Mark F. Foecking

Active steroidogenesis in the normal rat skin

Using the radiolabeled precursors of adrenal steroids (14)C-11-deoxycorticosterone (DOC) and (14)C-progesterone ((14)C-PROG) we demonstrate that rat skin can synthesize a number of steroids. TLC separation of labeled metabolites show that among the (14)C-steroid products, two co-migrate with corticosterone (B) and 11-dehydrocorticosterone (A) standards. Thus, normal rodent skin possesses steroidogenic activity that can be shown using progesterone or DOC as primary substrates.

METABOLISM OF PROGESTERONE TO DOC, CORTICOSTERONE AND 18OHDOC IN CULTURED HUMAN MELANOMA CELLS

We are now showing that cultured human melanoma cells can synthesize steroids such as corticosterone from progesterone or deoxycorticosterone. Corticosterone production is strongly responsive to deoxycorticosterone substrate addition (12‐fold increase), but unresponsive to the adrenal stimulating factors ACTH and angiotensin II. This is the first demonstration that skin cells (malignant melanocytes) have the capability to synthesize 11‐deoxycorticosterone, corticosterone, and 18‐hydroxydeoxycorticosterone.

Complete Genome Sequence of Mycoplasma bovis Type Strain PG45 (ATCC 25523)

ABSTRACT This complete and fully assembled genome sequence of Mycoplasma bovis type strain PG45 is the first available for this species and offers a framework for comparison with additional pathogenic isolates. The single circular chromosome of 1,003,404 bp reveals multiple gene sets and mechanisms involved in variable expression of surface antigens and the incursion of numerous and assorted mobile elements, despite its reduced size.

Endothelin receptor subtypes and stimulation of aldosterone secretion.

Endothelins (ETs) are 21-amino acid peptides with two disulfide bonds that have powerful vasoactive properties. We have previously shown the presence of a specific, high-affinity, saturable receptor for porcine or human endothelin (ET-1) in cultured calf zona glomeniiosa cells. ET-1 was a stimulator of aldosterone secretion although not as powerful as angiotensin II. Incubations of cultured calf zona glomeniiosa cells with Sarafotoxin S6b (S6b), a snake venom that has a structure highly homologous to ET-1, stimulated aldosterone secretion with a potency similar to that of ET-1. Binding of [125I]ET-1 to the adrenal receptor gave a kA of 0.17 ± 0.05 nM and a Bmax of 36 ± 8.5 fmol/well (n=4). Displacement of [125I]ET-1 by unlabeled ETs and S6b showed that the concentrations needed to displace 50% of the tracer were 0 3 nM for ET-1,03 nM for ET-2,10 nM for S6b, and 100 nM for ET-3. Binding of [125I]S6b to cultured adrenal cells revealed a receptor with a KA of 0.05 ± 0.01 nM and a Bmax of 8 ± 2 fmol/well (n=4). Displacement of [l25I]S6b by unlabeled ETs and S6b showed that the concentrations needed to displace 50% of the tracer were 0.03 nM for S6b, 0.06 nM for ET-1, 0.04 nM for ET-2, and 0.05 nM for ET-3. Unlabeled ET-1 and ET-2 preferentially down-regulated the binding of [125I]ET-1, and S6b preferentially down-regulated the binding of [125I]S6b. Labeled S6b (which is unlikely to exist in mammals) served as a tool to uncover the high-affinity receptor for ET-1 (ET-1αreceptor), which is likely responsible for stimulation of aldosterone secretion. The second receptor or lower affinity, higher capacity receptor (ET-10 receptor) has an unclear function at this time.

11β-Hydroxysteroid dehydrogenases of the choriocarcinoma cell line JEG-3 and their inhibition by glycyrrhetinic acid and other natural substances

Mineralocorticoid receptor (MR) selectivity for aldosterone is thought to be exerted by enzymes which inactivate competing glucocorticoids before they bind the receptor. Two different 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) have been described. 11 beta-HSD-1 is NADP(+)-dependent and has a Km in the micromolar range and bidirectional activity. 11 beta-HSD-2 is NAD(+)-dependent, has a Km in the nanomolar range, exhibits only oxidase activity, and colocalizes with the MR in the kidney, so is likely to serve as the gatekeeper for the MR. We have further characterized 11 beta-HSD activity in JEG-3 cells, a cell line derived from a human choriocarcinoma which was reported to have only the high affinity, NAD(+)-dependent 11 beta-HSD-2. We found that the Km for the conversion of corticosterone to 11-dehydrocorticosterone in intact cells and homogenates was about 16 nM. NAD(+)-dependent corticosterone conversion was equal in the nuclear and mitochondrial fractions and less, but significant, in the microsomal fraction. A high affinity, Km = 40 nM, NADP(+)-dependent enzyme was also found in homogenates. The subcellular distribution of this high affinity activity was greatest in the mitochondria, less in the nuclei, and even less, but still significant, in microsomes. Because of its cofactor dependency, high affinity, and different subcellular distribution, we suggest that this enzyme is neither the 11 beta-HSD-1 nor the 11 beta-HSD-2 and have named it 11 beta-HSD-3. Conversion of 11-dehydrocorticosterone to corticosterone did not occur in intact cells or in homogenates incubated with NADH or NADPH. Enzyme activity in intact cells was inhibited by glycyrrhetinic acid, carbenoxolone, progesterone, 5 beta-dihydroprogesterone, and 5 alpha-dihydroprogesterone, but not bile acids.

T Cell Immunity in Connective Tissue Disease Patients Targets the RNA Binding Domain of the U1-70kDa Small Nuclear Ribonucleoprotein

Although the T cell dependence of autoimmune responses in connective tissue diseases has been well established, limited information exists regarding the T cell targeting of self Ags in humans. To characterize the T cell response to a connective tissue disease-associated autoantigen, this study generated T cell clones from patients using a set of peptides encompassing the entire linear sequence of the 70-kDa subunit of U1 snRNP (U1-70kDa) small nuclear ribonucleoprotein. Despite the ability of U1-70kDa to undergo multiple forms of Ag modification that have been correlated with distinct clinical disease phenotypes, a remarkably limited and consistent pattern of T cell targeting of U1-70kDa was observed. All tested T cell clones generated against U1-70kDa were specific for epitopes within the RNA binding domain (RBD) of the protein. High avidity binding of the RBD with U1-RNA was preserved with the disease-associated modified forms of U1-70kDa tested. The high avidity interaction between the U1-RBD on the polypeptide and U1-RNA may be critical in immune targeting of this region in autoimmunity. The T cell autoimmune response to U1-70kDa appears to have less diversity than is seen in the humoral response; and therefore, may be a favorable target for therapeutic intervention.

A Major B Cell Epitope Present on the Apoptotic but Not the Intact Form of the U1-70-kDa Ribonucleoprotein Autoantigen

Apoptotically modified forms of autoantigens have been hypothesized to participate in lupus immunopathogenesis. This study identifies a major B cell epitope present on the apoptotic but not the intact form of the U1-70-kDa ribonucleoprotein lupus autoantigen (70k). Human autoimmune sera with strong recognition of apoptotic 70k and minimal recognition of intact 70k were identified and tested for reactivity to truncated forms of 70k by immunoblot and ELISA. Patient sera that preferentially recognized apoptotic 70k were specific for an epitope dependent on residues 180–205 of the protein. This epitope was also recognized by 19 of 28 (68%) intact anti-70k-positive autoimmune human sera with Abs also recognizing apoptotic but not the intact form 70k, but only 1 of 9 (11%) intact 70k-positive sera without such Abs (Fisher’s exact, p = 0.0055). Immunization of HLA-DR4-transgenic C57BL/6 mice with a peptide containing this epitope induced anti-70k immunity in 13 of 15 mice, including Abs recognizing apoptotic but not intact forms of autoantigens in 12 of 15 mice. Anti-70k responder mice also developed spreading of immunity to epitopes on the endogenous form of 70k, and proliferative lung lesions consistent with those described in patients with anti-70k autoimmunity. Thus, a major epitope in the B cell response to U1-70 kDa localizes to the RNA binding domain of the molecule, overlaps with the most common T cell epitope in the anti-70k response, and is not present on the intact form of the 70k molecule. Immunization of mice against this epitope induces an immune response with features seen in human anti-70k autoimmune disease.

Serum 18-Hydroxycortisol in Primary Aldosteronism, Hypertension, and Normotensives

This study reports the determination of plasma 18-hydroxycortisol (18-OHF) using a new and easy enzyme-linked immunosorbent assay (ELISA) method in primary aldosteronism and compares the values found in essential hypertensives and normotensive controls. In primary aldosteronism, we evaluated usefulness of plasma 18-OHF determination and the dexamethasone suppression test in the diagnosis of glucocorticoid-remediable aldosteronism using the genetic test as the gold standard. We studied 31 primary aldosteronism patients, 101 essential hypertensives, and 102 healthy normotensive controls. The plasma 18-OHF was measured using a biotin-avidin enzyme-linked assay by a new and purified polyclonal antibody. The 18-OHF value in primary aldosteronism was 6.3±8.05 nmol/L; this value is significantly higher than the value found in essential hypertensives and normotensive controls (2.81±1.42 and 2.70±1.41 nmol/L, respectively;P <0.0005). In primary aldosteronism, 4 of 31 patients had 18-OHF levels that were 10 times higher than the normal upper limit (2.983 nmol/L). The dexamethasone suppression test in primary aldosteronism patients was positive (serum aldosterone <4 ng/dL) in 13 of 31 cases. A chimeric CYP11B1/CYP11B2 gene was demonstrated in 4 primary aldosteronism patients, corresponding to the same cases that had higher level of 18-OHF. In conclusion, plasma 18-OHF determination by this ELISA method is reliable for detecting glucocorticoid-remediable aldosteronism, and it does so better than the dexamethasone suppression test.

Steroidogenesis in the human skin : 21-hydroxylation in cultured keratinocytes

We have evaluated the metabolism of radiolabeled progesterone (P) by the microsomal fraction isolated from HaCaT keratinocytes. P was widely metabolized to different compounds that included DOC (5-7% conversion) thus demonstrated 21-hydroxylase (21-OHase) activity, a key step in adrenal synthesis of gluco- and mineralocorticoids. However, RT-PCR amplification for the CYPc21 transcript of the corresponding gene showed no evidence for gene expression in HaCaT cells suggesting that the 21-OHase enzyme present in keratinocytes is different from that described in adrenal gland. Further characterization showed that whereas estradiol stimulated markedly P metabolism by HaCaT microsomes, with generation of new unidentified compounds, Lineweaver-Burk analysis of keratinocyte 21-OHase activity showed that the K(m) and V(max) were unaffected by estrogen. The apparent K(m) was 0.6 microM without estradiol and 0.7 microM with estradiol, while the respective V(max) values were 60 and 76 nmol/l/min. To conclude, we found extensive metabolism of P in human keratinocytes, we also provide the first demonstration of 21-OHase activity in this cell system and further showed that it is coded by a gene different from the adrenal CYPc21.

Cloning and expression of the rat adrenal cytochrome P-450 11B3 (CYP11B3) enzyme cDNA: Preferential 18-hydroxylation over 11β-hydroxylation of DOC

Abstract The biosynthesis of glucocorticoids and mineralocorticoids in the rat adrenal cortex requires the action of two different cytochrome P450 11β-hydroxylases, CYP11B1 and CYP11B2, which are distributed in the zona fasciculata and glomerulosa, respectively. The existence of another cytochrome P450-11β gene, CYP11B3, was recently reported. Although CYP11B3 has similar gene structure and great homology to the CYP11B1 and -B2 genes, the CYP11B3 mRNA was not originally detected by reverse transcription-polymerase chain reaction (RT-PCR) and has only recently been cloned and detected from neonatal rat adrenals. Herein we demonstrate RT-PCR detection of CYP11B3 mRNA expressed in adult rat adrenal and brain tissues. The whole coding region of the CYP11B3 enzyme cDNA was cloned and sequenced. When transiently expressed in COS-7 cells the CYP11B3 converted deoxycorticosterone (DOC) to corticosterone and 18-hydroxydeoxycorticosterone, but not to 18-hydroxycorticosterone or aldosterone. It produced more 18-OH-DOC than corticosterone. A single mutation in CYP11B3 in which Gly-59 was replaced by Ser, reduced the enzymatic activity 5–6-fold. Furthermore, CYP11B3 mRNA expression is greater in neonatal, compared to adult rat adrenal glands.