BMC Genomics 2004, 5:46 http://www.biomedcentral.com/1471-2164/5/46 adhesion complexes [6,9,10], and directed cell migration [9,11,12]. NHE1-dependent increases in pHi also play an essential role in cell transformation and the development of malignant progression [13,14] and NHE1-deficient cells have a markedly reduced capacity for tumor growth in vivo [15]. In this study we used cDNA microarray analysis to deter- mine changes in steady-state gene expression in fibrob- lasts stably a mutant NHE1 lacking ion translocation activity compared with fibroblasts stably expressing wild- type NHE1. Consistent with a role for NHE1 in cell growth regulation, the unbiased microarray analysis indi- cated that in the absence of NHE1 activity there are signif- icant changes in the expression pattern of genes related to growth factor signaling, growth and oncogenesis, and DNA synthesis and cell cycle control. Results and Discussion Global gene profiling Recent evidence indicates that in addition to the function of NHE1 in ion translocation and pHi homeostasis, the exchanger also acts as a scaffold to assemble signaling complexes and as a plasma membrane anchor for the actin-based cytoskeleton [3,6]. To selectively impair only ion translocation by NHE1, we engineered an ion translo- cation-defective NHE1 containing an isoleucine substitu- tion for glutamine 266 (NHE1-E266I). In cells expressing NHE1-E266I, the scaffolding and actin anchoring func- tions of NHE1 are retained, but ion translocation is absent [5,6]. Wild-type NHE1 (LAPN cells) and NHE1-E266I (LAPE cells) were stably expressed in NHE1-null LAP1 cells, which are derived from NHE1-expressing Ltk-mouse muscle fibroblasts [16,17]. As previously reported [5] NHE1 expression in LAPN and LAPE cells, as determined by immunoblotting, is similar and steady-state pHi in the continuous presence of serum and HCO3- is ~7.35 for LAPN cells and ~7.10 for LAPE cells. The presence of HCO3- allowed the function of anion exchangers contrib- uting to pHi homeostasis in the absence of ion transloca- tion by NHE1. For DNA microarray analysis, significant regulation of genes in LAPE cells compared with LAPN cells was defined as a fold change > 1.5 with a p value of < 0.05 from five independent cell preparations and microarray hybridiza- tions. Of the 6,500 probe sets, 198 or 3.05% were signifi- cantly different in LAPE cells. Two widely used approaches to analyze DNA microarray data include hierarchial clus- tering of genes with similar expression patterns [18] and grouping of biologically related genes into processes or pathways [19,20]. We used the latter strategy to group genes regulated by NHE1 activity into related biological pathways or processes. Genes were grouped according to key-words representing functional categories and Gen- MAPP, developed by the Conklin laboratory at the Uni- versity of California, San Francisco [20,21], was used to visualize gene expression data on maps representing bio- logical pathways. The advantage of a pathway-based anal- ysis is that it provides a global perspective of functionally- related genes. Pathway-based grouping indicated a sub- stantial number of differentially expressed genes associ- ated with growth factor/hormone signaling and growth and oncogenesis (Fig. 1). The caveat of pathway-based analyses is that based on key-word representation, some genes are implicated in multiple biological processes. Hence, we listed all genes exhibiting significant changes in LAPE cells compared with LAPN cells (Table 1). Data in Table 1 are grouped according to biological function with absolute changes indicated. Growth factor and oncogenic signaling The expression of a substantial number of genes encoding proteins related to growth factor/hormone signaling and growth and oncogenesis was altered in LAPE cells com- pared with LAPN cells. A schematic cascade of growth fac- tor signaling indicates that a number of genes regulated in LAPE cells function in Ras-dependent signaling (Fig 2A). Activation of many of these signaling proteins, including serotonin (5HT) [22] and thrombin (PAR2) [23] recep- tors, Ras [13], Raf [24], PLCγ1 [25], and Erk [26,27] stim- ulates NHE1 activity. Increased NHE1 activity and a resulting intracellular alkalinization are thought to be necessary for oncogenic transformation [14] and tumor development [15]. Immunoblot analysis confirmed increased protein expression of Fyn, PLCγ1, and ERK2 in LAPE cells compared with LAPN cells (Fig. 2B), In LAPE cells, the global increased expression of a number of genes involved in growth factor and oncogenic signaling sug- gests a feedback response to acidic or osmotic stress. Alter- natively, because the proliferative response is suppressed in LAPE cells [5] (Fig. 3), increased growth factor signaling could result from a feedback mechanism to maintain cell proliferation. Expression of a number of transcription factor genes act- ing downstream of growth factor signaling was also differ- entially regulated in LAPE cells compared with LAPN cells (Fig. 2A). Fra1 and Fra2 are Fos proteins and components of the AP1 transcription factor. They form dimmers with Jun transcription factors to regulate a number of cell proc- esses including differentiation, proliferation, and onco- genic transformation [28]. The regulated expression of two members of the C/EBP (CAAT/enhancer binding pro- tein) family of transcription factors was confirmed. RT- PCR was used to confirm decreased expression of C/EBPδ (Fig. 2C), which dimerizes with C/EBPβ in response to Ras-ERK signaling to regulate adipocyte [29,30] and epi- dermal [31] differentiation. Increased expression of GADD153 (CHOP), which is a transcription factor in the Page 2 of 13 (page number not for citation purposes)