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Biomedical subjects

M Hewison

Publications and source records attributed to M Hewison.

At least 37 records · Page 2Linked to original sources
PubMed2001

Extrarenal expression of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase.

The mitochondrial enzyme 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (1 alpha-hydroxylase) plays an important role in calcium homeostasis by catalyzing synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D(3), in the kidney. However, enzyme activity assays indicate that 1 alpha-hydroxylase is also expressed in a variety of extrarenal tissues; recent cloning of cDNAs for 1 alpha-hydroxylase in different species suggests that a similar gene product is found at both renal and extrarenal sites. Using specific complementary ribonucleic acid probes and antisera to 1 alpha-hydroxylase, we have previously reported the distribution of messenger ribonucleic acid and protein for the enzyme along the mouse and human nephron. Here we describe further immunohistochemical and Western blot analyses that detail for the first time the extrarenal distribution of 1 alpha-hydroxylase in both normal and diseased tissues. Specific staining for 1 alpha-hydroxylase was detected in skin (basal keratinocytes, hair follicles), lymph nodes (granulomata), colon (epithelial cells and parasympathetic ganglia), pancreas (islets), adrenal medulla, brain (cerebellum and cerebral cortex), and placenta (decidual and trophoblastic cells). Further studies using psoriatic skin highlighted overexpression of 1 alpha-hydroxylase throughout the dysregulated stratum spinosum. Increased expression of skin 1alpha-hydroxylase was also associated with sarcoidosis. In lymph nodes and skin from these patients 1 alpha-hydroxylase expression was observed in cells positive for the surface antigen CD68 (macrophages). The data presented here confirm the presence of protein for 1 alpha-hydroxylase in several extrarenal tissues, such as skin, placenta, and lymph nodes. The function of this enzyme at novel extrarenal sites, such as adrenal medulla, brain, pancreas, and colon, remains to be determined. However, the discrete patterns of staining in these tissues emphasizes a possible role for 1 alpha-hydroxylase as an intracrine modulator of vitamin D function in peripheral tissues.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2001

Modulation of 11beta-hydroxysteroid dehydrogenase isozymes by proinflammatory cytokines in osteoblasts: an autocrine switch from glucocorticoid inactivation to activation.

Tissue damage by proinflammatory cytokines is attenuated at both systemic and cellular levels by counter anti-inflammatory factors such as corticosteroids. Target cell responses to corticosteroids are dependent on several factors including prereceptor regulation via local steroidogenic enzymes. In particular, two isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), by interconverting hormonally active cortisol (F) to inactive cortisone (E), regulate the peripheral action of corticosteroids 11beta-HSD1 by converting E to F and 11beta-HSD2 by inactivating F to E. In different in vitro and in vivo systems both 11beta-HSD isozymes have been shown to be expressed in osteoblasts (OBs). Using the MG-63 human osteosarcoma cell-line and primary cultures of human OBs, we have studied the regulation of osteoblastic 11beta-HSD isozyme expression and activity by cytokines and hormones with established roles in bone physiology. In MG-63 cells, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) potently inhibited 11beta-HSD2 activity (cortisol-cortisone conversion) and messenger RNA (mRNA) levels in a dose-dependent manner while stimulating reciprocal expression of 11beta-HSD1 mRNA and activity (cortisone-cortisol conversion). A similar rise in 11beta-HSD1 reductase activity also was observed in primary cultures of OBs treated with 10 ng/ml TNF-alpha. Pretreatment of MG-63 cells with 0.1 ng/ml IL-1beta resulted in increased cellular sensitivity to physiological glucocorticoids as shown by induction of serum and glucocorticoid-inducible kinase (SGK; relative increase with 50 nM F but no IL-1beta pretreatment 1.12 +/- 0.34; with pretreatment 2.63 +/- 0.50; p < 0.01). These results highlight a novel mechanism within bone cells whereby inflammatory cytokines cause an autocrine switch in intracellular corticosteroid metabolism by disabling glucocorticoid inactivation (11beta-HSD2) while inducing glucocorticoid activation (11beta-HSD1). Therefore, it can be postulated that some of the effects of proinflammatory cytokines within bone (e.g., periarticular erosions in inflammatory arthritis) are mediated by this mechanism.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2001

Expression and putative role of 11 beta-hydroxysteroid dehydrogenase isozymes within the human eye.

PURPOSE: The human eye is an important target tissue for steroid hormones, and glucocorticoids have been implicated in the pathogenesis of ocular disease, including glaucoma. In peripheral tissues, corticosteroid hormone action is regulated at a prereceptor level through the activity of the 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) isozymes: an oxo-reductase (11 beta-HSD1) that activates cortisol (F) from cortisone (E) and a dehydrogenase (11 beta-HSD2) that inactivates F to E. The purpose of this study was to analyze the expression and putative role of 11 beta-HSD within the human eye. METHODS: Immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) studies were performed on sections of human ocular tissues, surgical trabecular meshwork (TM) specimens and a ciliary nonpigmented epithelial (NPE) cell-line. Free F and E concentrations in aqueous humor were determined by gas chromatography-mass spectrometry (GC/MS). IOP was measured in eight male volunteers before and after oral ingestion of carbenoxolone (CBX), a known inhibitor of 11 beta-HSD. RESULTS: 11 beta-HSD1 was expressed in the basal cells of the corneal epithelium and the NPE. 11 beta-HSD2 was restricted to the corneal endothelium. RT-PCR revealed mRNA for only the glucocorticoid receptor (GR) in the TM specimens, whereas GR, mineralocorticoid receptor and 11 beta-HSD1 mRNAs were all present in the NPE cell line. The demonstration of free F in excess of E (F/E 14:1) in the aqueous humor suggested predominant 11 beta-HSD1 activity. Compared with baseline (14.7 +/- 1.06 mm Hg, mean +/- SD), the IOP decreased significantly on both the third and seventh days of CBX ingestion (12.48 +/- 1.11 mm Hg, P < 0.0001 and 11.78 +/- 1.50 mm Hg, P < 0.0001, respectively). CONCLUSIONS: These results suggest that the 11 beta-HSD1 isozyme may modulate steroid-regulated sodium transport across the NPE, thereby influencing IOP.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2000

Expression and functional consequences of 11beta-hydroxysteroid dehydrogenase activity in human bone.

Glucocorticoids have an essential role in skeletal development and function but are detrimental in excess. In several tissues, glucocorticoid action is dependent upon the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) isozymes, which interconvert active cortisol (F) and inactive cortisone (E). We previously demonstrated the expression of 11beta-HSD isozymes in human osteosarcoma cell lines, osteoblast cultures, and fetal bone. We now characterize 11beta-HSD expression in adult human bone using specific antihuman 11beta-HSD antibodies, riboprobes, and enzyme activity studies. In addition, the effect of 11beta-HSD on bone metabolism in vivo was assessed using the 11beta-HSD inhibitor carbenoxolone in eight normal male volunteers. In fresh normal human bone tissue, both 11beta-dehydrogenase (cortisol-to-cortisone conversion) and reductase (cortisone-to-cortisol conversion) activities were demonstrated. There was considerable interindividual variation in the dehydrogenase, but not reductase, activity. In bone homogenates, activity was NADP-dependent with a K(m) for F of 4.8 +/- 1.2 micromol/L, suggesting the presence of 11beta-HSD1. This was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Immunohistochemistry and in situ hybridization studies demonstrated 11beta-HSD1 isozyme expression in cells of the osteoblast lineage and in osteoclasts. The 11beta-HSD2 isozyme was expressed, but only in osteoblasts and at a low level. Ingestion of 300 mg of carbenoxolone by eight normal volunteers for 7 days resulted in a significant decrease in the bone resorption markers, pyridinoline (Pyr) and deoxypyridinoline (DPyr) (change in urinary Pyr/creatinine -1.55 +/- 0.55 [mean +/- SE], for DPyr/creatinine -0. 4 +/- 0.14 nmol/mmol; p < 0.05 for both), with no overall change in the bone formation markers C- and N-terminal propeptides of type I collagen (PICP and PINP). These data suggest that local tissue metabolism of glucocorticoids is likely to be important in determining the sensitivity of both osteoblasts and osteoclasts to glucocorticoids. In particular, variation in 11beta-HSD isozyme expression and activity may explain individual variation in susceptibility to glucocorticoid-induced osteoporosis.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2000

Oestrogen inactivation in the colon: analysis of the expression and regulation of 17beta-hydroxysteroid dehydrogenase isozymes in normal colon and colonic cancer.

Epidemiological data suggest that oestrogen contributes to the aetiology of colonic cancer. Furthermore, recent studies have suggested that local hormone metabolism may play a key role in determining colonic responsiveness to oestrogen. To further clarify this mechanism we have characterized the expression and regulation of isozymes of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in vitro and in situ. Immunohistochemistry was used to confirm expression of the type 2 and 4 isozymes of 17beta-HSD (17beta-HSD2 and 4) in normal colonic epithelial cells. Parallel studies suggested that both isozymes were abnormally expressed in colonic tumours and this was confirmed by Western blot analyses. Abnormal expression of 17beta-HSD2 and 4 proteins was also observed in Caco-2, HT-29 and SW620 colonic cancer cell lines, although the overall pattern of oestrogen metabolism in these cells was similar to that seen in primary colonic mucosal tissue. The predominant activity (conversion of oestradiol to oestrone) was highest in Caco-2>SW620>HT-29, which correlated inversely with the rate of proliferation of the cell lines. Regulatory studies using SW620 cells indicated that the most potent stimulator of oestradiol to oestrone inactivation was the antiproliferative agent 1,25-dihydroxyvitamin D3 (1,25D3), whilst oestradiol itself inhibited 17beta-HSD activity. Both oestradiol and 1,25D3 decreased mRNA for 17beta-HSD2 and 4. Data indicate that the high capacity for inactivation of oestrogens in the colon is associated with the presence of 17beta-HSD2 and 4 in epithelial cells. Abnormal expression of both isozymes in colonic cancer cells and the stimulation of oestrogen inactivation by the antiproliferative agent 1,25D3 highlights a possible role for 17beta-HSD isozymes as modulators of colonic cell proliferation.

17-Hydroxysteroid Dehydrogenases↗
PubMed2000

Expression of 25-hydroxyvitamin D3-1alpha-hydroxylase along the nephron: new insights into renal vitamin D metabolism.

Renal synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is a pivotal step in calcium and phosphate homeostasis. Production of 1,25(OH)2D3 is catalyzed by the mitchondrial cytochrome P450, 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-HYD). As a consequence of the tight regulation of vitamin D metabolism during normal physiology, studies of the expression and regulation of 1alpha-HYD have proved remarkably difficult. However, the recent cloning of the gene for 1alpha-HYD has enabled a more comprehensive analysis of the tissue distribution of 1alpha-HYD, as well as the mechanisms involved in controlling 1,25(OH)2D3 production. In particular, an understanding of site-specific expression and regulation of 1alpha-HYD along the nephron might help to elucidate a more versatile role for 1,25(OH)2D3 in renal physiology.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2000

1alpha-Hydroxylase and the action of vitamin D.

The active form of vitamin D, 1,25-dihydroxvitamin D(3) (1, 25(OH)(2)D(3)), is a pleiotropic hormone whose actions include the regulation of calcium homeostasis, control of bone cell differentiation and modification of immune responses. Synthesis of 1, 25(OH)(2)D(3) from the major circulating metabolite, 25-hydroxyvitamin D(3) (25(OH)D(3)), is catalysed by a mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase). Although 1alpha-OHase is expressed predominantly in the kidney, extra-renal production of 1,25(OH)(2)D(3) has also been demonstrated in tissues such as lymph nodes and skin. The tight regulation of 1alpha-OHase which occurs in both renal and peripheral tissues has made studies of the expression and regulation of this enzyme remarkably difficult. However, the recent cloning of mouse, rat and human cDNAs for 1alpha-OHase (CYP1alpha/Cyp1alpha) has enabled a more thorough characterization of this enzyme. In particular, analysis of the CYP1alpha gene has identified mutations causing the inherited disorder vitamin D-dependent rickets type 1, also known as pseudo-vitamin D deficiency rickets. Studies from our own group have focused on the distribution of 1alpha-OHase in both renal and extra-renal tissues. Data indicate that the enzyme is expressed throughout the nephron, suggesting discrete endocrine and paracrine/autocrine functions. Further immunohistochemical analyses have shown that the enzyme is widely distributed in extra-renal tissues, and this appears to be due to the same gene product as the kidney. Collectively, these observations have raised important new questions concerning the role of 1alpha-OHase in vitamin D signalling at a local level. The relationship between expression of protein for 1alpha-OHase and enzyme activity has yet to be fully characterized and may be dependent on membrane proteins such as megalin. Similarly, elucidation of the mechanisms involved in differential regulation of renal and extra-renal 1,25(OH)(2)D(3) production will be essential to our understanding of the tissue-specific functions of 1alpha-OHase. These and other issues are discussed in the current review.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1999

Estradiol formation by human osteoblasts via multiple pathways: relation with osteoblast function.

The importance of estrogens in bone metabolism is illustrated by the accelerated bone loss and increase in osteoporotic fractures associated with postmenopausal estrogen deficiency. In this study, the expression and activity of the enzymes involved in estrogen metabolism in human osteoblastic cells were investigated in relation to differentiation of these cells. PCR reactions using mRNA from an in vitro differentiating human cell line (SV-HFO) were performed to assess mRNA expression of the enzymes aromatase, different subtypes of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), and steroid sulfatase. Aromatase, sulfatase, and 17beta-HSD type 2 and 4 were found to be expressed throughout differentiation. Expression of 17beta-HSD type 3, however, was relatively weak, except for early time points in differentiation. Type 1 17beta-HSD expression was not detected. Aromatase activity decreased during differentiation, as was demonstrated by the conversion of androstenedione (A) and testosterone (T) into estrone (E(1)) and estradiol (E(2)), respectively. The 17beta-HSD isozymes catalysing a reductive reaction convert androstenedione and estrone into testosterone and estradiol, respectively. Their activity declined with differentiation. Analysis of 17beta-HSD activity indicated both oxidative (E(2) to E(1); T to A) and reductive (E(1) to E(2); A to T) metabolism at all stages of osteoblast differentiation. Both activities declined as cells moved toward a differentiating mineralizing phenotype. However, the oxidative reaction was increasingly in favor of the reductive reaction at all times during differentiation. Sulfatase activity, as demonstrated by the conversion of estrone-sulfate into estrone, was constant during differentiation. In conclusion, we have demonstrated that all enzymes necessary for estrogen metabolism are expressed and biologically active in differentiating human osteoblasts. The activity of aromatase and 17beta-HSD was found to be dependent on the stage of cell differentiation. In addition, human osteoblasts effectively convert estradiol into estrone. The efficacy of osteoblasts to synthesize estradiol may determine the ultimate change in rate of bone turnover after menopause, as well as the development of osteoporosis. Moreover, the enzymes involved in the metabolism of estradiol may form a target for intervention.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

The renal function of 25-hydroxyvitamin D3-1alpha-hydroxylase.

The active, hormonal form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has numerous pleiotropic actions including the regulation of calcium homeostasis, control of bone cell differentiation and modification of immune responses. Synthesis of 1,25(OH)2D3 from the major circulating metabolite, 25-hydroxyvitamin D3 (25(OH)D3), is catalysed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-HYD). Although 1alpha-HYD activity has been demonstrated at several ectopic sites, circulating levels of 1,25(OH)2D3 appear to reflect the expression of this enzyme in the kidney. The tight regulation of 1alpha-HYD in both renal and ectopic tissues has made studies of the expression and regulation of this enzyme remarkably difficult. However, the recent cloning of mouse, rat and human cDNAs for 1alpha-HYD has stimulated renewed interest in the molecular endocrinology of 1,25(OH)2D3 production. Analysis of the 1alpha-HYD sequence has revealed homology with the liver enzyme vitamin D-25-hydroxylase, and the ubiquitously expressed vitamin D-24-hydroxylase. Furthermore, mutations causing the inherited disorder vitamin D-dependent rickets type 1, also known as pseudo-vitamin D deficiency rickets have been described for the 1alpha-HYD gene and these have been mapped to chromosome 12q14 by linkage analysis. The availability of sequence information for the 1alpha-HYD gene has also facilitated the development of new molecular tools which will help to clarify key functions of the enzyme. Specific issues such as tissue distribution and regulatory pathways are discussed in this review, with particular emphasis on the role of 1alpha-HYD in renal calcium/phosphate homeostasis.

Animals↗
PubMed1999

Estrone potentiates myeloid cell differentiation: a role for 17 beta-hydroxysteroid dehydrogenase in modulating hemopoiesis.

Hormones such as 1 alpha, 25-dihydroxy vitamin D3 (D3), all-trans retinoic acid, and 9-cis retinoic acid stimulate differentiation of myeloid progenitor cells via their interaction with specific hormone receptors. However, the sensitivity of cells to these agents is not merely governed by the expression of their receptors and the availability of ligand to bind them. Recent studies from our group suggested that the actions of D3 and retinoids on myelopoiesis also are influenced by endogenous mechanisms involving other steroid hormones. In this study we examined the influence of local estrogen metabolism on the differentiation of HL60 cells and normal primitive myeloid progenitor cells. Quantitative thin-layer chromatography (TLC) analyses showed that HL60 and normal cells are able to generate estrone (E1) from estradiol (E2). Neither cell population generated significant amounts of E2 from E1. Reverse transcriptase polymerase chain reaction and Northern analyses confirmed that normal and leukemic myeloid progenitor cells expressed mRNA for the type I and IV isoforms of 17 beta-hydroxysteroid dehydrogenase. Conversion of E2 to E1 was upregulated within 24 hours when HL60 cells were treated with either all-trans retinoic acid or D3 at doses that induce their differentiation toward neutrophils or monocytes, respectively. Similarly, D3-induced monocyte differentiation of normal myeloid progenitor cells was associated with increased capacity to generate E1 from E2. When HL60 cells or normal myeloid progenitor cells were exposed to exogenous E1 they became more sensitive to the differentiation-inducing effects of D3. Data presented provide further evidence for the local modulation of myelopoiesis by intracrine mechanisms. In particular, our findings suggest that local metabolism of steroids by normal as well as leukemic myeloid cells influences their responsiveness to D3 and retinoids.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Constitutive expression of 25-hydroxyvitamin D3-1alpha-hydroxylase in a transformed human proximal tubule cell line: evidence for direct regulation of vitamin D metabolism by calcium.

Circulating levels of the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) are dependent on activity of the renal mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-hydroxylase). Production of 1,25-(OH)2D3 occurs predominantly in the renal proximal tubule, with 1alpha-hydroxylase activity being impaired in renal insufficiency and renal disease. The expression and activity of 1alpha-hydroxylase are tightly regulated in response to serum levels of PTH, calcium, phosphate, and 1,25-(OH)2D3 itself. As a consequence of this, the characterization of 1alpha-hydroxylase in human renal tissue has proved difficult. In this study we have characterized constitutive 1alpha-hydroxylase expression in a simian virus 40-transformed human proximal tubule cell line, HKC-8. Initial analyses of [3H]25-hydroxyvitamin D3 (25OHD3) metabolism in these cells using straight and reverse phase HPLC revealed product peaks that coincided with authentic 1,25-(OH)2D3 as well as 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3). Enzyme kinetic studies indicated that the Km for synthesis of 1,25-(OH)2D3 in HKC-8 cells was 120 nmol/liter 25OHD3, with a maximum velocity of 21 pmol/h/mg protein. This activity was inhibited by treatment with ketoconazole, but not diphenyl phenylenediamine. RT-PCR analysis of RNA from HKC-8 cells revealed a transcript similar in size to that observed in keratinocytes and primary cultures of human proximal tubule cells, and protein was detected by Western blot analysis. Synthesis of 1,25-(OH)2D3 was up regulated by treatment with forskolin (10 micromol/liter, 24 h) and was down-regulated by 1,25-(OH)2D3 (10 nmol/liter, 24 h). 1Alpha-hydroxylase activity in HKC-8 cells was also sensitive to the concentration of calcium. Cells grown in low calcium (0.5 mmol/liter) showed a 4.8-fold induction of 1alpha-hydroxylase, whereas treatment with medium containing high levels of calcium (2 mmol/liter) significantly inhibited 1,25-(OH)2D3 production. These data suggest that direct effects of calcium on proximal tubule cells may be an important feature of the regulation of renal 1,25-(OH)2D3 production.

24,25-Dihydroxyvitamin D 3↗
PubMed1999

Differentiation of adipose stromal cells: the roles of glucocorticoids and 11beta-hydroxysteroid dehydrogenase.

Glucocorticoids play an important role in determining adipose tissue distribution and function, with glucocorticoid excess states such as Cushing's syndrome resulting in central obesity. We have investigated the functional significance of local generation of cortisol within adipose tissue from inactive cortisone through the activity of the NADP(H)-dependent enzyme, 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1). In primary cultures of paired omental (om) and sc human adipose stromal cells (ASC; n = 34), 11betaHSD1 oxo-reductase activity was significantly higher in om ASC (median, 40.2 pmol/mg protein x h; 95% confidence interval, 1.8-105) compared with sc ASC (median, 11.4; 95% confidence interval, 0-48.1; P<0.001) despite similar endogenous NADPH/NADP concentrations. Both cortisol and insulin increased the differentiation of ASC to adipocytes (as assessed by glycerol-3-phosphate dehydrogenase expression), but only cortisol increased 11betaHSD1 activity and messenger RNA levels in a dose-dependent fashion. Cortisone (500 nM) was as effective as 500 nM cortisol in inducing ASC differentiation, but this stimulatory effect was inhibited by coincubation with the 11betaHSD1 inhibitor, glycyrrhetinic acid. The higher local conversion of cortisone to active cortisol through expression of 11betaHSD1 in om compared with sc ASC may explain the specific action of glucocorticoids on different adipose tissue depots. 11betaHSD1 expression in om ASC is regulated at a transcriptional level and is increased by glucocorticoids, but is not entirely dependent upon ASC differentiation. Inhibition of 11betaHSD1 within om ASC inhibits cortisone-induced ASC differentiation. These findings indicate that local metabolism of glucocorticoid may control differentiation of adipose tissue in a site-specific fashion. Specific inhibitors of 11betaHSD1 may offer a novel approach for the treatment of patients with central obesity.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1999

Loss of estrogen inactivation in colonic cancer.

Age and sex differences in the incidence of colonic cancer, together with epidemiological data on patients taking hormone replacement therapy, suggest the involvement of estrogens. Analogous to the role of aromatase in breast cancer, we postulated that steroid metabolism within the colon itself may be a crucial mechanism in regulating tissue exposure to estrogens. We have characterized expression of aromatase (responsible for converting C19 androgens to C18 estrogens) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) [responsible for interconversion of active estradiol (E2) to less potent estrone (E1)] in normal and neoplastic human colon from 24 patients undergoing tumor resection. Aromatase activity was similar in homogenates from normal mucosa, tissue adjacent to tumors, and the tumors themselves. Analysis of 17beta-HSD activity indicated that the predominant activity was oxidative (E2 to E1), and this conversion was significantly lower in colonic tumors [444 (90-1735); median (95% confidence interval) pmol/mg protein x h], compared with normal mucosa [1709 (415-13828), P < 0.001]. Northern blot analyses indicated expression of messenger RNAs (mRNAs) for the type 2 and 4 isozymes of 17beta-HSD in normal colon; messenger RNA for 17beta-HSD 4 was significantly lower in tumor tissue [0.75 +/- 0.22 (mean +/- SD) arbitrary U vs. 0.43 +/- 0.17, P < 0.01]. Studies in vitro, using three colonic cancer cell lines, indicated that there was an inverse correlation between 17beta-HSD oxidative activity and the rate of cell proliferation. In addition, E1, but not E2, was shown to significantly decrease proliferation when added exogenously to the colonic epithelial cell line, SW620 cells. Colonic mucosa can regulate estrogen hormone action in an intracrine fashion. The loss of estrogen inactivation may be an important mechanism in the pathogenesis of colonic cancer.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Characterization of 11beta-hydroxysteroid dehydrogenase activity and corticosteroid receptor expression in human osteosarcoma cell lines.

Studies in vitro and in vivo have shown that corticosteroids play an important role in bone physiology and pathophysiology. It is now established that corticosteroid hormone action is regulated, in part, at the pre-receptor level through the expression of isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), which are responsible for the interconversion of hormonally active cortisol to cortisone. In this report we demonstrate 11beta-HSD activity in human osteoblast (OB) cells. Osteosarcoma-derived OB cell lines TE-85, MG-63 and SaOS-2 and fibrosarcoma Hs913T cells express the type 2 isoform of 11beta-HSD, as determined by reverse transcription polymerase chain reaction (RT-PCR) and specific enzyme assays. Enzyme activity was shown to be strictly NAD dependent with a Km of approximately 71 nM; 11beta-HSD type 1 mRNA expression and enzyme activity were not detected. All four cell lines expressed mRNA for the glucocorticoid receptor (GR) and mineralocorticoid receptor, but specific binding was only detectable with radiolabelled dexamethasone (Kd=10 nM) and not aldosterone. MG-63 cells had two to three times more GR than the other OB cells, which correlated with the higher levels of 11beta-HSD 2 activity in these cells. In contrast to the osteosarcoma cell studies, RT-PCR analysis of primary cultures of human OB cells revealed the presence of mRNA for 11beta-HSD 1 as well as 11beta-HSD 2. However, enzyme activity in these cells remained predominantly oxidative, i.e. inactivation of cortisol to cortisone (147 pmol/h per mg protein at 500 nM cortisol) was greater than cortisone to cortisol (10.3 pmol/h per mg protein at 250 nM cortisone). Data from normal human OB and osteosarcoma cells demonstrate the presence of an endogenous mechanism for inactivation of glucocorticoids in OB cells. We postulate that expression of the type 1 and type 2 isoforms of 11beta-HSD in human bone plays an important role in normal bone homeostasis, and may be implicated in the pathogenesis of steroid-induced osteoporosis.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1999

Expression of 25-hydroxyvitamin D3-1alpha-hydroxylase in the human kidney.

The secosteroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a vital role in calcium metabolism, tissue differentiation, and normal bone growth. Biosynthesis of 1,25(OH)2D3 is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase). Although activity of this enzyme has been described in several tissues, the kidneys are recognized to be the principal site of 1,25(OH)2D3 production. To date, enzyme activity studies using vitamin D-deficient animals have suggested that 1alpha-hydroxylase is expressed exclusively in proximal convoluted tubules. With the recent cloning of 1alpha-hydroxylase, specific cRNA probes and in-house polyclonal antiserum have been used to determine the distribution of 1alpha-hydroxylase along the human nephron. Immunohistochemistry and in situ hybridization studies indicated strong expression of 1alpha-hydroxylase protein and mRNA in the distal convoluted tubule, the cortical and medullary part of the collecting ducts, and the papillary epithelia. Lower expression was observed along the thick ascending limb of the loop of Henle and Bowman's capsule. Weaker and more variable expression of 1alpha-hydroxylase protein and mRNA was seen in proximal convoluted tubules, and no expression was observed in glomeruli or vascular structures. These data show for the first time the distribution of alpha1-hydroxylase expression in normal human kidney. In contrast to earlier enzyme activity studies conducted in vitamin D-deficient animals, our data indicate that the distal nephron is the predominant site of 1alpha-hydroxylase expression under conditions of vitamin D sufficiency.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1999

Clofibric acid: a potential therapeutic agent in AML and MDS.

Differentiation therapy using retinoic acids (RAs) or 1alpha25-dihydroxyvitamin D3 (D3) is an attractive alternative to chemotherapy in acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS). However, with the exception of RA therapy for acute promyelocytic leukaemia (APL), RAs and D3 are not potent enough at doses that can be tolerated by patients. We demonstrate that clofibric acid (CA) enhances the response of HL60 cells to all-trans RA and D3. Our findings and those of others in the field lead us to suggest that combination therapy using all-trans RA and CA should be considered as potential therapy for AML and MDS.

Aged↗