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A central feature of glucocorticoid (GC)-induced osteoporosis is decreased bone formation, secondary to decreased numbers of functional osteoblasts. We find that ERK activity is essential for serum-induced osteoblast proliferation in vitro because inhibition of MAPK/ERK kinase activity by U0126 completely abolished both serum-induced activation of ERK and proliferation of mouse (MBA-15.4) and human (MG-63) osteoblast cell lines. Dexamethasone (Dex) rapidly (<2 h) inhibits the sustained phase of ERK activation, required for nuclear shift and mitogenesis. This inhibition is reversed by cotreatment with the protein synthesis inhibitor, cycloheximide, and by the GC receptor antagonist, RU486, suggesting a classical transcriptional mechanism. Phosphatase activity was up-regulated by Dex treatment, and inhibition of ERK activity by Dex was also reversed by the protein tyrosine phosphatase inhibitor, vanadate. Coupled with the rapidity of Dex action, this indicates immediate-early gene phosphatase involvement, and we therefore used quantitative, real-time PCR to examine expression profiles of the dual-specificity MAPK phosphatases, MKP-1 and MKP-3. MKP-1, but not MKP-3, mRNA expression was 10-fold up-regulated in both mouse and human osteoblast cell lines within 30 min of Dex treatment and remained elevated for 24 h. MKP-1 protein was also markedly up-regulated following 1-8 h of Dex treatment, and this correlated precisely with dephosphorylation of ERK. Cell proliferation was impaired by Dex treatment, and this was reversed by both RU486 and vanadate. Therefore, MKP-1 up-regulation provides a novel and rapid mechanism, whereby GCs inhibit osteoblast proliferation.

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South African Angora goats (Capra aegagrus) are susceptible to stress conditions, possibly due to adrenal cortex malfunction. Selection for mohair production may reduce adrenal function and decrease cortisol production. Secretion of cortisol by the adrenal cortex is essential for the induction of several gluconeogenic enzymes that enable animals to survive stressful conditions, and adrenocortical insufficiency, therefore, precipitates a vulnerability to stress. In this study, Angora goats were compared with two breeds generally accepted as hardy, Boer goats (Capra hircus) and Merino sheep (Ovis aries). Adrenal steroidogenesis was studied using subcellular fractions prepared from the adrenal glands of freshly slaughtered animals. Adrenal microsomes and mitochondria were incubated with the relevant steroid substrates, and products were analyzed and quantified with TLC, HPLC, or RIA. Subsequently, the activity of individual enzymes involved in this pathway were further investigated. The cytochrome P450 content in the preparations was also compared. The results from these studies indicated that the activity of the cytochrome P450c17 enzyme in Angora goats differed (P < .01) from that of the other species investigated. This difference may contribute to the cause of the observed hypoadrenocorticism in Angora goats.

Our objective was to identify the primary site of the reduced adrenal function in South African Angora goats (Capra aegagrus) that causes a decrease in cortisol production and leads to severe losses of Angora goats during cold spells. Angora goats, Boer goats (Capra hircus), and Merino sheep (Ovis aries) were assigned to three intravenous treatments: 1) insulin, 2) corticotropin-releasing factor (CRF), and 3) ACTH. Blood cortisol concentrations were determined over a 90-min period to determine any differences in the response of the experimental animals to these treatments. For both the insulin and ACTH treatments, cortisol concentrations were less in Angora goats than in the other experimental animals. The adrenal gland was subsequently investigated as a possible cause for the observed hypoadrenocorticism. Primary adrenal cell cultures were prepared from these species, subjected to different treatments, and the cortisol production determined. Upon pregnenolone (PREG) addition, all the experimental animals' cortisol production increased significantly, with the production in Boer goats higher (P<.01) when compared with that in the other species. The stimulation of cortisol biosynthesis by ACTH was only obtained for Boer goats and Merino sheep. The stimulation of cortisol production by forskolin and cholera toxin were compared with ACTH, and, for Angora goats, only cholera toxin caused a significant increase in cortisol production. For Boer goats, no difference (P>.05) between the PREG, ACTH, forskolin, or cholera toxin treatments were observed. The Merino adrenal cells were increasingly stimulated in the following order: PREG, ACTH, forskolin, and cholera toxin (forskolin and cholera toxin stimulated cortisol production to the same extent). This investigation of the hypothalamic-pituitary-adrenocortical axis, therefore, identified the adrenal gland as the primary site of the Angora's hypoadrenocorticism.

The production of glucocorticoids and mineralocorticoids in the endoplasmic reticulum (ER) of the mammalian adrenal cortex are, to a great extent, regulated by the relative activities of the steroid 21-hydroxylase (P450c21) and steroid 17 alpha-hydroxylase (P450c17) enzymes. Progesterone can be 17 alpha-hydroxylated to yield 17 alpha-hydroxyprogesterone which, under certain conditions and in certain species, can be further lyased to adrostenedione by the same enzyme. P450c21 can 21-hydroxylate 17 alpha-hydroxyprogesterone to yield cortisol but also converts progesterone to corticosterone. Cytochrome b5 (cyt b5) can also participate in the regulation of adrenal microsomal steroid hydroxylase activities by changing the rates of the P450c21 and P450c17 reactions or by affecting the 17 alpha-hydroxylation:17,20-lyase ratio of progesterone, by P450c17. We investigated the metabolism of progesterone by sheep adrenal microsomes to identify the products of the different steroid hydroxylase activities in the ER and to investigate the influence of cyt b5 on progesterone metabolism using purified ovine cyt b5 and anti-cyt b5. The P450c17-activity in sheep adrenal microsomes is inhibited by the addition of purified cyt b5 while anti-cyt b5 IgG stimulates the 17 alpha-hydroxylation of progesterone. No 17,21-lyase-activity towards progesterone could be detected in sheep adrenal microsomes.

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