Identification of a pH-responsive DNA region upstream of the transcription start site of human NBCe1-B.

Human SLC4A4 is located on chromosome 4q21 amongst a cluster of enamel and dentin genes, including ameloblastin (AMBN), enamelin (ENAM), amelotin (AMTN), dentin matrix acidic phosphoprotein 1 (DMP1), and dentin sialophosphoprotein (DSPP) (1). SLC4A4 codes for the sodium/bicarbonate (Na+/HCO3−) transporter protein NBCe1, and is essential for the normal development of enamel (1–3). Our previously published data showed that in the ameloblast-like LS8 cell line, the expression of mRNA for the NBCe1-B isoform is pH dependent, with the highest expression seen at a pH of < 7.0 (4). These results showed maximal expression of NBCe1-B mRNA at pH 6.0 (4). Since this paper (4) was published, we have repeated the experiment (at pH 6.0, 7.3, and 8.5) using real-time PCR, and obtained similar results (Fig. 1). Our findings imply that in LS8 cells the SLC4A4 gene promoter can respond to transcriptional factors in a pH-dependent manner. Fig. 1 Relative expression levels of NBCe1-B mRNA at acidic, physiologically neutral, and basic pH conditions assessed by real-time PCR. LS8 cells cultured for 24 h at pH 6.0 showed a 1.41 ± 0.22-fold (mean ± SE) increase in NBCe1-B mRNA transcripts ... Based on published literature it is apparent that no gene promoter analyses have been carried out for the SLC4A4 gene transcripts (referred to in this manuscript as NBCe1-A, NBCe1-B, and NBCe1-C). The literature is also scant with regards to identifying pH-dependent transcription factors for other bicarbonate transporter genes, of which there are many. One example is the solute carrier SLC26A4 gene coding for pendrin, an anion exchanger involved in HCO3−/Cl− exchange (5). A study on the molecular mechanisms of epithelial-specific expression and gene regulation of pendrin, using a luciferase reporter assay, has identified a DNA element responsive to changing pH within a 96-bp region of the pendrin promoter (5). This 96-bp region is ~ 1,000 bp upstream of the transcription start site (5). For pendrin, this 96-bp pH-responsive region is most responsive in basic conditions, rather than in acidic conditions (5). No other potential pH-dependent transcription factors have been directly associated with this 96-bp region; however, an in silico search for possible transcription factors that could bind to this region suggested that the mitogen-activated protein kinase (MAPK) signaling pathway was involved (5). In the current study we sought to define pH-dependent response elements in the NBCe1-B promoter region. Data presented identify potential NFKB1- and potential TP53-binding sites ~ 100–200 bp upstream of the presumed NBCe1-B transcriptional start site (TSS). Both NFKB1- and TP53-binding sites are required for reporter gene up-regulation when cells are exposed to acidic pH conditions.