AP2α controls the dynamic balance between miR-126&126* and miR-221&222 during melanoma progression.

Accumulating evidences have shown the association between aberrantly expressed microRNAs (miRs) and cancer, where these small regulatory RNAs appear to dictate the cell fate by regulating all the main biological processes. We demonstrated the responsibility of the circuitry connecting the oncomiR-221&222 with the tumor suppressors miR-126&126* in melanoma development and progression. According to the inverse correlation between endogenous miR-221&222 and miR-126&126*, respectively increasing or decreasing with malignancy, their enforced expression or silencing was sufficient for a reciprocal regulation. In line with the opposite roles of these miRs, protein analyses confirmed the reverse expression pattern of miR-126&126*-targeted genes that were induced by miR-221&222. Looking for a central player in this complex network, we revealed the dual regulation of AP2α, on one side directly targeted by miR-221&222 and on the other a transcriptional activator of miR-126&126*. We showed the chance of restoring miR-126&126* expression in metastatic melanoma to reduce the amount of mature intracellular heparin-binding EGF like growth factor, thus preventing promyelocytic leukemia zinc finger delocalization and maintaining its repression on miR-221&222 promoter. Thus, the low-residual quantity of these two miRs assures the release of AP2α expression, which in turn binds to and induces miR-126&126* transcription. All together these results point to an unbalanced ratio functional to melanoma malignancy between these two couples of miRs. During progression this balance gradually moves from miR-126&126* toward miR-221&222. This circuitry, besides confirming the central role of AP2α in orchestrating melanoma development and/or progression, further displays the significance of these miRs in cancer and the option of utilizing them for novel therapeutics.

The Notch2-Jagged1 interaction mediates stem cell factor signaling in erythropoiesis.

Stem cell factor (SCF), the ligand for the c-kit receptor, is essential for the production of red blood cells during development and stress erythropoiesis. SCF promotes erythroblast proliferation and survival, while delaying erythroid differentiation through mechanisms that are largely unknown. In cultures of primary human differentiating erythroblasts, we found that SCF induces an increase in the expression of Notch2, a member of the Notch family implicated in the control of cell growth and differentiation. Functional inhibition of either Notch or its ligand Jagged1 inhibited the effects of SCF on erythroid cell expansion. SCF also induced the expression of Hes-1 and GATA-2, which may contribute to transduce Notch2 signals in response to SCF. Transduction of primary erythroid precursors with a dominant-negative Notch2 mutant inhibited both basal and SCF-mediated erythroblast expansion, and counteracted the effects of SCF on erythroblast differentiation. These findings provide a clue to understand the effects of increased proliferation and delayed differentiation elicited by SCF on the erythroid compartment and indicate Notch2 as a new player in the regulation of red cell differentiation.

Effect of digoxin on the in vitro secretion of renin and angiotensin II/III immunoreactivity by the human adrenal gland.

Cardiac glycosides in man inhibit renin secretion, probably through a direct effect at the renal level (i.e. inhibition of juxtaglomerular cell Na/K ATPase). Since there is evidence that the human adrenal possesses an intrinsic renin-angiotensin system, we investigated the effect of digoxin on the in vitro generation of renin and angiotensin II/III, as well as of aldosterone, by the human adrenal gland. Minced normal adrenal tissues were studied in a superfusion system, measuring in the 15-min superfusate fractions active renin by immunoradiometric assay and angiotensin II/III and aldosterone by radioimmunoassay, respectively. In a first set of four experiments using different concentrations of digoxin in sequence for 45 min periods, digoxin 10(-5), but not 10(-8) and 10(-6) mol/l, significantly reduced renin and angiotensin II/III output from adrenals, while no change in aldosterone was observed. In a second set of three experiments, the addition of digoxin 10(-5) mol/l for 120 min caused a sustained reduction of renin and angiotensin II/III, but not of aldosterone. In the final experiment, the decrease of renin and angiotensin II/III during superfusion with digoxin 10(-5) mol/l was significantly greater than that observed during superfusion with digoxin in the presence of antidigoxin antibodies. Our data indicate that digoxin at high doses reduces renin and angiotensin II/III but not aldosterone secretion by the human adrenal gland. This suggests two different effects of digoxin, probably both mediated by inhibition of the Na/K ATPase activity, on the adrenal renin-angiotensin- and aldosterone-secreting cells.

In vitro evidence for local generation of renin and angiotensin II/III immunoreactivity by the human adrenal gland.

The adrenal gland of various mammalian species has been shown to contain all the components of a functional renin-angiotensin system. We investigated the existence of this local system in human adrenal tissues surgically obtained. Eight normal adrenals (cortex and medulla) and 6 aldosterone-producing adenomas (aldosteronomas) were examined. Minced tissues were superfused over 270 min, and 15-min fractions were collected. In the perfusates, active renin was measured by immunoradiometric assay with human anti-renin monoclonal antibodies; immunoreactive angiotensin II/III and aldosterone were measured by radioimmunoassay. Adrenal tissues, either normal or pathological, were found concomitantly to release renin, angiotensin II/III and aldosterone. The pattern of this spontaneous release exhibited a pulsatile character. The total amount of renin and angiotensin II/III secreted during superfusion clearly exceeded the tissue content (determined by extraction). Addition of the angiotensin-converting enzyme inhibitor quinaprilat (4 x 10(-6) mol/l) in the superfusion caused a concomitant decrease of angiotensin II/III and aldosterone secretion by 3 normal tissues, and no change in 2 aldosteronomas. These data provide evidence that the human adrenal gland in vitro generates and releases both renin and angiotensin II/III, and support the hypothesis that locally formed angiotensin II/III may play a role as a paracrine regulator of physiological aldosterone secretion.

Hypertensive congenital adrenal enzymatic defects detected by high-performance liquid chromatography of corticosteroids.

The simultaneous measurement of the adrenal deoxycorticosterone (DOC), 18-OH-DOC, corticosterone (B), 18-OH-B, 11-deoxycortisol (S) and cortisol (F) present in human plasma in cases of adrenal dysfunction was accomplished using a high-performance liquid chromatographic (HPLC) system with a UV detector and with a radioimmunoassay (RIA). After a solid-phase extraction, plasma samples were separated by HPLC using a gradient of water-acetonitrile-ethanol on a radial compressed reversed-phase column. In a 70-min cycle, a complete separation of adrenal steroids was accomplished. The UV detector allowed direct measurement of F in each plasma sample while in selected cases B and S were directly determined. It was therefore possible quickly to identify patients with hypertensive congenital adrenal enzymatic defects with this method: the 17-alpha-hydroxylase deficiency characterized by the absence of measurable levels of F with an evident peak corresponding to B and the 11-beta-hydroxylase deficiency in which high levels of S without F are detected. The RIA of DOC, B, 18-OH-DOC and 18-OH-B complete the characterization of the adrenal defect. Therefore, with this HPLC method it is possible to recognize the major hypertensive adrenal enzymatic deficiencies such as the defect of 17-alpha-hydroxylase or 11-beta-hydroxylase. With "RIA" detectors an almost complete spectrum of adrenal steroid secretion can be obtained.

Atrial natriuretic peptide in Cushing's disease.

Atrial Natriuretic Peptide (ANF), is secreted by atrial myocytes in response to atrial stretch. Its plasma levels have been found elevated in conditions leading to salt and fluid repletion and consequent atrial distention. Recently, it has been demonstrated that dexamethasone can enhance ANF secretion, by acting on ANF gene expression and mRNA synthesis. High plasma levels of ANF have been observed in normal man after administration of cortisol and ACTH. In the case of glucocorticoid excess, as in Cushing's disease, limited and conflicting data are available. Therefore, we measured ANF basal values and ANF response to postural changes and volume expansion in eight patients with Cushing's disease. In our patients ANF values were higher than normals. ANF responded to volume expansion, 47.8 +/- 5.1 pg/ml before sodium load and 69.9 +/- 7.0 pg/ml after sodium load, and changed minimally after postural manoeuvres, 47.3 +/- 3.2 pg/ml supine and 41.7 +/- 5.1 pg/ml upright. These data indicate that ANF secretion is enhanced in Cushing's disease, and its regulation is partially altered. Since in this condition hypervolemia has not been certainly demonstrated, a direct relationship between elevated ANF and glucocorticoid excess could be suggested.

A new family with dexamethasone-suppressible hyperaldosteronism: aldosterone unresponsiveness to angiotensin II.

A family with nine siblings in which three siblings have been shown to have dexamethasone-suppressible hyperaldosteronism was studied. All three showed no significant changes of plasma aldosterone during angiotensin II infusion at incremental rates under baseline conditions. After dexamethasone administration (2 mg/d for 4 weeks) plasma renin activity (PRA) rose to normal-supranormal range, while plasma and urinary aldosterone were maintained at low-normal levels. No restoration of aldosterone response to angiotensin II was observed on dexamethasone. Two other siblings were found to be hypertensive with normal baseline data; however, both showed plasma aldosterone hyperresponsiveness to ACTH. In the four normotensive siblings aldosterone response to ACTH was normal. The family pedigree was consistent with autosomal dominant transmission of the disorder. HLA typing showed haplotype A3 Bw35 in all five hypertensive sibs and in one normotensive. In conclusion, low aldosterone compared to PRA, and plasma aldosterone unresponsiveness to angiotensin II infusion before and during dexamethasone, show functional impairment, at least temporary, of the zona glomerulosa. These findings support the hypothesis that aldosterone may be derived from the zona fasciculata in this disorder.

Effect of captopril on inactive renin and contact phase of coagulation system.

It has been suggested that a Factor XII-plasma Prekallikrein dependent pathway might play an important role in the activation of inactive renin. Since Captopril has the potential to affect the kinin-kallikrein system, we have studied in a group of 16 patients with essential hypertension its acute effect both on the levels of active, inactive and total renin, and on the contact phase of the coagulation system. Our results show that a single dose of Captopril (25 mg) induces a rapid and persistent increase of active and total renin, while inactive renin tends to decrease. Together with blood pressure, plasma Prekallikrein(PK), Factor XII(FXII) and Factor XI(FXI) concomitantly decrease, although not significantly, and their values seem to return to basal levels soonafter. However, no correlation was found at any time between the levels of any of these coagulation factors, including PK, and those of inactive, active or the ratio inactive/total renin. In spite of that, it is still possible that an activation of PK, which is likely to occur under Captopril administration, may affect at least the conversion of vessel-bound prorenin rather than the circulating form.