-dependent manner after induction of neuronal differentiation. Expression was assessed at both the transcriptional and translation levels, and reached a maximum at 24 h post-induction. In addition, a marked inhibition of neurite extension and proper localization of a marker for synapse formation, synapsin I, were observed when MeCP2 expression was decreased by the addition of an antisense morpholino oligomer directed to the translational initiation site for MeCP2beta. The removal of the antisense oligomer allowed neurite extension to progress. However, the addition of antisense oligomer to previously differentiated PC12 cells did not affect established neurite processes. Taken collectively, our results indicate a role for MeCP2beta early in the events of neurite formation and that the relative levels of MeCP2alpha and MeCP2beta may be different in early differentiating neurons than is found in the adult brain. In addition, unique functions may exist for the two isoforms of MeCP2. Our results indicate that the inhibition of neurite elaboration caused by a reduction in MeCP2 may be reversible.
Subject(s)
Chromosomal Proteins, Non-Histone , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Neurites/physiology , Neurons/drug effects , Oligonucleotides, Antisense/pharmacology , Protein Biosynthesis/physiology , Repressor Proteins , Animals , Cell Differentiation , Cells, Cultured , Gene Expression Regulation , Methyl-CpG-Binding Protein 2 , PC12 Cells , RNA, Messenger/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Synapsins/metabolism , Transcription, GeneticABSTRACT
Caspase activation is indispensable for the proper execution of apoptosis. However, to date, little is known about other possible physiologic functions for this class of enzymes in addition to their well-defined role in apoptosis. In this report, we described an action of caspase-3 involving cell dispersion that is independent of cell death. Using an in vitro neuronal model system consisting of PC12 cells, we observed a transient activation of caspase-3 both in situ and by Western blot analysis that was evident at 1 h following plating, was maximal by 3 h, and was attenuated by 24 h. Preincubation of PC12 cells with either the caspase-3 inhibitor, DEVD, or antisense caspase-3 oligonucleotides caused cells to be more rounded in appearance and led to a failure of cells to disperse properly. Additional experiments demonstrated a possible target for caspase cleavage to be the cytoskeletal protein, tau. These data suggest a requirement for caspase activation and subsequent disassembly of the cytoskeleton during cell dispersion and represent a novel role for caspases that may allow for proper migration of neurons to target locations during development.
Subject(s)
Caspases/metabolism , Cell Death/physiology , Animals , Caspase 3 , Caspase Inhibitors , Cell Movement/physiology , Cysteine Proteinase Inhibitors/pharmacology , Enzyme Activation , Immunohistochemistry , Kinetics , Oligonucleotides, Antisense/pharmacology , PC12 Cells/cytology , PC12 Cells/physiology , RatsABSTRACT
Type XI collagen is a quantitatively minor yet essential constituent of the cartilage extracellular matrix. The amino propeptide of the alpha1 chain remains attached to the rest of the molecule for a longer period of time after synthesis than the other amino propeptides of type XI collagen and has been localized to the surface of thin collagen fibrils. Yeast two-hybrid system was used to demonstrate that a homodimer of alpha1(XI) amino propeptide (alpha1(XI)Npp) could form in vivo. Interaction was also confirmed using multi-angle laser light scattering, detecting an absolute weight average molar mass ranging from the size of a monomer to the size of a dimer (25,000-50,000 g/mol), respectively. Binding was shown to be saturable by ELISA. An interaction between recombinant alpha1(XI)Npp and the endogenous alpha1(XI)Npp was observed, and specificity for alpha1(XI)Npp but not alpha2(XI)Npp was demonstrated by co-precipitation. The interaction between the recombinant form of alpha1(XI)Npp and the endogenous alpha1(XI)Npp resulted in a stable association during the regeneration of cartilage extracellular matrix by fetal bovine chondrocytes maintained in pellet culture, generating a protein that migrated with an apparent molecular mass of 50-60 kDa on an SDS-polyacrylamide gel.