Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal β-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal β-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized β-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized β-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis.

These findings establish a critical role of IGF signaling in ACC pathophysiology and provide rationale for use of targeted IGF-1R antagonists to treat adrenocortical carcinoma in future clinical trials.

Incubation of HTC rat hepatoma cells with 8-bromocAMP results in a 3-fold increase in the rate of degradation of type-1 plasminogen activator inhibitor (PAI-1) mRNA. We have reported previously that the 3-most 134 nt of the PAI-1 mRNA is able to confer cyclic nucleotide regulation of message stability onto a heterologous transcript. R-EMSA and UV cross-linking experiments have shown that this 134 nt cyclic nucleotide-responsive sequence (CRS) binds HTC cell cytoplasmic proteins ranging in size from 38 to 76 kDa. Mutations in the A-rich region of the CRS both eliminate cyclic nucleotide regulation of mRNA decay and abolish RN-protein complex formation, suggesting that these RNA-binding proteins may be important regulators of mRNA stability. By sequential R-EMSA and SDS-PAGE we have purified a protein from HTC cell polysomes that binds to the PAI-1 CRS. N-terminal sequence analysis and a search of protein data bases revealed identity with two human sequences of unknown function. We have expressed one of these sequences in E. coli and confirmed that the recombinant protein interacts specifically with the PAI-1 CRS. Mutation of the A-rich portion of the PAI-1 CRS reduces binding by the recombinant PAI-1 RNA-binding protein.The amino acid sequence of this protein includes an RGG box and two arginine-rich regions, but does not include other recognizable RNA binding motifs. Detailed analyses of nucleic acid and protein data bases demonstrate that blocks of this sequence are highly conserved in a number of metazoans, including Arabidopsis, Drosophila, birds, and mammals. Thus, we have described a novel RNA-binding protein that identifies a family of proteins with a previously undefined sequence motif. Our results suggest that this protein, PAI-RBP1, may play a role in regulation of mRNA stability.Regulation of mRNA stability is an important component of the regulation of gene expression and is known to have a significant role in normal physiology and development (1-5). Our understanding of the regulation of message degradation has been enhanced by the identification of consensus cis-acting sequences that are involved in determining message stability and of some proteins that interact with them (4, 6). Although it is known that many stimuli alter mRNA stability and some cis-acting sequences responsible have been identified, in few cases have trans-acting factors been isolated (2, 7-10). In contrast, a broad spectrum of RNA-binding proteins that are involved in RNA processing, cellular localization, and translation have been identified, and structural domains involved in RNA recognition have been described (11,12). In many cases RNAbinding proteins contain short signature domains that bind RNA and anchor the protein such that functional domains align (13,14). Much less is known about the mechanisms by which RNA-binding proteins regulate mRNA stability.Plasminogen activators (PAs) 1 are serine proteases that catalyze the conversion of plasminogen to the broad spectrum protease, plasmin. Plasmin is the major fibrinolytic en...

Scientists have long hypothesized the existence of tissue-specific (somatic) stem cells and have searched for their location in different organs. The theory that adrenocortical organ homeostasis is maintained by undifferentiated stem or progenitor cells can be traced back nearly a century. Similar to other organ systems, it is widely believed that these rare cells of the adrenal cortex remain relatively undifferentiated and quiescent until needed to replenish the organ, at which time they undergo proliferation and terminal differentiation. Historical studies examining cell cycle activation by label retention assays and regenerative potential by organ transplantation experiments suggested that the adrenocortical progenitors reside in the outer periphery of the adrenal gland. Over the past decade, the Hammer laboratory, building on this hypothesis and these observations, has endeavored to understand the mechanisms of adrenocortical development and organ maintenance. In this review, we summarize the current knowledge of adrenal organogenesis. We present evidence for the existence and location of adrenocortical stem/progenitor cells and their potential contribution to adrenocortical carcinomas. Data described herein come primarily from studies conducted in the Hammer laboratory with incorporation of important related studies from other investigators. Together, the work provides a framework for the emerging somatic stem cell field as it relates to the adrenal gland.

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor selectively expressed in the adrenal cortex and gonads, where it mediates the hormonal stimulation of multiple genes involved in steroid hormone biosynthesis. SF-1 is the target of both phosphorylation and SUMOylation, but how these modifications interact or contribute to SF-1 regulation of endogenous genes remains poorly defined. We found that SF-1 is selectively SUMOylated at K194 in Y1 adrenocarcinoma cells and that although SUMOylation does not alter the subcellular localization of SF-1, the modification inhibits the ability of SF-1 to activate target genes. Notably, whereas SF-1 SUMOylation is independent of S203 phosphorylation and is unaffected by adrenocorticotropin (ACTH) treatment, loss of SUMOylation leads to enhanced SF-1 phosphorylation at serine 203. Furthermore, preventing SF-1 SUMOylation increases the mRNA and protein levels of multiple steroidogenic enzyme genes. Analysis of the StAR promoter indicates that blockade of SF-1 SUMOylation leads to an increase in overall promoter occupancy but does not alter the oscillatory recruitment dynamics in response to ACTH. Notably, we find that CDK7 binds preferentially to the SUMOylation-deficient form of SF-1 and that CDK7 inhibition reduces phosphorylation of SF-1. Based on these observations, we propose a coordinated modification model in which inhibition of SF-1-mediated transcription by SUMOylation in adrenocortical cancer cells is mediated through reduced CDK7-induced phosphorylation of SF-1.Steroidogenic factor 1 (SF-1) (also called NR5A1 or Ad4BP) is an orphan nuclear receptor that plays a crucial role in the regulation of steroid hormone biosynthesis, as well as in the endocrine development of both the adrenal gland and gonads (68). Several genes, including the CYP17, DAX-1, CYP19, CYP11A1, MIS, 3␤-HSD, CYP21, StAR, and Mc2R genes, have been identified as SF-1 target genes (8,9,38,39,43,45,62,69,70,73). Regulation of these genes involves the concerted action of SF-1 with multiple transcription factors with which it can synergize, such as Sox9 (18), Wt1 (31, 48), Gata4 (65), EGR1 (19,25), PITX1 (64), multiprotein bridging factor 1 (36), and . A number of coregulators, such as steroid receptor coactivator 1 (SRC-1) (16, 33), cyclic AMP response element-binding protein (CREB)-binding protein/p300 (47), transcriptional intermediary factor 2 (6), nuclear receptor corepressor (15), and ␤-catenin (46), have been reported to interact with SF-1 and likely participate in SF-1 gene activation. On the other hand, factors such as Dax-1 (34) and DP103 (50) appear to play an inhibitory role by limiting SF-1 function. The transcriptional capacity of SF-1 is influenced by posttranslational modifications, with phosphorylation at S203 playing a key stimulatory role (26). S203 phosphorylation serves to enhance coactivator binding and the transactivation potential of this receptor. Recent data indicate that SF-1 can be phosphorylated on residue S203 by either ERK1/2 or CDK7 (44). Given that CDK7 is a unique ...

Dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1 (Dax1) is an orphan nuclear receptor essential for development and function of the mammalian adrenal cortex and gonads. DAX1 was cloned as the gene responsible for X-linked AHC, which is characterized by adrenocortical failure necessitating glucocorticoid replacement. Contrary to these human data, young mice with genetic Dax1 knockout (Dax1(-/Y)) exhibit adrenocortical hyperfunction, consistent with the historic description of Dax1 as a transcriptional repressor that inhibits steroidogenic factor 1-dependent steroidogenesis. This paradox of molecular function and two apparently opposite phenotypes associated with Dax1 deficiency in mice and humans is compounded by the recent observations that under certain circumstances, Dax1 can serve as a transcriptional activator of steroidogenic factor 1. The recently revealed role of Dax1 in embryonic stem cell pluripotency, together with the observation that its expression in the adult adrenal is restricted to the subcapsular cortex, where presumptive undifferentiated progenitor cells reside, has led us to reexamine the phenotype of Dax1(-/Y) mice in order to reconcile the conflicting mouse and human data. In this report, we demonstrate that although young Dax1(-/Y) mice have enhanced steroidogenesis and subcapsular adrenocortical proliferation, as these mice age, they exhibit declining adrenal growth, decreasing adrenal steroidogenic capacity, and a reversal of their initial enhanced hormonal sensitivity. Together with a marked adrenal dysplasia in aging mice, these data reveal that both Dax1(-/Y) mice and patients with X-linked AHC exhibit adrenal failure that is consistent with adrenocortical subcapsular progenitor cell depletion and argue for a significant role of Dax1 in maintenance of these cells.

Interactions with co-factors provide a means by which HOX proteins exert specificity. To identify candidate protein interactors of HOXA13, we created and screened an E11.5–E12.5, distal limb bud yeast two-hybrid prey library. Among the interactors, we isolated the BMP-signaling effector Smad5, which interacted with the paralogous HOXD13 but not with HOXA11 or HOXA9, revealing unique interaction capabilities of the AbdB-like HOX proteins. Using deletion mutants, we determined that the MH2 domain of Smad5 is necessary for HOXA13 interaction. This is the first report demonstrating an interaction between HOX proteins and the MH2 domain of Smad proteins. HOXA13 and HOXD13 also bind to other BMP and TGF-β/Activin-regulated Smad proteins including Smad1 and Smad2, but not Smad4. Furthermore, HOXD13 could be co-immunoprecipitated with Smad1 from cells. Expression of HOXA13, HOXD13 or a HOXD13 homeodomain mutant (HOXD13IQN>AAA) antagonized TGF-β-stimulated transcriptional activation of the pAdtrack-3TP-Lux reporter vector in Mv1Lu cells as well as the Smad3/Smad4-activated pTRS6-E1b promoter in Hep3B cells. Finally, using mammalian one-hybrid assay, we show that transcriptional activation by a GAL4/Smad3-C-terminus fusion protein is specifically inhibited by HOXA13. Our results identify a new co-factor for HOX group 13 proteins and suggest that HOX proteins may modulate Smad-mediated transcriptional activity through protein–protein interactions without the requirement for HOX monomeric DNA-binding capability.

Primary cultures of rat hepatocytes produce tissue-type plasminogen activator (tPA) and plasminogen activator-inhibitor type 1 (PAI-1). Incubation of hepatocytes with 50 microM 8-(4-chlorophenylthio)cAMP (CPT-cAMP) results in a 4-fold increase in tPA activity, whereas the synthetic glucocorticoid dexamethasone (1 microM) causes a more than 90% decrease. In combination, dexamethasone completely overcomes the CPT-cAMP effect and markedly decreases PA activity. PAI-1 is induced by both CPT-cAMP and dexamethasone, and the effects of these agents are additive. Accumulation of tPA mRNA is increased more than 4-fold by CPT-cAMP and is greatly decreased by incubation with dexamethasone. Dexamethasone in combination with CPT-cAMP totally blocks this cAMP effect. The protein synthesis inhibitor cycloheximide does not prevent either the dexamethasone-induced decrease or the CPT-cAMP-induced increase in tPA message and, in fact, augments the cAMP-induced increase in tPA mRNA. Hepatocyte PAI-1 mRNA levels are increased 2-fold by incubation with either CPT-cAMP or dexamethasone; in combination, these effectors cause a 4-fold increase in PAI-1 mRNA. Cycloheximide alone causes a marked increase in PAI-1 mRNA, but does not block the induction by either CPT-cAMP or dexamethasone. We conclude that incubation of hepatocytes with CPT-cAMP induces tPA activity by increasing tPA mRNA accumulation and that dexamethasone causes a decrease in tPA activity by both decreasing tPA mRNA and increasing PAI-1 mRNA and activity. Concomitant protein synthesis is not required for the regulation of tPA or PAI-1 mRNA by either CPT-cAMP or dexamethasone, indicating a primary effect of these agents on gene transcription or mRNA stability.