11Beta-hydroxylase deficiency and other syndromes of mineralocorticoid excess as a rare cause of endocrine hypertension.

Hypertension represents a major public and global health problem, most of which likely can be improved by lifestyle changes including changing dietary habits with less consumption of processed and preserved foods, which generally contain higher amounts of salt than freshly prepared food items. Among causes for endocrine hypertension are syndromes of mineralocorticoid excess. This group of mostly monogenic and acquired disorders typically causes hypertension through activation of the mineralocorticoid receptor either directly or indirectly via hormonal mediators and from overactive amiloride-sensitive epithelial sodium channels located in the distal tubule and collecting ducts of the kidneys. Apart from primary aldosteronism, mineralocorticoid excess can be caused by congenital adrenal hyperplasia (CAH) due to mutations of the 11beta-hydroxylase and 17alpha-hydroxylase genes, by inactivating mutations of the glucocorticoid receptor gene (Chrousos syndrome), endogenous hypercortisolism (Cushing's syndrome), by mutations of the 11beta-hydroxysteroid dehydrogenase type 2 gene (apparent mineralocorticoid excess/AME) or licorice/carbenoxolone intake, mutations of the epithelial sodium channel genes (Liddle syndrome), mutations of the mineralocorticoid receptor gene (Geller syndrome), and by mutations in the WNK1, WNK4, KLHL3, CUL3 genes (pseudohypoaldosteronism type 2 or Gordon syndrome). Most of these conditions are treated by restricting dietary salt intake. However, some require special therapies including dexamethasone/hydrocortisone (CAH), spironolactone/eplerenone (AME), epithelial sodium channel inhibitors amiloride/triamterene (Liddle and Gordon syndrome), while in others spironolactone and MR antagonists may be contraindicated due to an abnormally structured MR (Geller syndrome). We here review the pathophysiology, diagnosis, and therapy of these rare conditions including the presentation of a patient with 11beta-hydroxylase deficiency.

Dimerization interfaces of v-erbA homodimers and heterodimers with retinoid X receptor alpha.

The oncoprotein v-ErbA, a member of the zinc finger transcription factor superfamily, is a mutated version of thyroid hormone receptor alpha1 that is virtually incapable of binding T3. v-ErbA and other members of this family can bind as homodimers and heterodimers with retinoid X receptors to specific DNA sequences arranged as direct, inverted, or everted repeats. At least two regions in the C-terminal domain, the I box (10 and 11 helices in v-ErbA and thyroid hormone receptors) and the 20-amino acid region are involved in dimerization. However, it has not been entirely understood how these receptors dimerize on differently oriented core motifs and whether the domain(s) responsible for homodimerization and heterodimerization are identical. Therefore, deletions of the entire 20-amino acid region, the 10 helix, the 11 helix, and point mutations within these regions of v-ErbA were made by site-directed mutagenesis. The mutant proteins were tested for their ability to form v-ErbA homodimers and heterodimers with retinoid X receptor alpha on differently oriented core motifs by electrophoretic mobility shift assay. Transient transfections were performed to determine the dominant negative activity of the v-ErbA mutants. The data indicate that different dimerization interfaces are used for v-ErbA homodimerization and heterodimerization with retinoid X receptor alpha, and different dimerization interfaces are used on differently oriented core motifs. The data are of general interest because the information improves our understanding of the role of these dimerization interfaces in the mechanism of action not only of v-ErbA but also of other members of the superfamily.

Characterization of the DNA-binding and dominant negative activity of v-erbA homodimers.

The oncoprotein v-erbA is a mutated form of thyroid hormone receptor alpha1 that is virtually incapable of binding T3. V-erbA is a dominant repressor of transcription induced by thyroid hormone receptors and retinoic acid receptors; however, the genetic targets of v-erbA that lead to oncogenesis are not known. Although v-erbA can bind as monomers and dimers to DNA containing the consensus sequence AGGTCA arranged as direct, inverted, or everted repeats, it is not known which sequence represents the optimal v-erbA-binding site. Determination of the DNA recognition properties of v-erbA would allow a better understanding of the repressor activity of this oncoprotein. The current studies, by using a random DNA selection strategy, have determined that the imperfect everted repeat 5'-TGACC(T/C)NT(A/G)AGGTCAC is the optimal v-erbA homodimer-binding site, where N represents any di- or trinucleotide. Functional studies show that everted repeats containing this sequence are substantially more potent v-erbA response elements than direct or inverted repeats, even though many classic T3 response elements are direct repeats. Thus, v-erbA represses only a subset of T3 response elements. In a similar fashion, v-erbA was found to repress a subset of vitamin D response elements. Of general interest, the data indicate that the two molecules of a transcription factor homodimer do not necessarily have identical DNA-binding specificities.

Retinoid X receptor alpha binds with the highest affinity to an imperfect direct repeat response element.

The regulation of gene expression by retinoids is mediated by two classes of receptors, retinoic acid receptors and retinoid X receptors (RXR). RXR can bind to specific target genes as homodimers, and these homodimers can activate gene expression in the presence of the ligand 9-cis-retinoic acid. A direct repeat of AGGTCA with a 1 base pair spacer (DR1) acts as a RXR homodimer response element in the presence of 9-cis-retinoic acid. However, it is not known if this represents the highest affinity binding site for the RXR homodimer. To investigate this question, we used a nonbiased strategy to isolate from a pool of random DNA those sequences that have the highest affinity for RXR alpha homodimers. The imperfect DR1 sequence 5'-GGGGTCAAAGGTCA displayed the highest in vitro binding affinity for RXR alpha homodimers. Transient transfection studies confirmed that this sequence is a more potent response element than is a perfect DR1 of either AGGTCA or GGGGTCA. The results also indicate that for RXR alpha homodimers, the receptor bound to the 5' half-site dislays different DNA binding specificity than that bound to the 3' half-site. Thus, DNA binding specificity is determined not only by the amino acid sequence of the protein but also by its protein-protein interactions and its position on the response element (5' vs. 3').

Comparison of the DNA binding specificity and function of v-ErbA and thyroid hormone receptor alpha 1.

The oncoprotein v-ErbA is a mutated version of thyroid hormone receptor alpha 1. Although the basis for the oncogenic action of v-ErbA is unknown, expression of this protein is known to inhibit thyroid hormone and retinoic acid induction of target genes. The DNA binding domain of v-ErbA differs from that of thyroid hormone receptor alpha 1 in two amino acids felt to be crucial for determining the specificity of DNA binding. However, the DNA binding properties of v-ErbA have not been examined independent of a comparison of binding to already known thyroid hormone response elements. In the current studies a non-biased strategy was used to select from a pool of random DNA those sequences that bind v-ErbA with high affinity. The highest affinity binding sequence was identified as the decamer 5'-T(A/G)AGGTCACG, which is closely related to the optimal thyroid hormone receptor alpha 1 binding sequence, TAAG-GTCA. Transfection studies demonstrate that among equal thyroid hormone responsive elements, those that contain the optimal v-ErbA consensus will be repressed by v-ErbA in preference to those that do not. These studies indicate that v-ErbA and thyroid hormone receptor alpha 1 regulate overlapping sets of response elements, and that all sequences that are highly responsive to thyroid hormone are not necessarily responsive to v-ErbA.

The interplay of half-site sequence and spacing on the activity of direct repeat thyroid hormone response elements.

Direct repeats of the hexamer AGGTCA can serve as response elements for vitamin D, thyroid hormone, or retinoic acid. The specificity of the response appears to reside in the spacing between the hexamers, with response elements for vitamin D restricted to direct repeats separated by a 3-base pair (bp) spacer, thyroid hormone a 4-bp spacer, and retinoic acid a 5-bp spacer (3-4-5 rule). Recently we have shown that the optimum thyroid hormone receptor binding site consists of an 8-bp sequence (TAAGGTCA), not a hexamer. Therefore we tested whether the 3-4-5 rule is valid for octamer sequence direct repeats. In transfection experiments octamer direct repeats with 3-, 4-, or 5-bp spacers conferred equivalently strong thyroid hormone responses, although a repeat with a 9-bp spacer was substantially weaker. For the 4- and 5-bp spacer constructs, the 5' half-site octamer had as strong an influence on thyroid hormone induction as did the 3' half-site octamer, although for the 3-bp spacer construct the 5' octamer was marginally less potent than the 3' octamer. Transfection and gel shift experiments did not suggest a simple correlation between the binding of thyroid hormone receptor-retinoid X receptor heterodimers and thyroid hormone induction from these response elements. We conclude that half-site sequence can override the effect of spacing in determining the hormone responsiveness of a direct repeat response element. In addition, the thyroid hormone response may not be due simply to the binding of thyroid hormone receptor-retinoid X receptor heterodimers to the DNA.

DNA binding specificity and function of retinoid X receptor alpha.

Retinoid X receptors are members of the erbA superfamily of ligand-inducible transcription factors. Similar to several other members of this gene family, retinoid X receptors are known to bind to the hexameric DNA sequence AGGTCA. After binding to a direct repeat of this hexamer with a one-base pair spacer, retinoid X receptor homodimers are able to activate transcription in the presence of the ligand 9-cis-retinoic acid. However, it is not known if AGGTCA represents the highest affinity binding site for retinoid X receptors. A combination of the electrophoretic mobility shift assay and polymerase chain reaction was used to isolate from a pool of random DNA those sequences that bind retinoid X receptors with highest affinity. This approach, combined with mutational analysis and DNA footprinting, led to the identification of the seven-base pair sequence GGGGTCA as the highest affinity retinoid X receptor binding site. A direct repeat of this sequence is substantially more active than a direct repeat of AGGTCA as a retinoid X response element.