Regulation of growth factor induced gene expression by calcium signalling: Integrated mRNA and protein expression analysis

Abstract

There is considerable indirect evidence that growth factor induced changes in the intracellular concentration of calcium play an important role in the regulation of the mammalian cell cycle. However, the precise mechanism by which this may be achieved remains unclear. Here we show that SKF-96365, an inhibitor of growth factor induced capacitative calcium entry (CCE), inhibits cell cycle progression by preventing entry into S phase. SKF-96365 changes the temporal profile of growth factor induced calcium signalling and recent studies have shown that alterations in the temporal and spatial patterns of calcium signalling can differentially regulate gene expression. We have therefore sought to examine the effect of inhibition of CCE on growth factor induced gene expression during G1. To achieve this we have initiated a combined transcriptomic and proteomic approach to measure CCE regulated gene expression using cDNA arrays and two-dimensional polyacrylamide gel electrophoresis, respectively. The initial results of this on-going analysis are reported here. They reveal that inhibition of CCE influences the expression of 29 genes at the mRNA level and 22 genes at the protein level. We report the identification of the mRNAs whose expression is altered by inhibition of CCE and describe the potential functional significance of some of these changes. The value of integrating a transcriptomic and two-dimensional gel electrophoresis based proteomic approach to studies of gene expression is discussed.

Gene expression profiles in HEK-293 cells with low or high store-operated calcium entry: can regulatory as well as regulated genes be identified?

“…A number of signaling genes whose levels of expression were altered in our studies were also found to be Ca 2ϩ regulated in other studies linking gene expression to SOCE (18,27). These include calcineurin, cAMP-dependent protein kinase catalytic-␤, FLAME-1, c-myc, frizzled homolog 7, Sine oculis homeobox homolog 2 (SIX2), and TGF-␤1.…”

“…Although there are a few genes in common between our study and the lymphocyte study, for the most part, SOCE appears to regulate different populations of genes in the two cell types, a possibility that we considered at the outset of the investigation (see INTRODUCTION). Compared with the data from the fibroblast study (27), the 150 genes regulated by a decrease in SOCE are much higher than the 29 genes seen to change. However, the microarray used in that study only represented 1,200 cDNA clones compared with the 22,000 cDNA clones represented on the Affymetrix microarray.…”

“…Gene expression profiles in HEK-293 cells with low or high store-operated calcium entry: can regulatory as well as regulated genes be identified?. Physiol Genomics 21: [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] 2005. First published December 28, 2004; doi:10.1152/physiolgenomics.…”

“…Therefore, the gene expression profiles produced by thapsigargin and ionomycin may vary significantly from those produced by activation of SOCE by receptor agonists. Another recent study utilized cDNA arrays (Atlas 1.2 mouse array from Clontech, which represents 1,200 genes) to demonstrate that inhibition of serum-stimulated 3T3 cells with SKF-96365, an inhibitor of SOCs, leads to the upregulation, or downregulation, of 29 genes (27). However, the interpretation of these SKF-96365 experiments is complicated by the lack of specificity of this reagent.…”

Cell Shape-dependent Control of Ca2+ Influx and Cell Cycle Progression in Swiss 3T3 Fibroblasts

“…Roles for Ca 2ϩ influx have been proposed for both early and late in G 1 (7). One attractive scenario is that Ca 2ϩ influx acting through calmodulin kinase regulates the biphasic activity of MAP kinase (60). G 1 progression requires an initial peak of MAP kinase activity upon mitogen stimulation that is followed by a lower sustained level of MAP kinase activity (1,2,22).…”