BMC Genomics 2004, 5:46 http://www.biomedcentral.com/1471-2164/5/46 EdFxiifgfpeurreresensti5oianllyorfecgyutolastkeedleintoLnAaPnEdceexlltsracellular matrix genes Expression of cytoskeleton and extracellular matrix genes differentially regulated in LAPE cells. A. Immu- noblotting for gelsolin (top panel) and zymography for type IV collagenase (MMP-9) activity (bottom panel) confirmed increases and decreases, respectively, in LAPE cells com- pared with LAPN cells observed with GeneChip data. B. Rel- ative TaqMan analysis indicated decreased p24p3 expression in LAPE cells, consistent with GeneChip data. is increased lactic acid production [47] and decreased lac- tate dehydrogenase in LAPE cells correlates with increased NHE1 activity being necessary for oncogenic transforma- tion [14] and tumor development [15]. Consistent with increased glycolysis and pyruvate produc- tion in LAPE cells, key enzymes favoring ATP production by electron transport and oxidative phosphorylation were increased (Fig. 4A). Increases in NADH-ubiquinone oxi- doreductase and cytochrome c oxidase would favor oxida- tion of NADH. Increases in subunits A, C, and D of ATP synthase would increase endergonic synthesis of ATP. ATP synthase in the inner mitochondrial membrane is a pro- ton translocator, extruding protons into the mitochon- drial matrix. Whether decreased cytosolic pH in LAPE cells compared with LAPN cells alters the pH of the mitochon- drial matrix and proton-electromotive force powering ATP synthesis remains to be determined. Collectively, the profile of gene expression in LAPE cells suggests equilib- rium towards increased glycolytic flux and ATP production. Cytoskeleton and extracellular matrix cDNA array analysis indicated that loss of NHE1 activity in LAPE cells was associated with the regulation of a number of genes involved in cytoskeleton organization, cell adhesion, and extracellular matrix assembly. The reg- ulation of several genes correlates with reported effects of NHE1 activity and pHi on cell shape determination [6], cell polarity [9], actin-filament bundling [9,48], cell-sub- strate adhesion [9,10] and cell migration and metastasis [9,11,14]. NHE1 acts as an anchor for actin filaments by binding directly members of the ERM (ezrin, radixin, moesin) family of actin binding proteins, and in LAPE cells, expression of NF2 (merlin), a tumor suppressor pro- tein and member of the ERM family, was downregulated. Consistent with a role for NHE1 activity in cell polarity and actin dynamics, loss of NHE1 activity was associated with decreased expression of myosin regulatory light chain, and increased expression of gelsolin (Fig. 5A), a pH-dependent actin severing and capping protein. Although regulation of the microtubule-based cytoskele- ton by NHE1 and pHi have previously not been reported, a number of microtubule-related genes, including Clip 170, KIF4, kinesin light chain, and a dynein heavy chain, were upregulated in LAPE cells. Consistent with NHE1-dependent cell adhesion and migration, LAPE cells had a marked (~26-fold) decrease in type IV collagenase (MMP-9) expression, and zymography confirmed that activity of MMP-9, but not activity of MMP-4, was selectively decreased in LAPE cells compared with LAPN cells (Fig. 5B). Correlating with a decrease in MMP-9 expression, LAPE cells also had similar marked (~24-fold) decrease in the expression of lipocalin 24p3, which was confirmed by TaqMan analysis (Fig. 5C). NGAL (Neutrophil Gelatinase Associated Lipocalin), the human homolog of mouse lipocalin, is covalently bound to MMP-9 and protects MMP-9 from degradation [49,50]. Lipocalins are transcriptionally regulated by C/EBPβ [51], which is likely suppressed by decreased expression of C/ EBPδ (Fig. 2C) and increased expression of GADD153 (Fig. 2D). Moreover, increased expression of MMP-9 [52] and lipocalins [53] is associated with tumor cell growth and invasion, which correlates with a role for NHE1 activ- ity in these processes [14,15]. Conclusions In summary, global profiling revealed genes regulated by loss of NHE1 activity and decreased pHi. A number of the differentially regulated genes involved in growth factor signaling, cell cycle progression, and cytoskeleton and extracellular matrix remodeling are consistent with previ- ously established roles of NHE1 activity and pHi in mitogenic responses, cell proliferation, and tumor metas- tasis and invasion. In contrast, some genes, including those regulating carbohydrate metabolism and microtubule dynamics, have previously not been linked to NHE1 activity. An important future direction is to determine primary and secondary effects of gene regula- tion by NHE1 and of particular interest is whether promoters within the genes differentially regulated in LAPE cells are pH-responsive. Page 10 of 13 (page number not for citation purposes)