The importance of nonvoltage-gated calcium in the regulation of cellular events continues to unfold, using CAI as a laboratory tool. The basic science focus of the laboratory is identification and characterization of the molecular targets of modulation of transmembrane calcium signaling. A two-pronged approach has been used. First, modulation of expression of known genes by alteration of calcium homeostasis has been investigated. We have demonstrated that CAI exposure reduces gelatinolytic activity of MMP-2 and MMP-9 in several cell types; this reduction in activity is due to reduction of MMP-2 gene expression. There is no known calcium-sensitive regulatory element for MMP-2, however MMP-1, MMP-9, and MT-MMT have AP-1 sites in their promoters. MMP-1 gene expression is also reduced with CAI exposure. Studies are ongoing to investigate the effects of CAI on MMP-9 and MT-MMP. The effect of CAI on calcium influx was also shown to regulate expression of a proliferation marker, VL-30, that is known to have a calcium regulatory component, previously linked to intracellular calcium release. The second approach is to identify and characterize novel genes which are regulated by CAI. CAI-resistant A2058 cells were produced with ability to grow in culture despite constant CAI pressures up to 40 microM. The cells have normal growth rate and phenotype with up to 20 microM resistance, however, their growth rate falls off with increasing chronic exposure to CAI. A subtractive hybridization yielded 35 independent clones selectively expressed in the resistant cells. Three have been further studied; two are expressed 2-3 fold above control and encode novel transcripts by GenBank analysis. The third is expressed up to 4-fold higher in the resistant cells and encodes a novel protein, CAIR-1. It is expressed normally in a number of tissues and by Southern zoo blot has homologs in mammals, birds, and some amphibians. Anti-peptide antibody immunoblots indicate a protein of approximately 78 kDa, consistent with the anticipated open reading frame. Further sequence and protein analysis studies are ongoing. New techniques are being tried to identify and characterize the cellular CAI binding site. These include trials of a recently synthesized biotinylated CAI.
