Evidence that PKA activity is constitutively activated in human GH-secreting adenoma cells in a patient with Carney complex harbouring a PRKAR1A mutation.

CONTEXT: The GHRH-protein kinase A (PKA) signalling pathway is essential for cell proliferation and GH synthesis/secretion in somatotrophs. An inactivating mutation of PRKAR1A is one of the causes of somatotrophinoma in Carney complex (CNC). The basal PKA activity of somatotroph adenoma cells from CNC has not been evaluated because of a limited amount of available tissue. OBJECTIVE: This study examined how the PRKAR1A mutation affects the PKA signalling pathway in a human somatotrophinoma with a PRKAR1A mutation. DESIGN AND SETTING: Somatotrophinoma cells from a 40-year-old male patient with CNC were used. The patient had a novel somatic heterozygous germline frameshift mutation (227delT) in PRKAR1A leading to a premature stop codon. The tumour showed loss of heterozygosity (LOH) at 17q23-24. Primary cultured adenoma cells were subjected to electrophysiological experiments to evaluate PKA signalling in individual cells. RESULTS: GHRH did not increase the nonselective cation current or the voltage-gated calcium current in these adenoma cells, in contrast to nonadenomatous somatotroph cells in which these currents increase through the PKA pathway. Application of a PKA inhibitor inhibited the basal currents in these adenoma cells, results that were not observed in nonadenomatous somatotrophs. These data indicate that the basal currents are already increased and cannot be further increased by GHRH. CONCLUSIONS: The results demonstrate that PKA is activated at the basal state in these adenoma cells. The data also show that both the nonselective cation current and the voltage-gated calcium current, vital regulators of GH secretion downstream of PKA, are maximally increased in these cells. These maximally increased currents probably account for the excessive GH secretion.

Protective effects of Peganum harmala extracts on thiourea-induced diseases in adult male rat.

Cancers and hepatoprotective prevention using traditional medicines have attracted increasing interest. The aim of our study was to characterize the putative protective effects of ethanol and chloroform extracts of Peganum harmala on thiourea-induced diseases in adult male rat. We seek to determine the effects of these plant extracts on body weight, thyroid and endocrine cancer parameters. In addition the putative hepatoprotective effect was checked by the determination of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and the bilirubin level in the blood. Our data show that ethanol and chloroform extracts of Peganum harmala protected the animal against the carcinogenic effects induced by thiourea since neuron-specific enolase (NSE) and thyroglobulin (TG) levels were back to the normal range. In addition, the observed-hepatocytotoxicity after thiourea treatment was greatly reduced (AST and ALT activities were respectively 270 IU/l and 60 IU/l and in the same order of magnitude as in the untreated rats) as well as the bilirubin levels (6 micromol/l) especially for animals receiving the choroform preparation. Therefore we may suggest that extracts of Peganum harmala are efficient to reduce the toxicity induced by thiourea in male rat as far as the above parameters are concerned.

Primary pigmented nodular adrenocortical disease reveals insulin-like growth factor binding protein-2 regulation by protein kinase A.

OBJECTIVE: Primary pigmented nodular adrenocortical disease (PPNAD) can occur as an isolated trait or part of Carney complex, a familial lentiginosis-multiple endocrine neoplasia syndrome frequently caused by mutations in PRKAR1A, which encodes the 1alpha regulatory subunit of protein kinase A (PKA). Because alterations in the insulin-like growth factor (IGF) axis, particularly IGF-II and IGF binding protein (IGFBP)-2 overexpression, have been implicated in sporadic adrenocortical tumors, we sought to examine the IGF axis in PPNAD. DESIGN: RNA samples and paraffin-embedded sections were procured from adrenalectomy specimens of patients with PPNAD. Changes in expression of IGF axis components were evaluated by real-time quantitative RT-PCR and immunohistochemistry. NCI-H295R cells were used to study PKA and IGF axis signaling in adrenocortical cells in vitro. RESULTS: IGFBP-2 mRNA level distinguished between the two genetic subtypes of this disease; increased IGFBP-2 expression in PRKAR1A mutation-positive PPNAD tissues was also confirmed by immunohistochemistry. Moreover, PKA inhibitors increased IGFBP-2 expression in NCI-H295R adrenocortical cells, and anti-IGFBP-2 antibody reduced their proliferation. CONCLUSIONS: IGFBP-2 expression is increased in PPNAD caused by PRKAR1A mutations, and in adrenocortical cancer cells. This is the first evidence for PKA-dependent regulation of IGFBP-2 expression in adrenocortical cells.

PRKAR1A mutations in primary pigmented nodular adrenocortical disease.

Primary Pigmented Nodular Adrenocortical Disease (PPNAD) is a rare primary bilateral adrenal defect causing corticotropin-independent Cushing's syndrome. It occurs mainly in children and young adults. Macroscopic appearance of the adrenals is characteristic with small pigmented micronodules observed in the cortex. PPNAD is most often diagnosed in patients with Carney complex (CNC), but it can also be observed in patients without other manifestations or familial history (isolated PPNAD). The CNC is an autosomal dominant multiple neoplasia syndrome characterized by the association of myxoma, spotty skin pigmentation and endocrine overactivity. One of the putative CNC genes has been identified as the gene of the regulatory R1A subunit of protein kinase A (PRKAR1A), located at 17q22-24. Germline heterozygous inactivating mutations of PRKAR1A have been reported in about 45% of patients with CNC, and up to 80% of CNC patients with Cushing's syndrome due to PPNAD. Interestingly, such inactivating germline PRKAR1A mutations have also been found in patients with isolated PPNAD. The hot spot PRKAR1A mutation termed c.709[-7-2]del6 predisposes mostly to isolated PPNAD, and is the first clear genotype/phenotype correlation described for this gene. Somatic inactivating mutations of PRKAR1A have been observed in macronodules of PPNAD and in sporadic cortisol secreting adrenal adenomas. Isolated PPNAD is a genetic heterogenous disease, and recently inactivating mutations of the gene of the phosphodiesterase 11A4 (PDE11A4) located at 2q31-2q35 have been identified in patients without PRKAR1A mutations. Interestingly, both PRKAR1A and PDE11A gene products control the cAMP signaling pathway, which can be altered at various levels in endocrine tumors.

A mouse model for the Carney complex tumor syndrome develops neoplasia in cyclic AMP-responsive tissues.

Carney complex is an autosomal dominant neoplasia syndrome characterized by spotty skin pigmentation, myxomatosis, endocrine tumors, and schwannomas. This condition may be caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A. To better understand the mechanism by which PRKAR1A mutations cause disease, we have developed conventional and conditional null alleles for Prkar1a in the mouse. Prkar1a(+/-) mice developed nonpigmented schwannomas and fibro-osseous bone lesions beginning at approximately 6 months of age. Although genotype-specific cardiac and adrenal lesions were not seen, benign and malignant thyroid neoplasias were observed in older mice. This spectrum of tumors overlaps that seen in Carney complex patients, confirming the validity of this mouse model. Genetic analysis indicated that allelic loss occurred in a subset of tumor cells, suggesting that complete loss of Prkar1a plays a key role in tumorigenesis. Similarly, tissue-specific ablation of Prkar1a from a subset of facial neural crest cells caused the formation of schwannomas with divergent differentiation. These observations confirm the identity of PRKAR1A as a tumor suppressor gene with specific importance to cyclic AMP-responsive tissues and suggest that these mice may be valuable tools not only for understanding endocrine tumorigenesis but also for understanding inherited predispositions for schwannoma formation.

A pleiomorphic GH pituitary adenoma from a Carney complex patient displays universal allelic loss at the protein kinase A regulatory subunit 1A (PRKARIA) locus.

Carney complex (CNC) is a familial multiple endocrine neoplasia syndrome associated with GH-producing pituitary tumours and transmitted as an autosomal dominant trait. Mutations of the PRKAR1A gene are responsible for approximately half the known CNC cases but have never found in sporadic pituitary tumours. Pituitary tissue was obtained from an acromegalic CNC patient heterozygote for a common (PRKARIA)i-inactivating mutation. Both immunohistochemistry and electron microscopy showed a highly pleiomorphic pituitary adenoma. The cell culture population appeared morphologically heterogeneous and remained so after more than 30 passages. The mixture was comprised of cells strongly immunostained for GH, spindle-shaped myofibroblast-like cells, and cuboid cells with large axonal projections (negative for GH). The population appeared to have both epithelial and mesenchymal cells. Both at baseline and at passage 30, cytogenetic analysis indicated the presence of normal 46, XY diploid karyotype, whereas losses of the PRKARIA(i) locus were demonstrated in more than 98% of the cells by fluorescent in situ hybridisation, supporting this gene's involvement in pituitary tumorigenesis. Allelic loss may have occurred in a single precursor cell type that differentiated and clonally expanded into several phenotypes. Epithelial-to-mesenchymal transition may also occur in CNC-associated pleiomorphic pituitary adenomas.

Protein kinase A and its role in human neoplasia: the Carney complex paradigm.

The type 1 alpha regulatory subunit (R1alpha) of cAMP-dependent protein kinase A (PKA) (PRKAR1A) is an important regulator of the serine-threonine kinase activity catalyzed by the PKA holoenzyme. Carney complex (CNC) describes the association 'of spotty skin pigmentation, myxomas, and endocrine overactivity'; CNC is in essence the latest form of multiple endocrine neoplasia to be described and affects the pituitary, thyroid, adrenal and gonadal glands. Primary pigmented nodular adrenocortical disease (PPNAD), a micronodular form of bilateral adrenal hyperplasia that causes a unique, inherited form of Cushing syndrome, is also the most common endocrine manifestation of CNC. CNC and PPNAD are genetically heterogeneous but one of the responsible genes is PRKAR1A, at least for those families that map to 17q22-24 (the chromosomal region that harbors PRKAR1A). CNC and/or PPNAD are the first human diseases to be caused by mutations in one of the subunits of the PKA holoenzyme. Despite the extensive literature on R1alpha and PKA, little is known about their potential involvement in cell cycle regulation, growth and/or proliferation. The presence of inactivating germline mutations and the loss of its wild-type allele in CNC lesions indicated that PRKAR1A could function as a tumor-suppressor gene in these tissues. However, there are conflicting data in the literature about PRKAR1A's role in human neoplasms, cancer cell lines and animal models. In this report, we review briefly the genetics of CNC and focus on the involvement of PRKAR1A in human tumorigenesis in an effort to reconcile the often diametrically opposite reports on R1alpha.

Human tumors associated with Carney complex and germline PRKAR1A mutations: a protein kinase A disease!

Carney complex (CNC) is a multiple neoplasia syndrome that consists of endocrine (thyroid, pituitary, adrenocortical and gonadal), non-endocrine (myxomas, nevi and other cutaneous pigmented lesions), and neural (schwannomas) tumors. Primary pigmented nodular adrenocortical disease (PPNAD) is the most common endocrine manifestation of CNC and the only inherited form of Cushing syndrome known to date. In the search of genes responsible for CNC, two chromosomal loci were identified; one (17q22-24) harbored the gene encoding the type I-alpha regulatory subunit (RIalpha) of protein kinase A (PKA), PRKAR1A, a critical component of the cAMP signaling pathway. Here we review CNC and the implications of this discovery for the cAMP and/or PKA's involvement in human tumorigenesis.

Novel succinate dehydrogenase subunit B (SDHB) mutations in familial phaeochromocytomas and paragangliomas, but an absence of somatic SDHB mutations in sporadic phaeochromocytomas.

Phaeochromocytomas arising in adrenal or extra-adrenal sites and paragangliomas of the head and neck, in particular of the carotid bodies, occur sporadically and also in a familial setting. In addition to mutations in RET and VHL in familial disease, germline mutations in SDHD and SDHB genes that encode subunits of mitochondrial complex II have also been associated with the development of familial phaeochromocytomas. To further investigate the role of SDHD and SDHB in the development of these tumours we determined the occurrence of germline SDHD and SDHB mutations in four patients with a family history of phaeochromocytoma with associated head and neck paraganglioma, one patient with a family history of phaeochromocytoma only and two patients with apparently sporadic extra-adrenal phaeochromocytoma, one of whom had early onset disease. Secondly, we investigated whether somatic SDHB mutations correlated with loss of heterozygosity at 1p36 in a subgroup of 11 sporadic and three MEN 2-associated RET-mutation-positive phaeochromocytomas. Novel SDHB mutations were identified in the probands from four families and two apparently sporadic cases (six of seven probands studied), including two missense mutations, a single nonsense and frameshift mutation, as well as two splice site mutations, one of which was shown to have partial penetrance resulting in 'leaky' splicing. Further, five intronic polymorphisms in SDHB were found. No SDHD mutations were identified. In addition, no somatic SDHB mutations were found in the remaining allele of the 11 sporadic adrenal phaeochromocytomas with allelic loss at 1p36 or the three MEN 2-associated RET-mutation-positive phaeochromocytomas. Therefore, we conclude that SDHB has a major role in the pathogenesis of familial phaeochromocytomas, but the possible role of SDHB in sporadic tumours showing allelic loss at 1p36 has yet to be ascertained.

Molecular analysis of the cyclic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adrenocortical disease (PPNAD) reveals novel mutations and clues for pathophysiology: augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD.

We studied 11 new kindreds with primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC) and found that 82% of the kindreds had PRKAR1A gene defects (including seven novel inactivating mutations), most of which led to nonsense mRNA and, thus, were not expressed in patients' cells. However, a previously undescribed base substitution in intron 6 (exon 6 IVS +1G-->T) led to exon 6 skipping and an expressed shorter PRKAR1A protein. The mutant protein was present in patients' leukocytes and tumors, and in vitro studies indicated that the mutant PRKAR1A activated cAMP-dependent protein kinase A (PKA) signaling at the nuclear level. This is the first demonstration of an inactivating PRKAR1A mutation being expressed at the protein level and leading to stimulation of the PKA pathway in CNC patients. Along with the lack of allelic loss at the PRKAR1A locus in most of the tumors from this kindred, these data suggest that alteration of PRKAR1A function (not only its complete loss) is sufficient for augmenting PKA activity leading to tumorigenesis in tissues affected by CNC.

Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit (PRKAR1A) in patients with the "complex of spotty skin pigmentation, myxomas, endocrine overactivity, and schwannomas" (Carney complex).

Carney complex (CNC) is a familial multiple neoplasia syndrome associated with abnormal skin and mucosal pigmentation. The complex has features overlapping those of McCune-Albright syndrome (MAS) and the other multiple endocrine neoplasias (MENs). CNC is inherited as an autosomal dominant trait, and the responsible genes have been mapped by linkage analysis to loci at 2p16 and 17q22-24. Because of its unusual biochemical features (e.g., paradoxical responses to various endocrine signals) and its clinical similarities to MAS, genes implicated in cyclic nucleotide-dependent signaling, including GNAS1 (which is responsible for MAS), had been considered likely candidates for causing CNC. The gene encoding the protein kinase A (PKA) type I-alpha regulatory subunit (RI alpha), PRKAR1A, had been mapped to 17q22-24; loss-of-heterozygosity (LOH) analysis using polymorphic markers from this region revealed consistent changes in tumors from patients with CNC, including those from one family previously mapped to 17q22-24. Investigation of a polymorphic site within the 5' of the PRKAR1A gene showed segregation with the disease and retention of the allele bearing the disease gene in CNC tumors. Mutations of the PRKAR1A gene were also found to have occurred de novo in sporadic cases of CNC; no mutations were found in kindreds mapping to 2p16. Thus, genetic heterogeneity in CNC was confirmed; in total, 41% of all patients with CNC had mutations in the PRKAR1A gene. All mutations were frameshifts, insertions, and deletions that led to nonsense mRNA and premature termination of the predicted peptide product. Functional studies in CNC tumors suggested that inactivating mutations of the PRKAR1A gene led to nonsense mRNA decay (the mutant peptide product was not present) and were associated with dysregulated PKA activity, increased responsiveness to cAMP, and excess of type-II PKA activity. We conclude that the PRKAR1A gene, coding for the RIalpha subunit of PKA, a critical cellular component of a number of cyclic nucleotide-dependent signaling pathways, is mutated in a subset of patients with CNC. In their tumors, there is LOH of the normal allele, suggesting that normal RI-alpha may have tumor suppression function in the tissues affected by CNC. An excess of type-II PKA activity was present in affected tissues, which may be responsible for the apparent tumorigenicity of PRKAR1A mutations in endocrine tissues.

The essential role of RI alpha in the maintenance of regulated PKA activity.

Cloning of the individual regulatory (R) and catalytic (C) subunits of the cAMP-dependent protein kinase (PKA) and expression of these subunits in cell culture have provided mechanistic answers about the rules for PKA holoenzyme assembly. One of the central findings of these studies is the essential role of the RI alpha regulatory subunit in maintaining the catalytic subunit under cAMP control. The role of RI alpha as the key compensatory regulatory subunit in this enzyme family was confirmed by gene knockouts of the three other regulatory subunits in mice. In each case, RI alpha has demonstrated the capacity for significant compensatory regulation of PKA activity in tissues where the other regulatory subunits are expressed, including brain, brown and white adipose tissue, skeletal muscle, and sperm. The essential requirement of the RI alpha regulatory subunit in maintaining cAMP control of PKA activity was further corroborated by the knockout of RI alpha in mice, which results in early embryonic lethality due to failed cardiac morphogenesis. Closer examination of RI alpha knockout embryos at even earlier stages of development revealed profound deficits in the morphogenesis of the mesodermal embryonic germ layer, which gives rise to essential structures including the embryonic heart tube. Failure of the mesodermal germ layer in RI alpha knockout embryos can be rescued by crossing RI alpha knockout mice to C alpha knockout mice, supporting the conclusion that inappropriately regulated PKA catalytic subunit activity is responsible for the phenotype. Isolation of primary embryonic fibroblasts from RI alpha knockout embryos reveals profound alterations in the actin-based cytoskeleton, which may account for the failure in mesoderm morphogenesis at gastrulation.

Expression of neuropeptides and other neuroendocrine markers in human phaeochromocytomas.

AIMS: Phaeochromocytomas may produce several neuropeptides as they are considered neuroendocrine tumours. Nevertheless, studies are scarce and no clear predictive biologic value has been stablished in the case of neuropeptides expression. METHODS: We have investigated immunohistochemically the neuropeptides expression of a serie of 36 phaeochromocytomas: 25 sporadic, seven familial type MEN (multiple endocrine neoplasm) and four familial phaeochromocytomas not associated with MEN syndrome. The reactivity for neuron-specific enolase (NSE), synaptophysin, vasoactive intestinal peptide (VIP), chromogranin A, calcitonin, ACTH, somatostatin and HMB-45 was tested according to the avidin-biotin complex (ABC) method using polyclonal antibodies. RESULTS: Phaeochromocytomas have a multiple synthetic activity as main neuroendocrine feature. Despite phaeochromocytoma tumour cells heterogeneity chromogranin and synaptophysin are the most common neuropeptides synthesised, as they are associated with the presence of neuroendocrine storage granules. We find a statistically significant higher synthesis of corticotrophin hormone in familial phaeochromocytomas than in sporadic forms, on the contrary the synthesis of VIP is statistically associated with sporadic forms of phaeochromocytomas. We also found a direct relation of ACTH and overexpression and malignant tumours and a positive relationship between NSE and benign forms of phaeochromocytomas.

A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma.

Multiple endocrine neoplasia type 2 (MEN 2) comprises three clinically distinct, dominantly inherited cancer syndromes. MEN 2A patients develop medullary thyroid carcinoma (MTC) and phaeochromocytoma. MEN 2B patients show in addition ganglioneuromas of the gastrointestinal tract and skeletal abnormalities. In familial MTC, only the thyroid is affected. Germ-line mutations of the RET proto-oncogene have recently been reported in association with MEN 2A and familial MTC. All mutations occurred within codons specifying cysteine residues in the transition point between the RET protein extracellular and transmembrane domains. We now show that MEN 2B is also associated with mutation of the RET proto-oncogene. A mutation in codon 664, causing the substitution of a threonine for a methionine in the tyrosine kinase domain of the protein, was found in all nine unrelated MEN 2B patients studied. The same mutation was found in six out of 18 sporadic tumours.

Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC.

Multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC) are dominantly inherited conditions which predispose to the development of endocrine neoplasia. Evidence is presented that sequence changes within the coding region of the RET proto-oncogene, a putative transmembrane tyrosine kinase, may be responsible for the development of neoplasia in these inherited disorders. Single strand conformational variants (SSCVs) in exons 7 and 8 of the RET proto-oncogene were identified in eight MEN 2A and four FMTC families. The variants were observed only in the DNA of individuals who were either affected or who had inherited the MEN2A or FMTC allele as determined by haplotyping experiments. The seven variants identified were sequenced directly. All involved point mutations within codons specifying cysteine residues, resulting in nonconservative amino acid changes. Six of the seven mutations are located in exon 7. A single mutation was found in exon 8. Variants were not detected in four MEN 2B families studied for all exon assays available, nor were they detectable in 16 cases of well documented sporadic medullary thyroid carcinoma or pheochromocytoma that were tested for exon 7 variants. Coinheritance of the mutations with disease and the physical and genetic proximity of the RET proto-oncogene provide evidence that RET is responsible for at least two of the three inherited forms of MEN 2. Neither the normal function, nor the ligand of RET are yet known. However, its apparent involvement in the development of these inherited forms of neoplasia as well as in papillary thyroid carcinoma suggest an important developmental or cell regulatory role for the protein.