Differential expression of Rho family GTP-binding proteins and protein kinase C isozymes during C6 glial cell differentiation.
The differential expression of Rho family of low molecular weight GTP-binding proteins and protein kinase C (PKC) isozymes were examined during differentiation of rat C6 glial cells to astrocytic phenotypes induced by dibutyryl cAMP (dbcAMP)/theophylline. The cells showed rapid and distinct morphological changes, resembling stellate astrocytes at 12 h after the treatment. The treated cells had a round cell body that extended several long processes each with a beaded appearance. In addition to morphological changes, Western blot analysis revealed that S-100 protein, known as a glial cell differentiation marker, increased and reached the maximal level (approximately 6-fold increase) at 24 h following the addition of dbcAMP. In the control experiments with cells cultured in the absence of serum but also without dbcAMP/theophylline, morphological changes were marginal and apparent increases of S-100 protein were not observed by Western blotting. In response to dbcAMP/theophylline treatment, RhoA showed increases in the mRNA level followed by the protein level, as inferred by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Rac1 and Cdc42 proteins were undetectable by Western blot analyses. In PKC isozymes, increases were observed in PKC beta 1, epsilon, and zeta by RT-PCR, and in beta 1 and epsilon by Western blotting. Among them, PKC epsilon showed the most distinct changes. Its mRNA level transiently increased from 3 to 6 h and then decreased even below the basal level at 18 h after the treatment. In contrast, Western blot analysis revealed that PKC epsilon gradually increased time-dependently to 24 h (approximately 6-fold increase), and remained elevated until 48 h. These results suggested that RhoA and PKC epsilon, and probably also PKC beta 1 and PKC zeta, were closely implicated in C6 cell differentiation.
Yoshimura
S
S
Department of Neurosurgery, Gifu University School of Medicine, Japan.
Sakai
H
H
Nakashima
S
S
Nozawa
Y
Y
Shinoda
J
J
Sakai
N
N
Yamada
H
H
D000818:Animals; D001253:Astrocytes / Q000166:cytology* / Q000187:drug effects; D001483:Base Sequence; D015415:Biomarkers; D003994:Bucladesine / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000096:biosynthesis*; D007527:Isoenzymes / Q000096:biosynthesis*; D007700:Kinetics; D008969:Molecular Sequence Data; D009457:Neuroglia / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000096:biosynthesis*; D064546:Protein Kinase C beta; D051744:Protein Kinase C-epsilon; D051381:Rats; D009418:S100 Proteins / Q000096:biosynthesis; D013806:Theophylline / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein; D000099221:Protein Kinase C zeta
D015415:Biomarkers; D003994:Bucladesine; D017931:DNA Primers; D019204:GTP-Binding Proteins; D007527:Isoenzymes; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051744:Protein Kinase C-epsilon; D009418:S100 Proteins; D013806:Theophylline; D020742:rhoA GTP-Binding Protein; D000099221:Protein Kinase C zeta; C497257:Prkce protein, rat