Non-competitive cyclic peptides for targeting enzyme-substrate complexes.

Affinity reagents are of central importance for selectively identifying proteins and investigating their interactions. We report on the development and use of cyclic peptides, identified by mRNA display-based RaPID methodology, that are selective for, and tight binders of, the human hypoxia inducible factor prolyl hydroxylases (PHDs) - enzymes crucial in hypoxia sensing. Biophysical analyses reveal the cyclic peptides to bind in a distinct site, away from the enzyme active site pocket, enabling conservation of substrate binding and catalysis. A biotinylated cyclic peptide captures not only the PHDs, but also their primary substrate hypoxia inducible factor HIF1-α. Our work highlights the potential for tight, non-active site binding cyclic peptides to act as promising affinity reagents for studying protein-protein interactions.

Pleomorphic liposarcoma of bone: a rare primary malignant bone tumour.

BACKGROUND: Liposarcoma is an extremely rare primary bone sarcoma. CASE PRESENTATION: We report a case of primary pleomorphic liposarcoma that arose in an 18 year old male in the metaphysis of the left tibia. Plain radiographs showed a partly sclerotic lesion and MR imaging a heterogeneous tumour predominantly isointense on T1- and high-signal on T2-weighted sequences with focal areas of increased T1 signal that suppressed with fat saturation. PET/CT showed marked FDG uptake (SUV = 17.1) in the primary tumour as well as a metastasis in the right distal femur and multiple small pulmonary metastases. Histologically, the tumour was a pleomorphic liposarcoma containing large tumour cells with vacuolated cytoplasm and hyperchromatic pleomorphic nuclei as well as numerous lipoblasts and scattered brown fat-like cells. Tumour cells strongly expressed FABP4/aP2, a marker of adipocyte differentiation, and UCP1, a marker of brown fat, but not S100. The case was treated with neoadjuvant MAP chemotherapy, resulting in extensive (> 95%) necrosis in the primary tumour and almost complete resolution of the femoral and pulmonary metastases. CONCLUSIONS: Pleomorphic liposarcoma can present as a sclerotic primary malignant bone tumour; markers of adipose differentiation are useful in histological diagnosis and neoadjuvant MAP chemotherapy results in significant tumor necrosis.

Utility of VS38c in the diagnostic and prognostic assessment of osteosarcoma and other bone tumours/tumour-like lesions.

BACKGROUND: VS38c is a monoclonal antibody that recognises a rough endoplasmic reticulum (rER) intracellular antigen termed cytoskeleton-linking membrane protein 63. rER is typically found in viable tumour cells and is abundant in osteosarcoma cells. The aim of this study was to determine the diagnostic and prognostic utility of VS38c in the histological assessment of osteosarcoma and other bone tumours/tumour-like leisons. METHODS: Immunohistochemical staining with VS38c was carried out on formalin-fixed specimens of osteosarcoma (pre/post-chemotherapy) and a wide range of benign and malignant bone lesions. In addition, VS38c staining of cultures of MG63 and Sa0S2 osteosarcoma cell cultures. (±cisplatin and actinomycin D-treatment) was analysed. RESULTS: VS38c strongly stained tumour cells in all low-grade and high-grade osteosarcomas and in undifferentiated sarcomas and high-grade chondrosarcomas. There was little or no VS38c staining of low-grade chondrosarcomas or chordomas and variable staining of Ewing sarcomas. Osteoblasts in benign bone-forming tumours and mononuclear stromal cells in chondroblastomas, giant cell tumours and non-ossifying fibromas strongly stained for VS38c. VS38c staining was absent in cisplatin and actinomycin D treated Sa0S2 and MG63 cells. In specimens of osteosarcoma post-neoadjuvant therapy, VS38c staining was absent in most morphologically necrotic areas of tumor although some cells with pyknotic nuclei stained for VS38c in these areas. Most tumour cells exhibiting atypical nuclear forms were not stained by VS38c. CONCLUSIONS: Our findings show that VS38c is a sensitive but not specific diagnostic marker of osteosarcoma. Staining with VS38c identifies viable osteosarcoma cells, a feature which may be useful in the assessment of percentage tumour necrosis post-neoadjuvant chemotherapy.

Dendritic and mast cell involvement in the inflammatory response to primary malignant bone tumours.

BACKGROUND: A chronic inflammatory cell infiltrate is commonly seen in response to primary malignant tumours of bone. This is known to contain tumour-associated macrophages (TAMs) and lymphocytes; dendritic cells (DCs) and mast cells (MCs) have also been identified but whether these and other inflammatory cells are seen commonly in specific types of bone sarcoma is uncertain. METHODS: In this study we determined the nature of the inflammatory cell infiltrate in 56 primary bone sarcomas. Immunohistochemistry using monoclonal antibodies was employed to assess semiquantitatively CD45+ leukocyte infiltration and the extent of the DC, MC, TAM and T and B lymphocyte infiltrate. RESULTS: The extent of the inflammatory infiltrate in individual sarcomas was very variable. A moderate or heavy leukocyte infiltrate was more commonly seen in conventional high-grade osteosarcoma, undifferentiated pleomorphic sarcoma and giant cell tumour of bone (GCTB) than in Ewing sarcoma, chordoma and chondrosarcoma. CD14+/CD68+ TAMs and CD3+ T lymphocytes were the major components of the inflammatory cell response but (DC-SIGN/CD11c+) DCs were also commonly noted when there was a significant TAM and T lymphocyte infiltrate. MCs were identified mainly at the periphery of sarcomas, including the osteolytic tumour-bone interface. DISCUSSION: Our findings indicate that, although variable, some malignant bone tumours (e.g. osteosarcoma, GCTB) are more commonly associated with a pronounced inflammatory cell infiltrate than others (e.g. chondrosarcoma. Ewing sarcoma); the infiltrate is composed mainly of TAMs but includes a significant DC, T lymphocyte and MC infiltrate. CONCLUSION: Tumours that contain a heavy inflammatory cell response, which includes DCs, TAMs and T lymphocytes, may be more amenable to immunomodulatory therapy. MCs are present mainly at the tumour edge and are likely to contribute to osteolysis and tumour invasion.

Dentine matrix protein 1 (DMP-1) is a marker of bone formation and mineralisation in soft tissue tumours.

Dentine matrix protein 1 (DMP-1) is a non-collagenous matrix protein found in dentine and bone. It is highly expressed by osteocytes and has been identified in primary benign and malignant osteogenic bone tumours. Bone formation and matrix mineralisation are seen in a variety of benign and malignant soft tissue tumours and tumour-like lesions, and in this study, we analysed immunohistochemically the DMP-1 expression in a wide range of soft tissue lesions (n = 254) in order to assess whether DMP-1 expression is useful in the histological diagnosis of soft tissue tumours. Matrix staining of DMP-1 was seen in all cases of myositis ossificans, fibro-osseous tumour of the digits, extraskeletal soft tissue osteosarcoma and in most cases of ossifying fibromyxoid tumour. DMP-1 was also noted in dense collagenous connective tissue of mineralising soft tissue lesions such as tumoural calcinosis, dermatomyositis and calcific tendinitis. DMP-1 was expressed in areas of focal ossification and calcification in synovial sarcoma and other soft tissue tumours. With few exceptions, DMP-1 was not expressed in other benign and malignant soft tissue tumours. Our findings indicate that DMP-1 is a matrix marker of bone formation and mineralisation in soft tissue tumours. DMP-1 expression should be particularly useful in distinguishing extraskeletal osteosarcoma and ossifying fibromyxoid tumour from other sarcomas and in identifying areas of osteoid/bone formation and mineralisation in soft tissue tumours.

The role of calcium and nicotinic acid adenine dinucleotide phosphate (NAADP) in human osteoclast formation and resorption.

Osteoclasts are specialised bone resorbing cells which form by fusion of circulating mononuclear phagocyte precursors. Bone resorption results in the release of large amounts of calcium into the extracellular fluid (ECF), but it is not certain whether changes in extracellular calcium concentration [Ca(2+)]e influence osteoclast formation and resorption. In this study, we sought to determine the effect of [Ca(2+)]e and NAADP, a potent calcium mobilising messenger that induces calcium uptake, on human osteoclast formation and resorption. CD14+ human monocytes were cultured with M-CSF and RANKL in the presence of different concentrations of calcium and NAADP and the effect on osteoclast formation and resorption evaluated. We found that the number of TRAP+ multinucleated cells and the extent of lacunar resorption were reduced when there was an increase in extracellular calcium and NAADP. This was associated with a decrease in RANK mRNA expression by CD14+ cells. At high concentrations (20 mM) of [Ca(2+)]e mature osteoclast resorption activity remained unaltered relative to control cultures. Our findings indicate that osteoclast formation is inhibited by a rise in [Ca(2+)]e and that RANK expression by mononuclear phagocyte osteoclast precursors is also [Ca(2+)]e dependent. Changes in NAADP also influence osteoclast formation, suggesting a role for this molecule in calcium handling. Osteoclasts remained capable of lacunar resorption, even at high ECF [Ca(2+)]e, in keeping with their role in physiological and pathological bone resorption.

Pleiotropic effect of somatic mutations in the E2F subunit DP-1 gene in human cancer.

Transcription factor E2F-1 and its interaction with pRb provide a key point of control in cell proliferation. E2F-1 participates in both cell cycle progression and apoptosis, and in cells exists with a DP dimerization partner protein, the most prominent being DP-1. By mining the tumor tissue and cancer cell line encyclopedia genomic databases, we identified the first somatic mutations in the DP-1 gene and describe 53 distinct mutation events here. The mutations are mostly missense mutations, but also include nonsense and frame-shift mutations that result in truncated DP-1 derivatives. Mutation occurs throughout the DP-1 gene but generally leaves protein dimerization activity intact. This allows the mutant derivatives to affect the properties of the E2F-1/DP-1 heterodimer through a transdominant mechanism, which changes the DNA binding, transcriptional activation and pRb-binding properties of the heterodimer. In particular, many DP-1 mutants were found to impair E2F-1-dependent apoptosis. Our results establish that somatic mutations in DP-1 uncouple normal control of the E2F pathway, and thus define a new mechanism that could contribute to aberrant proliferation in tumor cells.

Dentine matrix protein 1 (DMP-1) is a marker of bone-forming tumours.

Dentin matrix protein 1 (DMP-1) is highly expressed by osteocytes and is a non-collagenous matrix protein found in dentin and bone. In this study, we determined the expression of DMP-1 in mature and immature human bone and examined whether DMP-1 is useful in distinguishing osteoid/bone-forming tumours from other primary and secondary bone tumours. DMP-1 expression was immunohistochemically determined in paraffin sections of a wide range of benign and malignant primary bone tumours and tumour-like lesions (n = 353). DMP-1 mRNA expression was also examined in osteosarcoma and fibrosarcoma cell lines as well as bone tumour specimens (n = 5) using real-time PCR. In lamellar and woven bone, DMP-1 was expressed in the matrix around osteocyte lacunae and canaliculi; osteoblasts and other cell types in the bone were negative. Matrix staining of the osteoid and bone was seen in bone-forming tumours including osteoma, osteoid osteoma, osteoblastoma and osteosarcoma. DMP-1 staining was also seen in fibrous dysplasia, osteofibrous dysplasia and chondroblastoma and in reactive bone in solitary bone cysts and aneurysmal bone cysts. DMP-1 was not expressed in the tumour component of other bone neoplasms including Ewing sarcoma, chondrosarcoma, leiomyosarcoma, fibrosarcoma, giant cell tumour of bone and metastatic carcinoma. DMP-1 mRNA was expressed in osteosarcoma cell lines and tumour samples. DMP-1 is a matrix marker expressed around osteocytes in human woven and lamellar bone and is useful in identifying osteosarcoma and other bone-forming tumours.

Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder.

Methylmalonic aciduria and homocystinuria, cblC type, is the most common inborn error of cellular vitamin B12 metabolism. We previously showed that the protein carrying the mutation responsible for late-onset cblC (MMACHC-R161Q), treatable with high dose OHCbl, is able to bind OHCbl with wild-type affinity, leaving undetermined the disease mechanism involved [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. To assess whether the mutation renders the protein unstable, we investigated the thermostability of the wild-type and mutant MMACHC proteins, either unbound or bound to different cobalamins (Cbl), using differential scanning fluorimetry. We found that MMACHC-wt and MMACHC-R161Q are both very thermolabile proteins in their apo forms, with melting temperatures (T(m)) of 39.3+/-1.0 and 37.1+/-0.7 degrees C, respectively; a difference confirmed by unfolding of MMACHC-R161Q but not MMACHC-wt by isothermal denaturation at 35 degrees C over 120 min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (Delta T(m max)=8 degrees C, half-maximal effective ligand concentration, AC(50)=3 microM). We surveyed the effect of different cobalamins on the stabilization of the wild-type protein and found that AdoCbl was the most stabilizing, exerting a maximum increase in T(m) of approximately 16 degrees C, followed by MeCbl at approximately 13 degrees C, each evaluated at 50 microM cofactor. The other cobalamins stabilized in the order (CN)(2)Cbi>OHCbl>CNCbl. Interestingly, the AC(50)'s for AdoCbl, MeCbl, (CN)(2)Cbi and OHCbl were similar and ranged from 1-3 microM, which compares well with the K(d) of 6 microM for OHCbl [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. Unlike MMACHC-wt, the mutant protein MMACHC-R161Q is only moderately stabilized by OHCbl (Delta T(m max)=4 degrees C). The dose-response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC(50) of 7 microM. MMACHC-R161Q showed the same order of stabilization as MMACHC-wt, but each cobalamin stabilized this mutant protein less than its wild-type counterpart. Additionally, MMACHC-R161Q had a higher AC(50) for each cobalamin form compared to MMACHC-wt. Finally, we show that MMACHC-R161Q is able to support the base-off transition for AdoCbl and CNCbl, indicating this mutant is not blocked in that respect. Taken together, our results suggest that protein stability, as well as propensity for ligand-induced stabilization, contributes to the disease mechanism in late-onset cblC disorder. Our results underscore the importance of cofactor stabilization of MMACHC and suggest that even small increases in the concentration of cobalamin complexed with MMACHC may have therapeutic benefit in children with the late-onset, vitamin responsive cblC disease.

Dynamic protein methylation in chromatin biology.

Post-translational modification of chromatin is emerging as an increasingly important regulator of chromosomal processes. In particular, histone lysine and arginine methylation play important roles in regulating transcription, maintaining genomic integrity, and contributing to epigenetic memory. Recently, the use of new approaches to analyse histone methylation, the generation of genetic model systems, and the ability to interrogate genome wide histone modification profiles has aided in defining how histone methylation contributes to these processes. Here we focus on the recent advances in our understanding of the histone methylation system and examine how dynamic histone methylation contributes to normal cellular function in mammals.

Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes.

Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an alpha/beta folding pattern with a central beta sheet flanked by 2 - 3 alpha-helices from each side, thus a classical Rossmannfold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is determined by a variable C-terminal segment. Relationships exist with bacterial haloalcohol dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signalling.

Hep27, a member of the short-chain dehydrogenase/reductase family, is an NADPH-dependent dicarbonyl reductase expressed in vascular endothelial tissue.

Human Hep27 was originally isolated from growth-arrested HepG2 cells and identified as a member of the superfamily of short-chain dehydrogenases/reductases (SDR). Its substrate specificity has not been determined, but a cross-species comparison suggests that it occurs in widely divergent species, such as human, Cenorhabditis elegans, Drosophila and Arabidopsis thaliana. In this study, Hep27 was expressed as a His(6) fusion protein, and subjected to a substrate screen, using a compound library of SDR substrates, comprising steroids, retinoids, sugars and carbonyl compounds. Whereas no steroid dehydrogenase or retinoid activity was detected, it was found that Hep27 catalyzed the NADPH-dependent reduction of dicarbonyl compounds, like 3,4-hexanedione and 1-phenyl-1,2-propanedione with similar turnover numbers as DCXR (a mitochondrial dicarbonyl reductase/xylulose reductase). In contrast, Hep27 does not convert sugar substrates like xylulose or threose. Based on its substrate specificity and expression in endothelial tissues, it is suggested that Hep27 functions as a dicarbonyl reductase in enzymatic inactivation of reactive carbonyls, involved in covalent modification of cellular components.

Human and rodent type 1 11beta-hydroxysteroid dehydrogenases are 7beta-hydroxycholesterol dehydrogenases involved in oxysterol metabolism.

Interconversion between cortisone and the glucocorticoid receptor ligand cortisol is carried out by 11beta-hydroxysteroid dehydrogenase (11beta-HSD)isozymes and constitutes a medically important example of pre-receptor control of steroid hormones. The enzyme 11beta-HSD type 1 (11beta-HSD1) catalyzes the conversion of cortisone to its active receptor-binding derivative cortisol, whereas 11beta-HSD type 2 performs the reverse reaction. Specific inhibitors against the type 1 enzyme lower intracellular levels of glucocorticoid hormone, with an important clinical application in insulin resistance and other metabolic disorders. We report here on the in vitro oxysterol-metabolizing properties of human and rodent 11beta-HSD1. The enzyme, either as full-length, membrane-attached, or as a transmembrane domain-deleted, soluble form, mediates exclusively conversion between 7-ketocholesterol and 7beta-hydroxycholesterol with similar k(cat) values as observed with glucocorticoid hormones. Thus, human, rat, and mouse 11beta-HSD1 have dual enzyme activities like the recently described 7alpha-hydroxysteroid dehydrogenase/11beta-hydroxysteroid dehydrogenase from hamster liver, but differ fundamentally from the latter in that 7beta-OH rather than 7alpha-OH dehydrogenase constitutes the second activity. These results demonstrate an enzymatic origin of species differences in 7-oxysterol metabolism, establish the origin of endogenous 7beta-OH cholesterol in humans, and point to a possible involvement of 11beta-HSD1 in atherosclerosis.

Structural role of conserved Asn179 in the short-chain dehydrogenase/reductase scaffold.

Short-chain dehydrogenases/reductases (SDR) constitute a large family of enzymes found in all forms of life. Despite a low level of sequence identity, the three-dimensional structures determined display a nearly superimposable alpha/beta folding pattern. We identified a conserved asparagine residue located within strand betaF and analyzed its role in the short-chain dehydrogenase/reductase architecture. Mutagenetic replacement of Asn179 by Ala in bacterial 3beta/17beta-hydroxysteroid dehydrogenase yields a folded, but enzymatically inactive enzyme, which is significantly more resistant to denaturation by guanidinium hydrochloride. Crystallographic analysis of the wild-type enzyme at 1.2-A resolution reveals a hydrogen bonding network, including a buried and well-ordered water molecule connecting strands betaE to betaF, a common feature found in 16 of 21 known three-dimensional structures of the family. Based on these results, we hypothesize that in mammalian 11beta-hydroxysteroid dehydrogenase the essential Asn-linked glycosylation site, which corresponds to the conserved segment, displays similar structural features and has a central role to maintain the SDR scaffold.

Human type 10 17 beta-hydroxysteroid dehydrogenase: molecular modelling and substrate docking.

17 beta-hydroxysteroid dehydrogenases catalyze the oxidoreduction of hydroxy/oxo groups at position C17 of steroid hormones, thereby constituting a prereceptor control mechanism of hormone action. At present, 11 different mammalian 17 beta-hydroxysteroid dehydrogenases have been identified, catalyzing the cell- and steroid-specific activation and inactivation of estrogens and androgens. The human type 10 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD-10) is a multifunctional mitochondrial enzyme that efficiently catalyzes the oxidative inactivation at C17 of androgens and estrogens. However, it also mediates oxidation of 3 alpha-hydroxy groups of androgens, thereby reactivating androgen metabolites. Finally, it is involved in beta-oxidation of fatty acids by catalyzing the L-hydroxyacyl CoA dehydrogenase reaction of the beta-oxidation cycle. These features and expression profiles suggest a critical role of 17 beta-HSD-10 in neurodegenerative and steroid-dependent cancer forms. Since no three-dimensional structure of 17 beta-HSD-10 is available, homology modelling was carried out to understand the molecular basis of these substrate specificities. The structure obtained displays the properties of a one-domain, alpha/beta fold enzyme of the SDR family. The active site is located within a large, hydrophobic cleft, which forms optimal contacts with the different steroid surfaces. The data provide explanations for the substrate specificities toward the various classes of sex steroid hormones. The model is suitable to explore substrate and inhibitor characteristics that may be used in the development of novel strategies in the treatment of degenerative or malignant diseases.

Metabolic conversion as a pre-receptor control mechanism for lipophilic hormones.

The majority of physiological effects mediated by steroids, retinoids and thyroids is accomplished by binding to members of the nuclear receptor superfamily of ligand activated transcription factors. The complex specific effects of lipid hormones depend not only on receptor expression, distribution and interactions, but also on the availability and metabolic conversion of the hormone itself. The cell-specific metabolic activation of inactive hormone precursors introduces a further level of hormonal regulation, and constitutes an important concept in endocrinology. The metabolic reactions carried out are achieved by dehydrogenases/reductases, hydroxylases and other enzymes, acting on ligands of the steroid/thyroid/retinoic hormone receptor superfamily. The concept implies that these tissue- and cell-specific metabolic conversions contribute to lipid hormone action, thus pointing to novel targets in drug development. All components of this signalling system, the hormone compounds, the receptor proteins, and modifying enzyme families originate from an early metazoan date, emphasizing the essential nature of all elements for development and diversification of vertebrate life.

Forms and functions of human SDR enzymes.

Short-chain dehydrogenases/reductases (SDR) are defined by distinct, common sequence motifs but constitute a functionally heterogenous superfamily of enzymes. At present, well over 1600 members from all forms of life are annotated in databases. Using the defined sequence motifs as queries, 37 distinct human members of the SDR family can be retrieved. The functional assignments of these forms fall minimally into three main groups, enzymes involved in intermediary metabolism, enzymes participating in lipid hormone and mediator metabolism, and open reading frames (ORFs) of yet undeciphered function. This overview, prepared just before completion of the human genome project, gives the different human SDR forms and relates them to human diseases.

Novel enzymological profiles of human 11beta-hydroxysteroid dehydrogenase type 1.

The human enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the reversible oxidoreduction of 11beta-OH/11-oxo groups of glucocorticoid hormones. Besides this important endocrinological property, the type 1 isozyme (11beta-HSD1) mediates reductive phase I reactions of several carbonyl group bearing xenobiotics, including drugs, insecticides and carcinogens. The aim of this study was to explore novel substrate specificities of human 11beta-HSD1, using heterologously expressed protein in the yeast system Pichia pastoris. In addition to established phase I xenobiotic substrates, it is now demonstrated that transformed yeast strains catalyze the reduction of ketoprofen to its hydroxy metabolite, and the oxidation of the prodrug DFU-lactol to the pharmacologically active lactone compound. Purified recombinant 11beta-HSD1 mediated oxidative reactions, however, the labile reductive activity component could not be maintained. In conclusion, evidence is provided that human 11beta-HSD1 in vitro is involved in phase I reactions of anti-inflammatory non-steroidal drugs like ketoprofen and DFU-lactol.

Subcellular targeting analysis of SDR-type hydroxysteroid dehydrogenases.

Most mammalian hydroxysteroid dehydrogenases known thus far belong to the protein superfamilies of short-chain dehydrogenases/reductases (SDR) and aldo-keto reductases (AKR). Whereas members of the AKR family are soluble, cytoplasmic enzymes, SDR-type hydroxysteroid dehydrogenases are also located to other subcellular compartments, i.e. endoplasmic reticulum, mitochondria or peroxisomes. Differential localization might play an important role in influencing the reaction direction of hydroxy dehydrogenase/oxo reductase pathways by determining the available nucleotide cofactor pool. Targeting signals for different subcellular organelles in human hydroxysteroid dehydrogenases have been identified, however, in several enzymes localization signals remain to be determined.

Type 1 11beta -hydroxysteroid dehydrogenase mediates glucocorticoid activation and insulin release in pancreatic islets.

Metabolic transformation of glucocorticoid hormones constitutes a determinant of their cell-specific effects. The most important reaction for this class of steroids is the reversible C11 keto/beta-hydroxyl conversion between receptor-binding 11beta-OH steroids and the nonbinding 11-oxo compounds, carried out by 11beta-hydroxysteroid dehydrogenases (11beta-HSDs). In this study, we determined the role of glucocorticoid conversion by 11beta-HSD in pancreatic islets and its function in the regulation of insulin release. Pancreatic islets isolated from ob/ob mice display type 1 11beta-hydroxysteroid dehydrogenase activity, i.e. in intact cells the reductive reaction prevails, leading from dehydrocorticosterone to corticosterone. Expression of type 1 11beta-HSD mRNA was detected by reverse transcriptase-polymerase chain reaction in islets isolated from ob/ob mice and also from human tissue. Incubation of beta-cells in the presence of 11-dehydrocorticosterone leads to a dose-dependent inhibition of insulin release, indicating cellular activation of 11-dehydrocorticosterone to the receptor ligand, further confirmed by reporter gene assays. Inhibition of 11beta-HSD activity by carbenoxolone reverses inhibition of insulin release. The presence of 11beta-HSD in islets supports the concept that reactivation of inert circulating hormone precursors in a cell-specific manner plays a major role in glucocorticoid physiology in rodents and man.