Calmodulin is an ubiquitous Ca.sup.2+ -binding regulatory protein that mediates the action of Ca.sup.2+ in a diverse array of biological events including cell division, microtubular depolymerization and polymerization, cyclic nucleotide metabolism, Ca.sup.2+ transport, and protein phosphorylation .sup.6,8,31,38,43,72. The involvement of calmodulin in various nerve cell processes, together with its abundant distribution in brain tissue.sup.75, suggests that calmodulin plays an important role in the growth and differentiation of nerve cells.
The notion that calmodulin is a regulator of cell growth was initially suggested by studies showing that calmodulin antagonists inhibited cell division in vitro by blocking cell-cycle progression at the G1/S boundary and during G2/M.sup.6,15,60. Consistent with such results are the findings that calmodulin levels are altered during the various phases of the cell cycle.sup.7,42,60 and that calmodulin increases DNA synthesis in a variety of cells .sup.2,24,41,42. Using eukaryotic expression vectors to produce sense or antisense calmodulin RNA, it was demonstrated that increases or decreases in the intracellular levels of calmodulin result in increases and decreases, respectively, in the rates of proliferation of mouse C127 cells.sup.53,54. The role of calmodulin in cell growth is also supported by the findings of elevated levels of calmodulin in certain transformed cell types.sup.12,32,66,69.
Although there is evidence for the participation of calmodulin in cell differentiation, its role has not been precisely defined. Treatment of leukemic HL-60 cells with calmodulin antagonists resulted in an increase in cellular differentiation.sup.67 . On the other hand, the levels of calmodulin were found to increase during nerve growth factor (NGF) - induced differentiation of PC12 cells.sup.4.
Structural studies have revealed that the amino acid sequence of calmodulin is remarkably conserved among species.sup.50. In the rat genome, calmodulin is encoded by three genes expressing five transcripts, each of which encodes an identical protein, the sequence variations in the coding regions being within the limits of codon degeneracy .sup.47,48,50,63. These calmodulin mRNAs are heterogeneously distributed both in the newborns.sup.11,70 and adult rat brain.sup.18,56 and have been shown to change during ontogeny in a pattern which is paralleled by similar changes in the biological activity of calmodulin.sup.11, indicating that calmodulin is involved in neuronal development.
Using the PC12 clonal cell line.sup.21 as an in vitro model for investigating neuronal development, it was demonstrated that PC12 cells express, in varying levels, all five calmodulin gene transcripts.sup.4. Differentiation of PC12 cells induced by NGF or dibutyryl cyclic AMP was accompanied by characteristic and distinct changes in the levels of the various calmodulin mRNAs, demonstrating that the calmodulin genes can be differentially regulated.sup.34. Other studies showed that the multiple calmodulin transcripts have distinct subcellular distributions, some being found both in the cell bodies and neurites and others found only in the cell bodies.sup.74. Since the cellular location of calmodulin will determine its functional activity, these results suggested that the regulation of the levels of particular calmodulin mRNAs in different subcellular compartments may, in turn, be one means for controlling the different functional activities for calmodulin in nerve cells. However, it has not yet been determined if calmodulin gene expression plays a causal role in differentiation.