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Determination of the NFAT5/TonEBP transcription factor in the human and ovine placenta.

ABSTRACT: Osmotic stress results in the accumulation of osmolytes in tissues. Synthesis of these osmolytes is mediated by the transcription factor NFAT5/TonEBP in the human kidney. We tested for the presence of NFAT5 mRNA and protein in the human and ovine placenta and confirmed sorbitol and inositol osmolyte concentrations in these tissues. To determine NFAT5 protein, human and ovine placenta were tested for inositol, sorbitol and glucose using high performance liquid chromatography (HPLC). Additionally, RNA was extracted and cDNA was made from these tissues. PCR was performed and products were sequenced. Western blotting was used to assess the expression of the NFAT5 protein. Human and ovine placenta demonstrated: 1) high concentrations of sorbitol and inositol, 2) presence of NFAT5 mRNA, 3) confirmation by NFAT5 sequence identity, and 4) presence of NFAT5 protein. NFAT5 is present in the ovine and human placenta at the RNA and protein levels that suggest a role for this protein in the induction of these osmolytes. Further trophoblast studies of osmotic stress effects on osmolytes are planned.

SUBMITTER: Arroyo JA

PROVIDER: S-EPMC2900158 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Determination of the NFAT5/TonEBP transcription factor in the human and ovine placenta.

Arroyo Juan A JA Teng Cecilia C Battaglia Frederick C FC Galan Henry L HL

Systems biology in reproductive medicine 20090801 4

Osmotic stress results in the accumulation of osmolytes in tissues. Synthesis of these osmolytes is mediated by the transcription factor NFAT5/TonEBP in the human kidney. We tested for the presence of NFAT5 mRNA and protein in the human and ovine placenta and confirmed sorbitol and inositol osmolyte concentrations in these tissues. To determine NFAT5 protein, human and ovine placenta were tested for inositol, sorbitol and glucose using high performance liquid chromatography (HPLC). Additionally, ...[more]

PMID: 19886771

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Project description:Macular edema, a frequently encountered complication of diabetic retinopathy (DR), results from alterations of the blood retinal barrier (BRB) and leads to modifications of the retinal pigmented epithelium (RPE) functions. Osmolar changes of the surrounding medium could be responsible for modifications of the RPE functions leading to disturbance of retinal homeostasis. The expression, activation and function of the key hyperosmolar response factor Tonicity Enhancer Binding Protein (TonEBP also called nuclear factor of activated T-cell 5 - NFTA5) was investigated in ARPE-19 cells, derived from human RPE, in response to hyperosmolar stimulation.ARPE-19 cells were exposed to hyperosmolar medium. TonEBP mRNA and protein levels were quantified by qRT-PCR and semi-quantitative Western blot. TonEBP nuclear translocation was investigated by immunofluorescence. TonEBP transactivation activity was measured using a reported plasmid containing TonEBP binding sites.In response to hyperosmolar stimulation of ARPE-19 cells, a dose-dependent increase in TonEBP mRNA and protein levels, as well as TonEBP nuclear translocation were observed. TonEBP transactivation activity was further demonstrated using a reporter plasmid containing TonEBP binding sites. A dominant negative form of TonEBP abolished NaCl-induced increase in TonEBP transactivation activity, and inhibited the increase of the target genes aldose reductase and sodium-dependent taurine transporter mRNA levels. SB203580, an inhibitor of two of the p38 protein kinase's isoforms (p38? and p38?) inhibited the TonEBP nuclear translocation and transactivation activity in ARPE-19 cells exposed to hyperosmolar stimulation.Our data demonstrates the involvement of TonEBP in the mechanisms responsible for osmoadaptation to hyperosmolar stress in RPE cells. Given the emerging role of TonEBP in different pathological pathways, these data open new perspectives for the analysis of the mechanisms involved in the modification of functions of the RPE during macular edema.

Dataset Information

Determination of the NFAT5/TonEBP transcription factor in the human and ovine placenta.

Publications

Determination of the NFAT5/TonEBP transcription factor in the human and ovine placenta.

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