Changes in Zn homeostasis during long term culture of primary endothelial cells and effects of Zn on endothelial cell senescence.

Endothelial cell senescence and Zn nutritional status influence cardiovascular disease. The influence of Zn appears dichotomous, hence it is imperative to understand the relationship with cellular senescence to improve knowledge about the molecular and cellular basis of the disease. Here we aimed to determine: 1) the impact of chronic exposure to a moderately high dose of Zn on senescence of endothelial cells; 2) the changes in Zn homeostasis during the lifespan of primary cultured endothelial cells; and 3) the susceptibility of proliferating and senescent endothelial cells to cell death after short term exposure to increasing doses of Zn and of the Zn chelator TPEN. Chronic exposure to Zn accelerated senescence and untreated cells at later passages, where doubling time had increased, displayed relocation of labile Zn and altered expression of genes involved in the response to Zn toxicity, including SLC30A1, SLC39A6, SLC30A5, SLC30A10 and metallothioneins, indicating that senescent cells have altered zinc homeostasis. Most Zn-dependent genes that were expressed differently between early and late passages were correlated with changes in the expression of anti-apoptotic genes. Short-term treatment with a high dose of Zn leads to cell death, but only in the population of cells at both earlier and later passages that had already entered senescence. In contrast, Zn depletion led to death of cells at earlier but not later passages, which suggests that there are sub-populations of senescent cells that are resistant to Zn depletion. This resistant senescent cell population may accumulate under conditions of Zn deficiency and contribute to vascular pathology.

Hyperkalemia in both surgically and medically treated patients with primary aldosteronism.

Hyperkalemia is an important complication of adrenalectomy for patients with primary aldosteronism (PA). The frequency of hyperkalemia after medication using mineralocorticoid receptor antagonists (MRAs) for PA is unclear. The aim of this study is to investigate the frequency and the risk factors of hyperkalemia after surgery and medication for PA. The data of 376 patients with PA registered in a multicentre-collaborative study in Japan, including surgically treated patients (group A; n=142) and medically treated patients with MRAs (group B; n=234) were studied. The prevalence of hyperkalemic patients (serum potassium >5.0 mEq l-1) after treatment was higher in group A than group B (9.9 vs 3.8%, P<0.01). At diagnosis, the hyperkalemic patients were older and had a poorer renal function than the non-hyperkalemic patients in both groups (P<0.05). The hyperkalemic patients had severer PA in group A and milder PA in group B. The independent risk factor by a logistic regression analysis was only age in both groups. After treatment, the percentages of patients withdrawing antihypertensive drugs and the normalization of aldosterone renin ratio were not different between hyperkalemic and non-hyperkalemic patients in group A. The type and dose of MRAs and the combination of other antihypertensive drugs were not different between hyperkalemic and non-hyperkalemic patients in group B. In conclusion, the potential occurrence of hyperkalemia should be considered after medical as well as surgical treatment for PA, especially in patients with older age (>60 years) and impaired renal function (estimated glomerular filtration rate <70 ml min-1 per 1.73 m2) at diagnosis.

Significance of adrenal computed tomography in predicting laterality and indicating adrenal vein sampling in primary aldosteronism.

Although laterality assessed by computed tomography (CT) in primary aldosteronism (PA) is not always concordant with that assessed by adrenal vein sampling (AVS), it is unclear whether all patients diagnosed with PA should undergo AVS for subtype classification. The aim of the current study was to investigate the accuracy of CT in subtype classification and to develop a prediction score for bilateral subtype in patients without adrenal tumour. As part of the WAVES-J study, 393 patients with PA were analysed. Subtyping using CT was concordant with that using AVS in 68% (269/393) of patients in the total sample, and in 38% (68/156) of patients with unilateral tumours, 56% (5/9) of patients with bilateral tumours and 89% (204/228) of patients without tumour. In patients without tumour, female gender, plasma aldosterone concentration (pg ml-1) to plasma renin activity ratio ⩽550 and serum potassium ⩾3.8 mEq l-1 were shown to be independent predictors for bilateral subtype. A prediction score based on these three variables was constructed with one point attributed to each variable. A score of three points had 29% sensitivity and 96% specificity in a receiver operating characteristic curve analysis. The results suggest that although CT is not sufficiently accurate for subtype classification in patients with adrenal tumours, it is sufficient to determine bilateral subtype in patients without tumour. Moreover, using our clinical prediction score in patients without tumour could be useful in determining the necessity of AVS for subtype classification.

The zinc finger protein ZNF658 regulates the transcription of genes involved in zinc homeostasis and affects ribosome biogenesis through the zinc transcriptional regulatory element.

We previously identified the ZTRE (zinc transcriptional regulatory element) in genes involved in zinc homeostasis and showed that it mediates transcriptional repression in response to zinc. We now report that ZNF658 acts at the ZTRE. ZNF658 was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry of a band excised after electrophoretic mobility shift assay using a ZTRE probe. The protein contains a KRAB domain and 21 zinc fingers. It has similarity with ZAP1 from Saccharomyces cerevisiae, which regulates the response to zinc restriction, including a conserved DNA binding region we show to be functional also in ZNF658. Small interfering RNA (siRNA) targeted to ZNF658 abrogated the zinc-induced, ZTRE-dependent reduction in SLC30A5 (ZnT5 gene), SLC30A10 (ZnT10 gene), and CBWD transcripts in human Caco-2 cells and the ability of zinc to repress reporter gene expression from corresponding promoter-reporter constructs. Microarray analysis of the effect of reducing ZNF658 expression by siRNA uncovered a large decrease in rRNA. We find that ZTREs are clustered within the 45S rRNA precursor. We also saw effects on expression of multiple ribosomal proteins. ZNF658 thus links zinc homeostasis with ribosome biogenesis, the most active transcriptional, and hence zinc-demanding, process in the cell. ZNF658 is thus a novel transcriptional regulator that plays a fundamental role in the orchestrated cellular response to zinc availability.

Chronic diclofenac (DCF) exposure alters both enzymatic and haematological profile of African catfish, Clarias gariepinus.

Pharmaceuticals are used extensively in human and veterinary medicine to eradicate or prevent diseases. The residues of these drugs have been detected in aquatic ecosystem; nevertheless, their toxicological effects on Clarias gariepinus have not been critically investigated. In this study, the toxic effects of diclofenac (DCF), a non-steroid anti-inflammatory drug, were studied in C. gariepinus by acute and chronic static renewable bioassay. The 96 h LC50 of DCF to C. gariepinus was 25.12 mg/L. Exposure to acute toxicity resulted in abnormal behavior and mortality of some fish. Compared with the control, chronic exposure of the fish to concentration (1.57, 3.14 and 6.28 mg/L) showed significantly higher mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV) and white blood cell (WBC), with significantly lower haemoglobin (Hb), haematocrit, red blood cell (RBC) and mean corpuscular haemoglobin (MCH) with increase in the concentration of the drug. Furthermore, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and glucose values significantly increased while protein levels were reduced (p < 0.05) in serum and gills throughout the 42-day exposure period. The study reports that DCF-induced enzymatic and haematological changes in the fish and recommends that these parameters be used as potential biomarkers for assessing residual pharmaceuticals available in aquatic ecosystem.

Identification of the human zinc transcriptional regulatory element (ZTRE): a palindromic protein-binding DNA sequence responsible for zinc-induced transcriptional repression.

Many genes with crucial roles in zinc homeostasis in mammals respond to fluctuating zinc supply through unknown mechanisms, and uncovering these mechanisms is essential to understanding the process at cellular and systemic levels. We detected zinc-dependent binding of a zinc-induced protein to a specific sequence, the zinc transcriptional regulatory element (ZTRE), in the SLC30A5 (zinc transporter ZnT5) promoter and showed that substitution of the ZTRE abrogated the repression of a reporter gene in response to zinc. We identified the ZTRE in other genes, including (through an unbiased search) the CBWD genes and (through targeted analysis) in multiple members of the SLC30 family, including SLC30A10, which is repressed by zinc. The function of the CBWD genes is currently unknown, but roles for homologs in metal homeostasis are being uncovered in bacteria. We demonstrated that CBWD genes are repressed by zinc and that substitution of the ZTRE in SLC30A10 and CBWD promoter-reporter constructs abrogates this response. Other metals did not affect expression of the transcriptional regulator, binding to the ZTRE or promoter-driven reporter gene expression. These findings provide the basis for elucidating how regulation of a network of genes through this novel mechanism contributes to zinc homeostasis and how the cell orchestrates this response.

Withdrawal of pioglitazone in patients with type 2 diabetes mellitus.

BACKGROUND AND OBJECTIVE: The remodelling of the adipose tissue by pioglitazone may be associated with the sustained therapeutic effects. We studied the effects of withdrawal of pioglitazone after 3-month treatment on glucose, lipid and high-molecular weight (HMW) adiponectin levels as well as liver function in patients with type 2 diabetes mellitus. METHODS: Forty-nine Japanese patients with type 2 diabetes mellitus were randomly assigned into the withdrawal group after 3-month treatment with pioglitazone (15 or 30 mg daily) and the non-withdrawal group. RESULTS AND DISCUSSION: Three-month treatment with pioglitazone improved glycaemic control, homeostasis model assessment for insulin resistance (HOMA), dyslipidaemia and liver function tests in association with a marked increase in serum HMW adiponectin level. Three months later after the withdrawal of pioglitazone, however, fasting plasma glucose and HOMA increased, whereas serum HMW adiponectin decreased to the pretreatment levels. Dyslipidaemia also returned to the pretreatment level. On the other hand, liver enzymes at 3 months after the withdrawal remained lower after a mild rebound. In addition, the bone formation marker, serum bone-specific alkaline phosphatase, was significantly reduced by pioglitazone treatment in post-menopausal women. CONCLUSIONS: The present study suggests that 3-month treatment with pioglitazone has no sustained beneficial effects except in liver function tests in patients with type 2 diabetes mellitus.

The homeodomain protein Pbx1 is involved in cAMP-dependent transcription of human CYP17.

Pbx1 is a homeodomain transcription factor involved in cAMP-dependent transcriptional regulation of the bovine CYP17 gene. In this study, we have investigated the involvement of Pbx1 in the transcriptional regulation of the human CYP17 gene. Although a sequence identical to previously determined Pbx-binding sites is not present in the promoter region of the human CYP17 gene, three putative Pbx-binding sites are identified by sequence similarity analysis. Coexpression of Pbx1 and a catalytic subunit of protein kinase A (PKA) greatly enhances reporter gene transcription via the 5'-flanking region of the human CYP17 gene. Upon gel shift analysis utilizing nuclear extracts from human adrenal H295R cells, one of the three putative Pbx1-binding sites, -250/-241 bp, shows the typical intense doublet observed with other Pbx-binding sites. 5'-Deletion analyses of the reporter construct containing this Pbx-binding site showed approximately sixfold induction by coexpression of Pbx1 and PKA compared to the basal transcription, suggesting that Pbx1 binds the -250/-241 bp sequence and participates in cAMP-dependent regulation of the human CYP17 gene.

cAMP-dependent transactivation involving the homeodomain protein Pbx1.

Pbx1 is a DNA-binding homeodomain protein originally discovered in the t(1;19) chromosomal translocation associated with pediatric pre-B acute lymphoblastic leukemia. Previously we reported a cAMP-regulatory sequence (CRS1) in the promoter region of the bovine CYP17 gene encoding steroid 17alpha-hydroxylase cytochrome P450 (P450c17) to be the first endogenous Pbx1 binding site and that overexpression of Pbx1 in mouse adrenal Y1 tumor cells enhances cAMP-dependent transcription mediated by this element. Here we report further characterization of Pbx1 binding site in CRS1 and role of Pbx1 in cAMP-dependent, CRS1-mediated transcription. By gel shift analysis utilizing nuclear extracts from Y1 cells, a high-affinity Pbx-binding sequence has been determined to be TTGAT(T/G)GA(T/C)A which represents the 5' portion of CRS1. An artificial Pbx-binding sequence (PRS), previously determined by random PCR analysis, is similar to the Pbx1-binding sequence in CRS1 and by both gel shift analysis and transfection studies shows characteristics very similar to CRS1. Upon overexpression, Pbx1 is found capable of enhancing CRS1-mediated transcription in both steroidogenic (Y1, JEG3) and nonsteroidogenic (HepG2 and S194) cells when coexpressed with the catalytic subunit of cAMP-dependent protein kinase A. Thus even though Pbx1 has been found to be involved only in cAMP-dependent transcription of a gene involved in steroidogenesis (CYP17), Pbx1 is capable of participating in cAMP-dependent transcription of target genes without complex formation with steroidogenic tissue-specific nuclear factors.

Protein kinase A-dependent transactivation by the E2A-Pbx1 fusion protein.

The chimeric gene E2A-PBX1 is formed by the t(1;19) chromosomal translocation exclusively associated with pediatric pre-B cell acute lymphoblastic leukemia (pre-B ALL). The resultant fusion protein from this chimeric gene contains the DNA-binding homeodomain of Pbx1. The first and only functional Pbx1 binding site has been localized in bovine CYP17 to a sequence (CRS1) that participates in cAMP-dependent transcription of this gene encoding the steroid hydroxylase, 17 alpha-hydroxylase cytochrome P450. Because Pbx1 is not expressed in pre-B cells, it may be possible that the E2a-Pbx1 fusion protein expressed in pre-B cells having this translocation will activate, in response to cAMP, transcription of genes not normally expressed in these cells leading to arrest of differentiation at the pre-B cell stage. We have now shown that reporter genes comprising CRS1 are activated transcriptionally by protein kinase A (PKA) in the pre-B cell line 697, which endogenously expresses the fusion protein, and that overexpression of E2A-Pbx1 in additional cell lines enhances transcription of reporter genes in a PKA-dependent fashion. Thus, it seems plausible that arrest in the pre-B stage leading to pre-B ALL includes cAMP-dependent activation of E2A-Pbx1.

A cAMP-regulatory sequence (CRS1) of CYP17 is a cellular target for the homeodomain protein Pbx1.

Cytochrome P450c17 encoded by CYP17, whose expression is regulated by peptide hormones via cAMP, is required for cortisol and sex hormone biosynthesis thereby playing a key role in biological processes including sexual differentiation. Utilizing the cAMP-regulatory sequence CRS1 of the bovine CYP17 gene as an affinity ligand, four CRS1-binding proteins have been purified from nuclear extracts of mouse adrenocortical Y1 cells and shown to enhance the in vitro transcription of a reporter gene promoted by CRS1. Microsequencing of these four proteins established two of them to be the homeodomain proteins Pbx1a and Pbx1b, originally discovered by their involvement in the t(1;19) chromosomal translocation in pre-B-cell acute lymphoblastic leukemias. Overexpression of Pbx1 in Y1 cells enhances cAMP-dependent transcription of the CRS1-dependent reporter gene. These results identify the CRS1 of bovine CYP17 as a cellular target for Pbx1 and suggest that one role of this homeodomain protein is in the regulation of steroidogenesis and subsequently sexual development.

Acromegaly with hyperprolactinemia developed after bilateral adrenalectomy in a patient with Cushing's syndrome due to adrenocortical nodular hyperplasia.

A 27-yr-old woman was referred for evaluation of acromegaly and hyperprolactinemia. She had undergone left adrenalectomy at 12 and right adrenalectomy at 17 for Cushing's syndrome due to adrenocortical nodular hyperplasia. At this time a pituitary tumor was found by brain computerized tomography, but plasma levels of growth hormone (GH), prolactin (PRL) and adrenocorticotropin (ACTH) were normal. When she was 23, symptoms and signs of acromegaly and subsequently galactorrhea-amenorrhea had developed. Plasma GH and PRL were increased and she was followed up by the administration of bromocriptine (2.5 mg-12.5 mg/day, p.o.). However the plasma GH level had been increasing gradually. On admission, plasma GH and PRL were high (19.5 micrograms/L, 61.0 micrograms/L, respectively) and increased in response to thyrotropin releasing hormone (TRH, 500 micrograms i.v.). An intrasella mass, which had been detected when she was 17, had become enlarged and was removed by Hardy's operation. Microscopically, the resected tumor was an eosinophilic adenoma. Immunohistochemical studies showed GH, PRL and ACTH positive cells localized in the tumor. Immunoultrastructural analysis of the tumor confirmed that GH, PRL and ACTH were present in secretory granules and Golgi apparatus in the tumor cells. The patient was a rare case of acromegaly with hyperprolactinemia developed after bilateral adrenalectomy of Cushing's syndrome due to adrenocortical nodular hyperplasia, all of which manifestations may be caused by a GH, PRL and ACTH secreting pituitary adenoma.

A single amino acid substitution (Met786----Val) in the steroid-binding domain of human androgen receptor leads to complete androgen insensitivity syndrome.

Androgen receptors (ARs) in two Japanese siblings with complete androgen insensitivity syndrome were characterized, and their molecular bases were investigated. Androgen binding was undetectable in cultured pubic skin fibroblasts from the patients by whole cell assay. Sequence analysis of exons B-H, which encode the DNA- and steroid-binding domains, of the AR gene from these patients using polymerase chain reaction revealed a single nucleotide substitution in exon F, resulting in an amino acid change at 786 from methionine (ATG) to valine (GTG) within the steroid-binding domain of AR. Reconstruction of this mutation by site-directed mutagenesis into human AR cDNA followed by expression in COS-1 cells led to production of the same amount and the same molecular mass of immunodetectable AR protein as those found with expression of the normal human AR cDNA. However, in contrast to wild-type AR expressed in COS-1 cells, the mutant AR showed markedly low affinity of androgen binding by whole cell assay. These results suggest that androgen resistance in these patients is due to the point mutation in the steroid-binding domain of the AR.

Disordered expression of adrenal steroidogenic P450 mRNAs in incidentally discovered nonfunctioning adrenal adenoma.

In order to elucidate the steroidogenesis of clinically nonfunctioning adrenocortical adenoma, we studied the aldosterone, cortisol (F) and dehydroepiandrosterone (DHEA) content and the expression of mRNA of cytochrome P450 for side chain cleavage (P450scc), 17 alpha-hydroxylase (P450c17). 21-hydroxylase (P450c21) and 11 beta-hydroxylase (P450c11) in four clinically nonfunctioning adrenocortical adenomas discovered incidentally in asymptomatic patients (Cases 1, 2, 3 and 4). The results were compared with those in normal adrenal glands. In the adenomas from cases 1 and 2, the abundance of steroidogenic P450s mRNA were similar to those in normal adrenal glands, except P450c11 mRNA expression in the adenoma from case 1 which was slightly higher than normal. The steroid content was normal level, except for higher F in the adenoma from case 1 and lower aldosterone in case 2 adenoma than normal. The adenoma from case 3 contained much less P450scc, P450c17 and P450c21 mRNA, while the amount of P450c11 mRNA was slightly greater than in normal adrenals. The adenoma showed normal aldosterone, high F and low DHEA content compared with normal adrenal glands. In the adenoma from case 4, the accumulation of all four P450 mRNAs decreased, whereas aldosterone, F and DHEA content in the adenoma was similar to that of normal adrenal glands. These data indicated that nonfunctioning adrenocortical adenoma showed similar or decreased expression of steroidogenic P450 mRNAs that the normal adrenal gland. This decreased expression of steroidogenic P450 mRNAs may be at least partly concerned with the absence of clinical symptoms in patients with nonfunctioning adenoma.

The modification and expression of 21-hydroxylase gene in normal human adrenal gland and adrenal cancer.

Genomic DNA and total RNA from three adrenal cancer tissues were analyzed by hybridization with human 21-hydroxylase gene. Southern blot analysis with restriction enzyme Eco RI revealed major fragments at 18, 13 and 9 kilobase (kb) in normal adrenal glands. In two of three adrenal cancers, however, the band at 18 kb was either absent or decreased and the 9 kb fragment showed an increase in its intensity. Normal liver, kidney and leucocytes has only 18 and 13 kb while lacking the 9 kb fragment. Using Taq I, Bam HI or Bgl II, we found no difference in restriction fragment patterns between adrenal cancer and normal tissues. Cleavage with Hpa II after digestion with Bgl II showed that significantly more DNA was digested into low-molecular weight fragments in adrenal cancer and the normal adrenal gland than those in other normal tissues. By Northern blot analysis, there was difference of signal intensity of hybridizing mRNA between the adrenal cancers and normal adrenal glands. These results suggest that the Eco RI site in the flanking region of the 21-hydroxylase gene may be modified in adrenal cancer tissue, and that inadequate 21-hydroxylase is present in some forms of adrenal cancers.

Markedly increased expression of cytochrome P-450 17 alpha-hydroxylase (P-450c17) mRNA in adrenocortical adenomas from patients with Cushing's syndrome.

We studied the contents of cortisol (F) and dehydroepiandrosterone (DHEA) and the expression of mRNA of cytochrome P-450 for side-chain cleavage (P-450scc), 17 alpha-hydroxylase (P-450c17), 21 alpha-hydroxylase (P-450c21) and 11 beta-hydroxylase (P-450c11) in adrenocortical adenomas from three patients with Cushing's syndrome. The F content was significantly higher in adrenocortical adenomas than in normal adrenal glands, while the DHEA level was similar to that in normal adrenal glands. The adrenal adenomas showed a markedly higher level of P-450c17 mRNA, and a slightly but not significantly increased level of P-450c21 mRNA, compared with normal adrenal glands. The expression of P-450scc and P-450c11 mRNA in the adenomas was similar to that in normal adrenal glands. These results suggest that the overproduction of cortisol in adrenocortical adenomas associated with Cushing's syndrome results from an increased expression of P-450c17 and P-450c21 mRNA.

Expression of cytochrome P-450 mRNAs in steroidogenesis of adrenocortical adenomas from patients with primary aldosteronism.

We studied the contents of aldosterone and cortisol (F) and the expression of mRNA of cytochrome P-450 for side-chain cleavage (P-450scc), 17 alpha-hydroxylase (P-450c17), 21-hydroxylase (P-450c21) and 11 beta-hydroxylase (P-450c11) in adrenocortical adenomas from three patients with primary aldosteronism. The aldosterone content was significantly higher in adrenocortical adenomas than in normal adrenal glands, while F content in adenomas was similar to the level in normal adrenal glands. The aldosterone-producing adenomas showed a markedly higher level of P-450c11 mRNA, a slightly but not significantly increased level of P-450c21 mRNA and a significantly decreased level of P-450c17 mRNA, compared with those in normal adrenal glands. The expression of P-450scc mRNA in adenomas was similar to the level in normal adrenal glands. These results suggested that the renin-independent overproduction of aldosterone in adrenocortical adenomas from the patients with primary aldosteronism results from increasing expression of the mRNA for P-450c11 and decreasing expression of the mRNA for P-450c17.

Decreased expression of cytochrome P450 17 alpha-hydroxylase mRNA in senescent bovine adrenal gland.

To investigate the mechanisms of age-related decline in synthesis of adrenal androgen, we studied the contents of cortisol (F) and dehydroepiandrosterone (DHEA) and the amounts of cytochrome P450 17 alpha-hydroxylase (P450c17) mRNA and cytochrome P450 11 beta-hydroxylase (P450c11) mRNA in young (1 year old) and senescent (10-12 years old) bovine adrenal glands. We also examined effects of ACTH (10(-7) M) on the secretion of F and DHEA and on the induction of P450c17 and P450c11 mRNA expression in cultured adrenal cells from young and aged cows. The content of DHEA in adrenal glands and the secretion of DHEA in response to ACTH in cultured adrenal cells from senescent cow were lower than those from young cow, while the content and ACTH-stimulated secretion of F in senescent adrenals were of a similar level to those in young adrenals. The adrenal gland from aged cow showed a significantly lower level of P450c17 mRNA compared with young bovine adrenal. However, P450c11 mRNA was expressed in senescent adrenal glands at the same level as that in young adrenals. The induction of P450c17 mRNA by ACTH (10(-7) M for 24 h) in cultured adrenal cells from aged cow also showed a decline compared with that from young cow, although there was no difference the ACTH-induced accumulation of P450c11 mRNA in cultured adrenal cells between young and senescent cow. These results suggested that the expression of P450c17 mRNA decreased in aged bovine adrenal, which may cause the age-associated decline in biosynthesis of adrenal androgen.