Gene expression pattern in PC12 cells with reduced PMCA2 or PMCA3 isoform: selective up-regulation of calmodulin and neuromodulin

Abstract

Cellular calcium homeostasis is controlled predominantly by the plasma membrane calcium pump (PMCA). From four PMCA isoforms, PMCA1 and PMCA4 are ubiquitous, while PMCA2 and PMCA3 are found in excitable cells. We have previously shown that suppression of neuron-specific PMCAs in non-differentiated PC12 cells changed the cell morphology and triggered neuritogenesis. Using the microarrays, real-time PCR and immunodetection, we analyzed the effect of PMCA2 or PMCA3 reduction in PC12 cells on gene expression, with emphasis on calmodulin (CaM), neuromodulin (GAP43) and MAP kinases. In PMCA-suppressed lines total CaM increased, and the calm I and calm II genes appeared to be responsible for this effect. mRNA and protein levels of GAP43 were increased, however, the amount of phosphorylated form was lower than in control cells. Localization of CaM/GAP43 and CaM/pGAP43 differed between control and PMCA-reduced cells. In both PMCA-modified lines, amounts of ERK1/2 increased. While pERK1 decreased, the pERK2 level was similar in all examined lines. PMCA suppression did not change the p38 amount, but the p-p38 diminished. JNK2 protein decreased in both PMCA-reduced cells without changes in pJNK level. Microarray analysis revealed distinct expression patterns of certain genes involved in the regulation of cell cycle, proliferation, migration, differentiation, apoptosis and cell signaling. Suppression of neuron-specific PMCA isoforms affected the phenotype of PC12 cells enabling adaptation to the sustained increase in cytosolic Ca(2+) concentration. This is the first report showing function of PMCA2 and PMCA3 isoforms in the regulation of signaling pathways in PC12 cells.

Plasma Membrane Ca2+-ATPase Isoforms Composition Regulates Cellular pH Homeostasis in Differentiating PC12 Cells in a Manner Dependent on Cytosolic Ca2+ Elevations

“…Cells were cultured as described previously [25] and differentiated with 1 mM dibutyryl-cAMP for 48 h. All the results presented here were obtained following 2-day differentiation process. For pH measurements, mitoSypHer probe was transfected to undifferentiated antisense-carrying PC12 lines with TurboFect Transfection reagent and 2 days later cells were differentiated as described above.…”

The Dynamic Proteome of Oligodendrocyte Lineage Differentiation Features Planar Cell Polarity and Macroautophagy Pathways

“…Although GAP43 is a calmodulin (CaM)-binding protein, like ATP2B2, GAP43 phosphorylation reduces its affinity for CaM. Hence, the interaction between GAP43 and CaM controls the availability of CaM [ 162 ], which then regulates the calcium pump activity of ATP2B2, and thus GAP43 takes part in the regulation of OPC maintenance and differentiation [ 163–165 ]. PS 14 is another subset of STSPs that promotes this prolonged differentiation process into its final OL target.…”

The Effect of Coatings and Nerve Growth Factor on Attachment and Differentiation of Pheochromocytoma Cells

“…Various substratum coatings used in the formation of PC12 neuronal processes have been reported, e.g., glycoproteins, collagen (type IV from human placenta, type I) [ 8 , 9 , 10 , 11 ], laminin (Lam) [ 12 ], and fibronectin (Fn) [ 3 ]. In addition, it was shown that polyornithine [ 3 , 13 , 14 , 15 , 16 ], poly-D-lysine, and poly-L-lysine (PLL) can be used to enhance the attachment of PC12 cells [ 8 , 9 , 10 , 11 ]. Although Tomaselli et al [ 17 ] have shown that Lam and collagen type IV coated surfaces promoted PC12 adhesion to a greater degree than Fn, no systematic assessment of the suitability of various coatings that would enhance the attachment and differentiation of PC12 cells has been performed.…”