Human 11β-hydroxysteroid dehydrogenase: Studies on the stably transfected isoforms and localization of the type 2 isozyme within renal tissue

Bujalska, Iwona; Shimojo, Masako; Howie, Alex; Stewart, Paul M.

doi:10.1016/s0039-128x(96)00163-8

Cited by 96 publications

(60 citation statements)

References 29 publications

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“…Production of 11-␤-Hydroxysteroid Dehydrogenase 1 Expression Vectors-The human 11␤-HSD1 cDNA (3) was subcloned into pCDNA3.1 (16). This was further subcloned into pET21b(ϩ) and pET32b(ϩ) E. coli expression vectors (Novagen).…”

Section: Methodsmentioning

confidence: 99%

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Functional Expression, Characterization, and Purification of the Catalytic Domain of Human 11-β-Hydroxysteroid Dehydrogenase Type 1

Walker¹,

Clark

Hewison³

et al. 2001

Journal of Biological Chemistry

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11-␤-hydroxysteroid dehydrogenase type 1 catalyzes the conversion of cortisone to hormonally active cortisol and has been implicated in the pathogenesis of a number of disorders including insulin resistance and obesity. The enzyme is a glycosylated membrane-bound protein that has proved difficult to purify in an active state. Extracted enzyme typically loses the reductase properties seen in intact cells and shows principally dehydrogenase activity. The C-terminal catalytic domain is known to contain a disulfide bond and is located within the lumen of the endoplasmic reticulum, anchored to the membrane by a single N-terminal transmembrane domain. We report here the functional expression of the catalytic domain of the human enzyme, without the transmembrane domain and the extreme N terminus, in Escherichia coli. Moderate levels of soluble active protein were obtained using an N-terminal fusion with thioredoxin and a 6xHis tag. In contrast, the inclusion of a 6xHis tag at the C terminus adversely affected protein solubility and activity. However, the highest levels of active protein were obtained using a construct expressing the untagged catalytic domain. Nonreducing electrophoresis revealed the presence of both monomeric and dimeric disulfide bonded forms; however, mutation of a nonconserved cysteine residue resulted in a recombinant protein with no intermolecular disulfide bonds but full enzymatic activity. Using the optimal combination of plasmid construct and E. coli host strain, the recombinant protein was purified to apparent homogeneity by single step affinity chromatography. The purified protein possessed both dehydrogenase and reductase activities with a K m of 1.4 M for cortisol and 9.5 M for cortisone. This study indicates that glycosylation, the N-terminal region including the transmembrane helix, and intermolecular disulfide bonds are not essential for enzyme activity and that expression in bacteria can provide active recombinant protein for future structural and functional studies.

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Section: Methodsmentioning

confidence: 99%

“…Expression of human (and squirrel monkey) clones of 11␤-HSD1 has been achieved in COS cells (11,12), HEK cells (16), and the yeast Pichia pastoris (13,17) using a variety of vectors. This has led to ambiguous kinetic results with over 10-fold variation in K m values and often significant differences in activity between whole cells and lysates.…”

mentioning

confidence: 99%

Functional Expression, Characterization, and Purification of the Catalytic Domain of Human 11-β-Hydroxysteroid Dehydrogenase Type 1

Walker¹,

Clark

Hewison³

et al. 2001

Journal of Biological Chemistry

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“…Dehydrogenase activity (cortisol/cortisone conversion) was assessed using 100 nM (3$625!10 K5 mg/ml) unlabelled cortisol (Sigma) diluted in serum-free medium and trace amounts (1$5 nM) of [ 3 H] cortisol (specific activity 78$4 Ci/mmol; NEN, Boston, MA, USA) at 37 8C for 24 h. Conversion of cortisone to cortisol (oxo-reductase) was analysed by incubating cells with 100 nM (3$604!10 K5 mg/ml) unlabelled cortisone and trace amounts of [ 3 H]cortisone (50 000 c.p.m.) synthesised in-house as described previously (Bujalska et al 1997). Whole tissue and primary cultures were incubated with GC substrates as described earlier and a 100-fold excess (4$707!10 K3 mg/ml) of glycyrrhetinic acid (GE; Sigma), an inhibitor of 11b-HSDs (Monder et al 1989).…”

Section: B-hsd1 Enzyme Assaysmentioning

confidence: 99%

Characterisation of the prereceptor regulation of glucocorticoids in the anterior segment of the rabbit eye

Onyimba¹,

Vijapurapu²,

Curnow³

et al. 2006

Journal of Endocrinology

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The prereceptor regulation of glucocorticoids (GCs) by 11b-hydroxysteroid dehydrogenase type-1 (11b-HSD1), a bidirectional isozyme that interconverts active (cortisol) and inactive (cortisone) GCs, is an established determinant of GC function in tissues such as liver, adipose and bone. Although the therapeutic use of GCs is abundant in ophthalmic practice, where GC interactions with nuclear receptors modulate gene transcription, the prereceptor regulation of endogenous cortisol is not well described in ocular tissues. Recent descriptive studies have localised 11b-HSD1 to the human corneal epithelium and non-pigmented epithelium (NPE) of the ciliary body, indicating a link to corneal epithelial physiology and aqueous humour production. In this study, we characterise the functional aspects of the autocrine regulation of GCs in the anterior segment of the rabbit eye. Using our in-house generated primary antibody to human 11b-HSD1, immunohistochemical analyses were performed on paraffin-embedded sections of whole New Zealand white albino rabbits, (NZWAR) eyes. As in human studies, 11b-HSD1 was localised to the corneal epithelium and the NPE. No staining was seen in the albino 'pigmented' ciliary epithelium. Specific enzyme assays for oxo-reductase (cortisone/cortisol) and dehydrogenase (cortisol/cortisone) activity indicated predominant 11b-HSD1 oxo-reductase activity from both the intact ciliary body tissue (nZ12, median 2$1 pmol/mg per h and range 1$25-2$8 pmol/mg per h; PZ0$006) and primary cultures of corneal epithelial cells (nZ12, median 3$0 pmol/mg per h and range 1$0-7$4 pmol/mg per h, PZ0$008) compared with dehydrogenase activity (median 1$0 pmol/mg per h and range 0$5-2$0 pmol/mg per h; median 0$5 pmol/mg per h and range 0$25-1$9 pmol/mg per h respectively). These findings were supported by expression of 11b-HSD1 protein as visualised by Western blotting of ciliary body tissue and immunocytochemistry of corneal epithelial cells. Reduction of corneal epithelial cell proliferation was seen after primary cultures were co-incubated with cortisol and cortisone. 11b-HSD1 activity was not demonstrated in naïve conjunctival fibroblasts or corneal stromal keratocytes. Our results indicate that the distribution of 11b-HSD1 in the rabbit resembles that of the human eye and activates cortisone to cortisol in both corneal and uveal tissues. The NZWAR provides a suitable in vivo model for the further evaluation of 11b-HSD1 activity in the eye, especially its role in corneal epithelial and ciliary body physiology.

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“…The control reaction mixtures had the vehicle alone added. For experiments using CBX as an inhibitor, CBX (2 mM) was dissolved in the reaction buffer [7,9]. The effect of adding both pyridine nucleotide co-substrates (1 mM β-NADP + plus 1mM β-NAD + ) to an incubation mixture was compared to reaction mixtures used to measure CS and 7α-OH-DHEA oxidation with pig kidney microsomes (PKMc) and nuclei (PKN).…”

Section: Metabolism Assaysmentioning

confidence: 99%

“…11βHSD activity has been observed in nuclei, mitochondrial and microsomal organelles from normal human and rat placenta [5] and choriocarcinoma cells [6]. In human kidney nuclei, immuno-reactive human 11βHSD2 accounted for 40% of total cellular 11βHSD2 protein content [7,8]. In kidney from rat and sheep, a third dehydrogenase (11βHSD3) was shown to be present in membranes of Golgi apparatus and mitochondria, while 11βHSD2 is preferentially located in the nuclear membrane [9,10].…”

Section: Introductionmentioning

confidence: 99%

Interactions between dehydroepiandrosterone and glucocorticoid metabolism in pig kidney: Nuclear and microsomal 11β-hydroxysteroid dehydrogenases

Robinzon

Prough

2005

Archives of Biochemistry and Biophysics

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The 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) activates glucocorticoids (GC) by reversibly converting 11-keto-GC to 11-hydroxy-GC, while 11βHSD2 and 11βHSD3 only catalyzes the reverse reaction. Recently, rat and human 11βHSDs were shown to interconvert 7α-and 7β-hydroxy-dehydroepiandrosterone (7α-or 7β-OH-DHEA) with 7-oxo-DHEA. We report that pig kidney microsomes (PKMc) and nuclei (PKN) oxidize 7α-OH-DHEA to 7-oxo-DHEA at higher rates with NAD + , than with NADP + . Corticosterone (CS), dehydrocoticosterone (DHC), 11α-and 11β-hydroxyprogesterone, and carbenoxolone completely inhibited these reactions, while 7-oxo-DHEA only inhibited the NAD + -dependent reaction. Conversely, CS oxidation was not inhibited by 7α-OH-DHEA or 7-oxo-DHEA. PKMc and PKN did not convert 7-oxo-DHEA to 7-OH-DHEA with either NADPH or NADH. Finally, PKN contained a high affinity, NADPH-dependent 11βHSD that reduces DHC to CS. The GC effects on interconversion of DHEA metabolites may have clinical significance, since DHEA and its 7-oxidized derivatives have been proposed for treatment of human autoimmune and inflammatory disorders.

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Human 11β-hydroxysteroid dehydrogenase: Studies on the stably transfected isoforms and localization of the type 2 isozyme within renal tissue

Cited by 96 publications

References 29 publications

Functional Expression, Characterization, and Purification of the Catalytic Domain of Human 11-β-Hydroxysteroid Dehydrogenase Type 1

Functional Expression, Characterization, and Purification of the Catalytic Domain of Human 11-β-Hydroxysteroid Dehydrogenase Type 1

Characterisation of the prereceptor regulation of glucocorticoids in the anterior segment of the rabbit eye

Interactions between dehydroepiandrosterone and glucocorticoid metabolism in pig kidney: Nuclear and microsomal 11β-hydroxysteroid dehydrogenases

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