The two SIBLING (Small Integrin Binding Ligand N-linked Glycoproteins), bone sialoprotein (BSP) and osteopontin (OPN) are expressed in osteoblasts and osteoclasts. In mature BSP knockout (KO, -/-) mice, both bone formation and resorption as well as mineralization are impaired. OPN-/- mice display impaired resorption, and OPN is described as an inhibitor of mineralization. However, OPN is overexpressed in BSP-/- mice, complicating the understanding of their phenotype. We have generated and characterized mice with a double KO (DKO) of OPN and BSP, to try and unravel their respective contributions. Despite the absence of OPN, DKO bones are still hypomineralized. The SIBLING, matrix extracellular phosphoglycoprotein with ASARM motif (MEPE) is highly overexpressed in both BSP-/- and DKO and may impair mineralization through liberation of its ASARM (Acidic Serine-Aspartate Rich MEPE associated) peptides. DKO mice also display evidence of active formation of trabecular, secondary bone as well as primary bone in the marrow-ablation repair model. A higher number of osteoclasts form in DKO marrow cultures, with higher resorption activity, and DKO long bones display a localized and conspicuous cortical macroporosity. High bone formation and resorption parameters, and high cortical porosity in DKO mice suggest an active bone modeling/remodeling, in the absence of two key regulators of bone cell performance. This first double KO of SIBLING proteins thus results in a singular, non-trivial phenotype leading to reconsider the interpretation of each single KO, concerning in particular matrix mineralization and the regulation of bone cell activity.

In the normal human adrenal gland, steroid secretion is regulated by a complex network of autocrine/paracrine interactions involving bioactive signals released by endothelial cells, nerve terminals, chromaffin cells, immunocompetent cells, and adrenocortical cells themselves. ACTH can be locally produced by medullary chromaffin cells and is, therefore, a major mediator of the corticomedullary functional interplay. Plasma ACTH also triggers the release of angiogenic and vasoactive agents from adrenocortical cells and adrenal mast cells and, thus, indirectly regulates steroid production through modulation of the adrenal blood flow. Adrenocortical neoplasms associated with steroid hypersecretion exhibit molecular and cellular defects that tend to reinforce the influence of paracrine regulatory loops on corticosteroidogenesis. Especially, ACTH has been found to be abnormally synthesized in bilateral macronodular adrenal hyperplasia responsible for hypercortisolism. In these tissues, ACTH is detected in a subpopulation of adrenocortical cells that express gonadal markers. This observation suggests that ectopic production of ACTH may result from impaired embryogenesis leading to abnormal maturation of the adrenogonadal primordium. Globally, the current literature indicates that ACTH is a major player in the autocrine/paracrine processes occurring in the adrenal gland in both physiological and pathological conditions.

L'hyperplasie congénitale des surrénales (HCS) par déficit en 21-hydroxylase a pour conséquence des taux plasmatiques d'aldostérone et de cortisol très bas et une hypersécrétion d'androgènes. Les patients doivent suivre un traitement médical substitutif et freinateur afin de prévenir le risque d'insuffisance surrénal aiguë et de freiner la production d'androgènes. Cependant, ces traitements sont difficiles à optimiser et une augmentation de la mortalité chez ces patients a été démontrée. Face à ce constat, nous avons développé un modèle murin de transplantation de cellules cortico-surrénaliennes en sous-cutanée dans des chambres pré-implantées. Nous avons préparé des cellules cortico-surrénaliennes humaines issues de deux patients âgés de 2 et 43 ans avec une HCS qui avaient dû bénéficier d'une surrénalectomie bilatérale. Suite à leur mise en culture, ces cellules produisent de la 17-OH progestérone mais pas de cortisol même sous stimulus ACTH. Le transfert du gène CYP21A2 codant pour la 21-hydroxylase à l'aide de particules lentivirales restaure la capacité de ces cellules à secréter du cortisol. Les cellules transfectées produisent aussi de l'aldostérone ce qui représente un intérêt non négligeable dans la perspective d'une application humaine. La transplantation de ces cellules dans les chambres pré-implantées chez les souris Scid surrénalectomisées a permis la survie de ces animaux qui, sans transplantation, décèdent. La fonction des greffons a été validée par la présence de cortisol et d'aldostérone dans le plasma. Nos résultats montrent dans un modèle pré-clinique qu'une alternative à la supplémentation hormonale des patients atteints d'une HCS semble possible.

Adrenal carcinoma (ACC) is a rare endocrine neoplasm, notorious for its aggressive behavior. Aberrant expressions of β-catenin have been found in a third of ACC. Here, we studied its effects on the growth of the human ACC cell line H295R. The cells were infected with short hairpin RNA (shRNA)-mediated silencing β-catenin. Two shRNAs used induced down-regulation of β-catenin protein levels. The expression of these shRNAs decreased cell growth and increased H295R cells in S and G2/M phases. This cytostatic effect is due in part to a decrease of phosphorylated MAPK and to an up-regulation expression of the cyclin-dependent kinase inhibitors p57KIP2, p21WAF1/CIP1 and p27KIP1. In addition, the knockdown of β-catenin decreased phosphorylated Akt level and increased apoptosis. Finally, loss of β-catenin was sufficient to induce the reversal of the epithelial-to-mesenchymal transition. We then transplanted these genetically modified H295R cells in Scid mice. The animals were sacrificed 46 days following transplantation. Tumor growth suppression was achieved by the two shRNAs showing in vitro efficacy. Proliferation was not significantly reduced in silenced tumors compared to control ones. In contrast, p57, p27 and p21 proteins were found expressed at high levels in silenced tumors, which might modulate cell cycle progression. Moreover, the depletion of β-catenin induced an increase in apoptotic cells. Taken together, these findings indicate that the blockade of β-catenin inhibits tumor growth of β-catenin-activated tumor cells and finally, shRNA technology may have potential therapeutic use in ACC.

Adrenal carcinoma (ACC) is a rare neoplasm with a poor outcome. Aberrant expression of β-catenin has been found in approximatively 30% of ACC. We herein studied its effects on the growth of the human ACC cell line H295R. The cells were infected with short hairpin RNA (shRNA)-mediated silencing β-catenin. Two shRNAs used induced down-regulation of β-catenin protein levels. The expression of these shRNAs decreased cell growth and increased H295R cells in S and G2/M phases. This cytostatic effect is due to a decrease of phosphorylated MAPK and to an up-regulation expression of the cyclin-dependent kinase inhibitors p57KIP2, p21CIP and p27KIP1. In addition, the knockdown of β-catenin decreased phosphorylated Akt and increased apoptosis. Finally, loss of β-catenin was sufficient to induce the reversal of the epithelial-to-mesenchymal transition. We then transplanted these genetically modified H295R cells in Scid mice. Tumor growth suppression was achieved by the two shRNAs showing in vitro efficacy. Proliferation was not reduced in silenced tumors. In contrast, p57, p27 and p21 proteins were found expressed at high levels in silenced tumors along with an increase in apoptotic cells. These findings indicate that β-catenin loss in H295R cells inhibits tumor growth by inducing transcriptional and functional changes.

Sporadic adrenocortical carcinomas (ACC) are rare endocrine neoplasms with a dismal prognosis. By contrast, benign tumors of the adrenal cortex are common in the general population. Whether benign tumors represent a separate entity or are in fact part of a process of tumor progression ultimately leading to an ACC is still an unresolved issue. To this end, we have developed a mouse model of tumor progression by successively transducing genes altered in adrenocortical tumors into normal adrenocortical cells. The introduction in different orders of the oncogenic allele of Ras (H-Ras(G12V)) and the mutant p53(DD) that disrupts the p53 pathway yielded tumors displaying major differences in histological features, tumorigenicity, and metastatic behavior. Whereas the successive expression of Ras(G12V) and p53(DD) led to highly malignant tumors with metastatic behavior, reminiscent of those formed after the simultaneous introduction of p53(DD) and Ras(G12V), the reverse sequence gave rise only to benign tumors. Microarray profiling revealed that 157 genes related to cancer development and progression were differentially expressed. Of these genes, 40 were up-regulated and 117 were down-regulated in malignant cell populations as compared with benign cell populations. This is the first evidence-based observation that ACC development follows a multistage progression and that the tumor phenotype is directly influenced by the order of acquisition of genetic alterations.

Context: Stimulation of cortisol secretion through abnormally expressed G protein-coupled receptors (GPCRs) is a frequent feature of ACTH-independent macronodular adrenal hyperplasia (AIMAH). This has opened a pharmacological strategy that targets GPCRs for the treatment of Cushing's syndrome in AIMAH. However, only few drugs are available for the presently described GPCRs. Objective: The objective of the study was to identify new GPCR targets for the pharmacological treatment of adrenal Cushing's syndrome. Design and Patients: We designed a cDNA chip containing 865 nucleotidic sequences of GPCRs. mRNAs were extracted from three normal adrenals, 18 AIMAHs, four adrenals from Cushing's disease patients, and 13 cortisol-secreting adenomas. A set of GPCR mRNAs that showed significantly higher or lower expression in AIMAH than in normal adrenal were studied by quantitative RT-PCR analysis. Analysis of protein expression and function were performed on selected GPCRs. Setting: The study was conducted at a tertiary care center and basic research laboratories. Results: The ACTH MC2 receptor showed a low expression in 15 of 18 AIMAHs samples, whereas several previously undescribed GPCR genes were found highly expressed in a subset of AIMAH, such as the receptors for motilin (MLNR; three of 18 AIMAHs) and γ-aminobutyric acid (GABBR1; five of 18 AIMAHs), and the α2A adrenergic receptor (ADRA2A; 13 of 18 AIMAHs), on which we focused our attention. Western blot and immunochemistry analyses showed expression of ADRA2A protein in AIMAH but not in normal adrenal cortex. The ADRA2A agonist clonidine enhanced both basal and stimulated cortisol production. Clonidine-induced increase in basal cortisol levels was blocked by the ADRA2A antagonist yohimbine. Conclusion: ADRA2A is a potential target for pharmacological treatment of Cushing's syndrome linked to AIMAH.

Whereas benign adrenocortical tumors are frequent in the population, adrenocortical carcinoma (ACC) is a rare cancer. Significant advances in the understanding of the pathogenesis of sporadic ACCs have been possible through the study of hereditary syndromes responsible for ACCs. The genetic alterations involved in these syndromes have also been found in sporadic ACCs. Several specific genes have been shown to be altered in sporadic ACCs. Despite these progresses, the underlying sequence(s) of events remains to be elucidated. Progressive transformation of a normal tissue into a benign tumor and ultimately into a carcinoma occurs via accumulation of genetic and epigenetic alterations. Likewise, a multistage model has been proposed for the adrenal tumor development. This review summarizes the molecular alterations likely involved in the multistage tumorigenesis and describes a mouse model which allows us to evaluate the effect of individual genes or combination of genes in the development of adrenocortical tumors.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and its homolog Bombina variegata (Bv8), also termed prokineticin-1 and -2 (PK1 and PK2) respectively, are newly identified peptides with specific mitogenic activity on endocrine gland-derived endothelial cells. In the present study, we analyzed the sites of expression of EG-VEGF/PK1, Bv8/PK2, and their receptors (PKR1 and PKR2) in the adrenal cortex and checked for new biological functions of these factors on the endocrine cell compartment. RT-PCR and immunostaining analyses revealed that glomerulosa and fasciculata cells express both factors and both receptors. EG-VEGF/PK1 had no effect on the steroidogenic activity of both bovine glomerulosa and fasciculata cells but appeared to be mitogenic for both cell types. Binding of EG-VEGF/PK1 to fasciculata cells stimulated the phosphorylation of ERK1/2. Pretreatment with pertussis toxin suppressed this effect, indicating that it was Gi mediated. EG-VEGF/PK1 also increased the phosphorylation of Akt in endocrine cells of the adrenal cortex. EG-VEGF/PK1 and Bv8/PK2 thus represent new regulatory peptides acting as autocrine mitogens for endocrine cells.

Cushing's syndrome due to ACTH-independent macronodular adrenal hyperplasia (AIMAH) can be associated with abnormal responses of aberrantly expressed adrenocortical receptors. This study aimed to characterize in vitro the pathophysiology of hypercortisolism in a beta-blocker-sensitive Cushing's syndrome due to AIMAH. Cortisol secretion profile under aberrant receptors stimulation revealed hyperresponsiveness to salbutamol (beta2-adrenoceptor agonist), cisapride (5-HT4 receptor agonist), and vasopressin in AIMAH cultured cells, but not in normal adrenocortical cells. By RT-PCR, AIMAH tissues revealed beta2-adrenoceptor overexpression rather than ectopical expression. MC2R expression was similar in both AIMAH and normal adrenocortical tissues. Curiously, cortisol levels of AIMAH cells under basal condition were 15-fold higher than those of control cells and were not responsive to ACTH. Analysis of culture medium from AIMAH cells could detect the presence of ACTH, which was immunohistochemically confirmed. Finally, the present study of AIMAH cells has identified: a) cortisol hyperresponsiveness to catecholamines, 5-HT4 and vasopressin in vitro, in agreement with clinical screening tests; b) abnormal expression of beta2-adrenoceptors in some areas of the hyperplastic adrenal tissue; c) autocrine loop of ACTH production. Altogether, the demonstration of aberrant responses to hormonal receptors and autocrine hormone production in the same tissue supports the assumption of multiple molecular alterations in adrenal macronodular hyperplasia.

Aberrant expression of ectopic G protein-coupled receptors (GPCRs) in adrenal cortex tissue has been observed in several cases of ACTH-independent macronodular adrenal hyperplasias and adenomas associated with Cushing's syndrome. Although there is clear clinical evidence for the implication of these ectopic receptors in abnormal regulation of cortisol production, whether this aberrant GPCR expression is the cause or the consequence of the development of an adrenal hyperplasia is still an open question. To answer it, we genetically engineered primary bovine adrenocortical cells to have them express the gastric inhibitory polypeptide receptor. After transplantation of these modified cells under the renal capsule of adrenalectomized immunodeficient mice, tissues formed had their functional and histological characteristics analyzed. We observed the formation of an enlarged and hyperproliferative adenomatous adrenocortical tissue that secreted cortisol in a gastric inhibitory polypeptide-dependent manner and induced a mild Cushing's syndrome with hyperglycemia. Moreover, we show that tumor development was ACTH independent. Thus, a single genetic event, inappropriate expression of a nonmutated GPCR gene, is sufficient to initiate the complete phenotypic alterations that ultimately lead to the formation of a benign adrenocortical tumor.

CONTEXT:Aberrant expression of LH/human chorionic gonadotropin (hCG) receptor has been suggested in several cases of bilateral macronodular adrenal hyperplasia with Cushing's syndrome. The cortisol production is then directly controlled by endogenous secretion of LH/hCG. However, the direct involvement of this aberrant LH/hCG receptor expression in the development of the hyperplasia has not been demonstrated. Moreover in most cases, whenever investigated, the aberrant expression of LH/hCG receptor has been associated with the ectopic expression of other G protein-coupled receptors such as gastric inhibitory polypeptide, serotonin, or vasopressin receptors.OBJECTIVE:The aim of this study was to explore the action of LH/hCG receptor on the development of adrenal hyperplasia.RESULTS:The ectopic expression of this single nonmutated gene transduced into bovine adrenocortical cells was sufficient to induce not only the aberrant cortisol secretion but also hyperproliferation and benign transformation. The cells were transplanted beneath the kidney capsule of adrenalectomized immunodeficient mice. Only the cells expressing the LH/hCG receptor gene formed an enlarged tissue with a high proliferation rate. The tissue expressing LH/hCG receptor was responsible for elevated plasma cortisol and decreased plasma ACTH levels in transplanted mice. These animals displayed physiological changes similar to those of patients with Cushing's syndrome, including muscle atrophy, thin skin, spleen atrophy, and hyperglycemia.CONCLUSIONS:These results demonstrate that a single genetic event such as the inappropriate expression of the nonmutated LH/hCG receptor gene is sufficient to initiate the phenotypic changes that cause the development of a benign adrenocortical tumor.

The adrenal cortex is a complex tissue comprising different cell types, including endocrine, stromal, nerve, perivascular, and endothelial cells. The pituitary hormone ACTH controls the coordinated development of the vasculature and the endocrine tissue mass. This suggests that paracrine secretions between steroidogenic cells and capillary endothelial cells participate in the control of adrenocortical homeostasis. In this paper, we present data demonstrating that ACTH deprivation-induced adrenal atrophy results from alterations of both endocrine and endothelial compartments. These data support the concept that, in addition to its protective effect on endocrine cells, ACTH controls adult adrenal cortex trophicity through an additional paracrine mechanism implying maintenance of the vasculature by VEGF.

The adrenal cortex is a highly vascularized endocrine tissue. A dense network of blood capillaries centripetally irrigates the adrenal gland, allowing every endocrine cell to be in contact with an endothelial cell. The pituitary hormone ACTH controls the coordinated development of the vasculature and the endocrine tissue mass. This suggests that paracrine secretions between steroidogenic adrenocytes and capillary endothelial cells participate in the control of adrenocortical homeostasis. Besides its effect on the vascular tone of arteries, ACTH induces the expression of the angiogenic cytokine VEGF‐A (vascular endothelial growth factor‐A) in primary cultures of adrenocortical cells. This growth factor is a specific mitogen for endothelial cells and is likely to mediate the hormonal control of adrenocortical vascularization through a paracrine mechanism. The newly discovered angiogenic factor EG‐VEGF (endocrine‐gland‐derived vascular endothelial growth factor), the expression of which is restricted to endocrine glands and which is preferentially mitogenic for endocrine tissue‐derived endothelial cells, is another candidate mediator of great potential interest. Microsc. Res. Tech. 61:247–251, 2003. © 2003 Wiley‐Liss, Inc.

Adrenocortical cell transplantation is a powerful technique for the investigation of the regulation of adrenocortical structure and function. Some classical organ and tissue transplantation experiments suggest that the success of transplantation depends on the activity of the pituitary gland and other endocrine systems, and is therefore influenced by the host animals’ own adrenal glands. For this reason, our experiments have usually been performed on adrenalectomized animals. However, we show here that cell transplantation experiments, involving the introduction of bovine adrenocortical cells into scid mice, do produce transplant tissues in the presence of the host animals’ adrenal glands. However, the tissue that forms is small and its cells also smaller than usual. When the adrenals of such animals are removed in a second surgical procedure, the transplants show a rapid increase in steroidogenic function and a slower increase in size, over several weeks. We conclude that the initial process by which transplanted adrenocortical cells organize into a tissue structure is not affected by the presence of the host animals’ adrenal glands, but the growth of the transplants is limited until the adrenal glands are removed.

Cell transplantation provides a way to study genes that may be important in human tissue aging. Studies on gene action in human cells are usually restricted to cell culture investigations and clinical observations. Differences in human and rodent cellular biology, particularly with respect to telomere dynamics, show the need for new systems for investigating aging that use human cells or cells of other large, long-lived mammals, such as bovine cells. The system we describe uses human and bovine adrenocortical cells transplanted into scid (severe combined immunodeficiency) mice. They form a vascularized tissue structure that can replace the essential functions of the animals' own adrenal glands. The cells may be genetically modified before introduction into the animal. Using hTERT (telomerase reverse transcriptase) and oncoproteins, we show the potential for investigating gene action in genetically modified tissues created by cell transplantation.