NHE1 Transcript Research Articles

Cellular distribution and regulation of NHE-1 isoform of the NA-H exchanger in the avian osteoclast.

The sodium-hydrogen exchanger (NHE) has been implicated in bone resorption by osteoclasts. We have studied expression of NHE-1, an isoform of the NHE, in chicken bone marrow mononuclear phagocyte precursors during differentiation into the osteoclast phenotype in culture. A monoclonal anti-body raised against the carboxy-terminus of NHE-1 detected the presence of a 100 kDa protein (similar to the mammalian form of NHE-1) in the osteoclasts. Laser scanning confocal microscopy revealed association with the alpha(v)beta(3) integrin and focal adhesion kinase (pp(125)FAK) at the basolateral membrane (BLM) of the osteoclast in addition to a more generalized cellular distribution. A fragment of avian NHE-1 cDNA was obtained by polymerase chain reaction cloning, and it was used to characterize expression of NHE-1 transcripts in cultured chicken osteoclast precursors. The avian NHE-1 message was a 3.9 kB band on Northern analysis, which differed from the mammalian message. Retinoic acid (RA) elicited an increase in the steady-state intracellular pH (pH(1)) from 6.87 to 7.10 in the absence of bicarbonate and was inhibited by ethylisopropylamiloride, an inhibitor of Na-H exchange. Using ribonuclease protection assays, we found that NHE-1 transcripts are induced as cells differentiate in vitro and in response to 13-cis-RA. Western blot analysis indicated that protein levels also increased in response to 13-cis-RA. Our results demonstrate expression of NHE-1 in avian osteoclasts with a complex cellular distribution in culture, and NHE-1 expression is induced as cells differentiate into mature osteoclasts in response to 13-cis-RA.

Specific Activation of the Na+/H+ Exchanger Gene during Neuronal Differentiation of Embryonal Carcinoma Cells

We examined the regulation of the Na+/H+ exchanger gene during differentiation of the P19 mouse embryonal carcinoma cells. Treatment of P19 cells with retinoic acid induces the development of neurons, astroglia, and microglia cells. Upon retinoic acid-induced differentiation of P19 cells, there was an early and rapid 10-fold increase in NHE1 transcription. A proximal cis-acting AP-2 site of the NHE1 promoter was sufficient for stimulation of transcription of the gene by differentiation. Bandshift experiments demonstrated that in retinoic acid-treated cells there was an elevated level of AP-2 transcription factor binding to the AP-2 consensus site of the Na+/H+ exchanger gene. In the differentiation defective mutant RAC65, the effect of differentiation on Na+/H+ exchanger gene expression was reduced by 60%. Examination of Na+/H+ exchanger activity showed that retinoic acid-treated P19 cells recovered from an acid load at a rate approximately three times greater than untreated cells. The increases in gene expression and protein activity preceded major changes in cell morphology, suggesting that the initiation of differentiation is linked to NHE1 gene expression. Our findings show for the first time that the NHE1 gene is activated early in cell differentiation and that this activation may play an important role in the process of neuronal cell differentiation.

Proximal regulatory elements and nuclear activities required for transcription of the human Na +/H + exchanger (NHE-1) gene

We herein demonstrate competence of the 5′ upstream region −1374 to +16 of the human growth factor-activatable Na +/H + exchanger (NHE-1) gene to promote transcription of the chloramphenicol acetyltransferase gene in cells of hepatic origin (HepG2), vascular-smooth-muscle origin (VSM A7r5) and fibroblasts (3T3). We also describe the mapping of the regulatory elements required for such transcription. Sequential 5′ end-deletions indicated that the 5′ boundary of the positive regulatory elements of NHE-1 transcription is localized downstream of nucleotide −252 in both HepG2 and VSM A7r5 cells but downstream of nucleotide −654 in 3T3 cells. Footprinting analysis of the 0.25-kb promoter fragment using rat liver nuclear extracts identified 4 protected regions as follows: A, −31 to −9; B, −108 to −65; C, −124 to −111; and D, −239 to −215. Internal deletion and nucleotide substitutions within regulatory element D revealed its essential role for transcription of the human NHE-1 gene in HepG2 and VSM A7r5 cells. DNA binding and competition assays using rat liver nuclear extracts indicated that regulatory element D is recognized by 5 nuclear activities. Four of these activities (designated as NHE-1D1-4) are competed out completely by oligonucleotides containing the binding sites of transcription factors CREB, AP3, NFY, and other CCAAT box-binding proteins (C/EBPα or related proteins). This competition profile might be explained by the presence of homology between regulatory element D and the consensus sequence of C/EBP as well as the other competitor oligonucleotides. The actual relationship between these nuclear activities and the C/EBP family of proteins (or other transcription factors) remains to be determined.