Hormonal sensitivity of PGF_2α production in rodent adrenocortical cells and involvement of AKR1B7 protein in PGF_2α production.

<p><i>A</i>, Western-blot analysis of AKR1Bs and COX2 proteins accumulation in stably transfected Y1 cell clones expressing AKR1B7 (empty vector EV2) or devoid of AKR1B7 (antisense vector AS19) untreated or treated with 10⁻⁵ M forskolin (Fsk) for 6 h. Molecular weight markers are shown on the right. <i>B</i>, ELISA quantification of PGF_2α in media from stably transfected Y1 cell clones expressing AKR1B7 (EV2) or devoid of AKR1B7 (AS19), untreated or treated with 10⁻⁵ M forskolin for 6 h. <i>C</i>, Differential expression of AKR1Bs and COX2 proteins in primary cultures of rat cortical cells treated with vehicle (ctrl) or with 10⁻⁹ M ACTH for 6 h or 12 h. <i>D</i>, ELISA quantification of PGF_2α release in media from rat adrenocortical primary cells, cultured in the absence or presence of 10⁻⁹ M ACTH for 6 h. Values are the mean of 3 experiments ± S.D. *, <i>P</i><0.05, ** <i>P</i><0.01.</p

Functional cellular coupling between COX2 and AKR1B7.

<p><i>A</i>, Western blot analysis of six different HEK293 clones stably transfected with COX2 expression vector in combination with empty vector (COX2, clones 3, 4, 5) or AKR1B7 expression vector (COX2-AKR1B7, clones 3, 7, 14). <i>B</i>, ELISA quantification of PGF_2α in media from stably transfected HEK293 cell clones, expressing COX2 alone (COX2-3, -4, -5) or in combination with AKR1B7 (COX2-AKR1B7-3, -7, -14). Cells were stimulated for 30 min with 10 µM A23187 ionophore and culture media were used for PGF_2α quantification. Values were expressed as the mean of 4 experiments ± S.D. <i>Asterisks</i> point values significantly different from the release of PGF_2α by the COX2-4 cell clone. *, <i>P</i><0.05, **, <i>P</i><0.01. <i>C</i>, Subcellular localization of AKR1B7, AKR1B3, AKR1B8 and COX2 in Y1 adrenocortical cells was analysed by western-blot. Protein extracts (40 µg/lane) from nuclear (Nuc), heavy membrane (HM), light membrane (LM) and cytosolic fractions (Cyt) of Y1 adrenocortical cells untreated or treated with 10⁻⁵ M forskolin for 6 h were subjected to western blot analysis. StAR and SF1 signals were used as markers of heavy membrane fraction and nuclear fraction respectively. Molecular weight markers are shown on the right.</p

Role of PGF_2α in adrenal endocrine functions.

<p><i>A</i>, ELISA quantification of PGF_2α release by chromaffin MPC862L cells untreated (control) or treated with 10⁻⁶ M dexamethasone for 12 h (dex). <i>B</i>, HPLC quantification of dopamine secretion by MPC862L cells cultured in absence or presence of 10⁻⁷ M cloprostenol (clo) (PGF_2α analogue) used either alone or in combination with 10⁻⁶ M dexamethasone for 12 h (clo+dex). Values are the mean of 3 experiments ± S.D. *, <i>P</i><0.05, ** <i>P</i><0.01. <i>C</i>, Typical perifusion profiles illustrating the effects of increasing concentrations of PGF_2α (0.1 µM to 10 µM) and cloprostenol (0.1 nM to 1 µM) on corticosterone secretion. Horizontal bars indicate the start point and duration of PGF_2α or cloprostenol infusions. <i>D</i>, Semi-logarithmic plot showing the effect of increasing concentrations of PGF_2α and cloprostenol on the inhibition of corticosterone secretion. Results are expressed as a percentage of the basal secretory rate. Experimental values were calculated from data similar to those presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007309#pone-0007309-g005" target="_blank">Fig. 5</a><i>C</i>. Each curve represents the mean ± SEM of 6 to 12 independent experiments. After stabilization, the mean secretion rate of corticosterone in basal condition was 251±14 pg/min per adrenal. The concentration-response curve was fitted using the Prism program (GraphPad Software, Inc., San Diego, CA). **<i>P<0.001</i>.</p

Differential expression of AKR1Bs, COXs and FP receptor proteins in cell cultures from adrenal cortex and from adrenal <i>medulla</i>.

<p><i>A</i>, Time-dependent response to 10⁻⁵ M forskolin treatment of StAR, AKR1Bs, COXs and FP receptor proteins levels in adrenocortical Y1 cell line was analyzed by western blot. Whole adrenal protein extract was used as a positive control, (ad.). <i>B</i>, Expression levels of AKR1Bs, COXs and FP receptor proteins were analyzed by western-blot in the chromaffin MPC862L cell line either untreated (ctrl) or treated with 10⁻⁶ M dexamethasone (dex) for 12 h. These levels were compared to the levels observed in Y1 cells stimulated by 10⁻⁵ M forskolin (Fsk) for 6 h (ns, non specific signal). <i>C</i>, FP receptor expression in primary cell cultures of rat adrenal cortical and medullary cells. AKR1B7 and TH positive signals were used as markers of steroidogenic and chromaffin identity, respectively. Molecular weights are indicated on the right.</p

Hormonal regulation and differential expression of AKR1Bs, COXs and FP receptor proteins in adrenal glands.

<p><i>A</i>, Differential expression of AKR1Bs, COXs and FP receptor proteins in response to ACTH in mouse adrenal glands. Protein extracts (30 µg/lane) from pooled adrenal glands (3 to 6 animals per condition) treated with vehicle alone (5 days, control), dexamethasone (5 days, dex) or dexamethasone (5 days) plus ACTH for time ranging from 2 to 17 h (dex ACTH) were subjected to western blot analysis. Molecular weight markers are shown on the right. COX2 multiple molecular species are a consequence of the heterogeneous glycosylation of the protein (ns, non specific signal). <i>B</i>, Immunolocalization of AKR1B7, COX2 and StAR in mouse adrenal sections. Sections of adrenal glands from male mice treated 5 days with vehicle (control), 5 days with dexamethasone (dex) or with dexamethasone plus ACTH for the last 6 h (dex + ACTH) were immunostained with anti-AKR1B7 (L3 antiserum), anti-COX2 and anti-StAR (steroidogenic cell marker) antibodies (<i>B</i>, <i>bar</i>, 100 µm). <i>C</i>, immunolocalization of AKR1B1, COX2, TH and StAR in human adrenal sections. Sections of normal human adrenal glands were incubated with anti-AKR1B1 (L3 antiserum), anti-COX2, anti-StAR (steroidogenic cell marker) and anti-tyrosine hydroxylase (TH) (chromaffin cell marker) antibodies (<i>C</i>, <i>bar</i>, 200 µm). <i>A, artery, M, medulla, ZG, zona glomerulosa, ZF, zona fasciculata, ZR, zona reticularis, C, capsule</i>.</p

Proposed model to illustrate the integrated role of PGF_2α and PGFS of the AKR1B family in adrenal endocrine functions and cortico-medullary interactions.

<p>Free arachidonic acid (AA) is metabolized into PGH₂ by COX enzymes and then converted into PGF_2α by PGFS of the AKR1B family. FP receptor expression is restricted to the medullary zone. PGF_2α synthesized in both the cortex and <i>medulla</i> thus signals in an autocrine/paracrine manner on chromaffin cells. This inhibits catecholamines production. Catecholamines produced in the <i>medulla</i> normally stimulate glucocorticoids release by the cortex. Decreased catecholamine production in response to PGF_2α stimulation thus results in a decrease in glucocorticoids production. The differential expression and regulation of both COX and AKR1B enzymes within the adrenal zones could allow the adjustment of PGF_2α production to limit stress response or control basal steroidogenesis by finely tuning glucocorticoid secretion. In basal conditions, chromaffin cells of the medullary zone constitutively secrete PGF_2α, through the functional coupling between COX1 and possibly the PGFS AKR1B3. Under stress conditions, the resulting ACTH surge induces COX2 expression and sustains AKR1B7 levels in the cortex. Here, we demonstrated that the functional coupling between COX2 and AKR1B7 triggers a PGF_2α surge that could act as a local paracrine feedback to limit catecholamine-mediated glucocorticoid release. After the stress response has ended, COX2 returns to undetectable levels. The coupling between AKR1B7 and COX2 does not take place. AKR1B7 then functions only as a detoxifying enzyme of the harmful aldehydes produced under chronic/basal stimulation of steroidogenesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007309#pone.0007309-LefrancoisMartinez1" target="_blank">[23]</a>.</p