Overexpression of Ca2+/calmodulin-dependent protein kinase II in PC12 cells alters cell growth, morphology, and nerve growth factor-induced differentiation

Abstract

To examine the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in cell differentiation and neuronal functions, stable transformants of PC12 cells were established that expressed levels of the alpha-subunit of CaMKII (alpha CaMKII) equivalent to mammalian neurons. The expression of the transfected alpha CaMKII gene or the endogenous beta CaMKII gene was monitored by RNase protection assays, and alpha CaMKII protein expression was determined by Western blots. Several PC12-derived clones expressed amounts of alpha CaMKII mRNA and alpha CaMKII protein similar to that of hippocampal tissues and several orders of magnitude greater than untransfected PC12 cells. CaMKII catalytic activity was four times higher in extracts from alpha CaMKII-overexpressing compared with untransfected PC12 cells. All clones overexpressing alpha CaMKII displayed altered cellular growth and adhesion properties including increased cell-to-substrate adhesion, decreased cell-to-cell adhesion, enhanced contact inhibition, and prolonged survival at confluency. Furthermore, the alpha CaMKII activity in overexpressing PC12 cells inhibited neurite elongation during NGF-induced differentiation. Inhibition of CaMKII activity in vivo with KN-62 caused the morphological phenotypes of alpha CaMKII-overexpressing cells to partially revert to that of untransfected PC12 cells. These results show that alpha CaMKII catalytic activity affects growth, morphology, and NGF-induced differentiation of PC12 cells.

Fig. 1.

Analysis of αCaMKII mRNA and protein expression in PC12 cells and hippocampus. A, RNase protection analysis of αCaMKII (a) and βCaMKII (b) mRNAs in normal and αCaMKII-overexpressing PC12 cells or hippocampal tissue from adult rat brain. Total cellular RNA from two different PC12 cultures, two independent clones overexpressing αCaMKII, or hippocampi were analyzed. Equivalent amounts of RNA were hybridized with subunit-specific cRNA probes and digested with RNase A + T1 (see Materials and Methods). Autoradiograms show cRNA fragments protected by mRNAs from αCaMKII- overexpressing cells (lanes 1, 2), untransfected cells (lanes 3, 4), and hippocampus (lanes 5, 6). B, Western blot analysis of αCaMKII (top) and actin (bottom) in untransfected PC12 cells (lane 2), αCaMKII-overexpressing PC12 cells (lanes 1, 4–8), and rat brain hippocampus (lane 3). Equivalent amounts of total cellular protein (50 μg) were analyzed with monoclonal antibodies specific for αCaMKII or actin.

Fig. 2.

General morphology of normal (A) and αCaMKII-overexpressing (B) PC12 cells. Phase-contrast micrographs were obtained on living cells plated at low density on untreated tissue culture plastic and grown for 5 d in standard medium containing 15% serum. Scale bar, 100 μm.

Fig. 3.

Cellular growth curves of untransfected (dashes) and two independent clones of αCaMKII-overexpressing PC12 cells (a,b). After trypsinization of confluent cultures, equivalent numbers of cells (5 × 10⁵) were plated on tissue culture plastic and grown for the indicated times; half of the medium was replaced with fresh medium every 2 d. Cells were trypsinized, pelleted, and resuspended in PBS plus Trypan blue for counting. The number of total viable cells are given as the mean of triplicate cultures for each condition. Error bars indicate SEM.

Fig. 4.

Phase-contrast morphology of αCaMKII-overexpressing (A, C,D) and untransfected (B) PC12 cells after NGF differentiation under the following conditions: NGF differentiation for 2 d (A), NGF differentiation for 5 d (B), NGF differentiation in KN-62 (10 μm) for 2 d (C), and NGF differentiation in KN-62 (10 μm) for 5 d (D). Scale bar, 100 μm.

Fig. 5.

Time-lapse microscopy of growth cone dynamics. Phase-contrast micrographs of αCaMKII-overexpressing PC12 cells after culture in NGF for 2 d. Sequential micrographs of the same cells every 3 hr showed that growth cones are not stable during a 6 hr period (white arrowheads); after growth cone retraction/collapse, they reappear at the same location on the cell surface (black arrowheads). Scale bar, 50 μm.