We examined factors important in regulation of expression of the Na+/H+exchanger gene in NIH/3T3 cells. A stable fibroblast cell line was generated that contained a 1.1-kb proximal fragment of the mouse NHE1 promoter. The addition of serum to serum-starved cells resulted in an increase in activity of the NHE1 promoter. The mitogenic agonists insulin, thrombin, and epidermal growth factor also increased transcription from the NHE1 promoter. Phorbol esters also increased NHE1 promoter-directed transcription, whereas the serine/threonine protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine inhibited this stimulation. The protein kinase inhibitors GF-109203X, PD-98059, and genistein all stimulated promoter activity. Promoter deletion analysis and gel mobility shift assays showed that a region between 0.9 and 1.1 kb from the start site was involved in mediating the effect of mitogenic stimulation. The results show that a variety of mitogenic factors can activate the NHE1 promoter during cell growth and proliferation.

The genes encoding the first two enzymes of the peroxisomal ␤-oxidation pathway, acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), contain upstream cis-acting regulatory regions termed peroxisome proliferator response elements (PPRE). Transcription of these genes is mediated through the binding of peroxisome proliferator-activated receptor ␣ (PPAR␣), which binds to a PPRE as a heterodimer with the 9-cis-retinoic acid receptor (RXR␣). Here we demonstrate that the HD-PPRE is also a target for the constitutive androstane receptor ␤ (CAR␤). In vitro binding analysis showed that CAR␤ bound the HD-PPRE, but not the AOx-PPRE, as a heterodimer with RXR␣. Binding of CAR␤/RXR␣ to the HD-PPRE occurred via determinants that overlap partially with those required for PPAR␣/RXR␣ binding. In vivo, CAR␤/RXR␣ activated transcription from an HD-PPRE luciferase reporter construct. Interestingly, CAR␤ was shown to also modulate PPAR␣/RXR␣-mediated transactivation in a response element-specific manner. In the presence of the peroxisome proliferator, Wy-14,643, CAR␤ had no effect on PPAR␣/RXR␣-mediated transactivation from the HD-PPRE but antagonized transactivation from the AOx-PPRE in both the presence and the absence of proliferator. Our results illustrate that transcription of the AOx and HD genes is differentially regulated by CAR␤ and that the HD gene is a specific target for regulation by CAR␤. Overall, this study proposes a novel role for CAR␤ in the regulation of peroxisomal ␤-oxidation.

SummaryObjectives: To determine whether functional antithrombin III (AT-III) levels measured by a factor Xa inhibition (AT-III-Xa) assay identifies AT-III deficient individuals more reliably than functional AT-III levels measured by a thrombin inhibition (AT-III-IIa) assay.Study design: Cross-sectional study.Patient population: Sixty-seven members of a large family with type 2 AT-III deficiency.Intervention: DNA analysis was used as the reference diagnostic standard for AT-III status and subjects were classified as AT-III deficient or non deficient according to these results. Functional AT-III levels were measured in all subjects using: 1) a chromogenic substrate for thrombin and added human thrombin (AT-III-IIa), and 2) a chromogenic substrate for factor Xa and added bovine factor Xa (AT-III-Xa). Functional heparin cofactor II (HC-II) levels were measured using a commercially available kit. The proportions of 125I-α-thrombin complexed to AT-III and HC-II were measured by polyacrylamide gel electrophoresis and autoradiography.Results: Thirty-one (46%) individuals were classified as AT-III deficient and 36 (54%) as AT-III non deficient. AT-III-Xa assay measured a significantly lower mean AT-III value and a narrower range for individuals classified as AT-III deficient than the AT-III-IIa assay. Using the AT-III-IIa assay, six subjects had borderline AT-III levels compared to none with the AT-III-Xa assay. Thrombin inhibition by HC-II likely accounts for the AT-III-IIa assay giving higher values than the AT-III-Xa assay since 1) there was a significant correlation between the difference in AT-III-IIa and AT-III-Xa levels and HC-II levels, 2) the mean level of HC-II was significantly higher for individuals who had a positive difference between AT-III-IIa and AT-III-Xa levels compared to those who had a negative difference and 3) there was a significant correlation between the difference in AT-III-IIa and AT-III-Xa levels and the percentage of 125I-α-thrombin complexed to HC-II.Conclusion: The AT-III-Xa assay is a better discriminant between AT-III deficient and AT-III non deficient individuals than the AT-III-IIa assay.

The chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors involved in regulation of embryonic development and neuronal cell fate determination. We identified a target of COUP-TF involved in cell proliferation and cell differentiation. Using reporter assays, footprint analysis, and electrophoretic mobility shift assays, we showed that a nuclear hormoneresponsive element located at 2841/2800 nt of the mouse Na 1 /H 1 exchanger (NHE) promoter binds COUP-TF with enhancer activity. Mutation at 2829/2824 nt (and secondarily at 2837/2833) prevents COUP binding and activation of the NHE promoter. In vivo expression of COUP isoforms in NIH 3T3 or CV1 cells transactivates from the nuclear hormone-responsive element and from the entire NHE1 promoter. Transactivation is greater for COUP-TFII, is increased for either COUP isoform by the presence of high serum concentrations, and is greatly reduced by mutations preventing COUP binding. In vivo COUP expression in NIH 3T3 cells results in increased synthesis of NHE. Expression of COUP-TFII induced by either retinoic acid or dimethyl sulfoxide in differentiating P19 cells increases NHE expression. The results show that COUP-TF regulates expression of the NHE and provide a mechanism that may be important in physiological and pathological situations linked to its upregulation.