Group 13 HOX proteins interact with the MH2 domain of R-Smads and modulate Smad transcriptional activation functions independent of HOX DNA-binding capability

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 (HOXD13(IQN>AAA)) antagonized TGF-β-stimulated transcriptional activation of the pAdtrack-3TP-Lux reporter vector in Mv1Lu cells as well as the Smad3/Smad4-activated pTRS(6)-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

Insulin resistance in H-35 rat hepatoma cells is mediated by post-receptor mechanisms

Incubation of H-35 cells with 300 ng/ml (50 nM) of insulin causes a 3-4-fold induction of tyrosine aminotransferase at 4-6 h of incubation. At 24 h the activity of transaminase returns to basal levels despite the presence of sufficient insulin to stimulate a maximal response. Furthermore, addition of 300 ng/ml of fresh insulin fails to stimulate the induction of transaminase. In contrast, the addition of 0.1 [mu]M dexamethasone to insulintreated cells stimulates the induction of tyrosine aminotransferase, indicating that the loss of responsiveness is specific to insulin action. Incubation of H-35 cells with insulin also causes a 25-30% decrease in insulin binding. This modest decrease in receptor binding is not sufficient to explain the virtually complete loss of insulin responsiveness. Hence, in H-35 hepatoma cells insulin-induced desensitization to insulin action is mediated primarily by post-receptor events.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25115/1/0000548.pd

Induction of tyrosine aminotransferase and amino acid transport in rat hepatoma cells by insulin and the insulin-like growth factor, multiplication-stimulating activity : Mediation by insulin and multiplication-stimulating activity receptors

Insulin stimulates a 2-fold increase in the amount of tyrosine aminotransferase and a 5-10-fold increase in the rate of amino acid transport in dexamethasone-treated rat hepatoma cells. In order to determine whether these effects are mediated by insulin receptors or receptors for insulin-like growth factors, we have examined the binding of 125I-labeled insulin and 125I-labeled multiplication-stimulating activity, a prototype insulin-like growth factor, and compared the biological effects of these polypeptides. Insulin and multiplication-stimulating activity cause an identical increase in transaminase activity and transport velocity; half-maximal biological effects were observed at 35 ng/ml (5.5 nM) insulin and 140 ng/ml multiplication-stimulating activity. The hepatoma cells display typical insulin receptors of appropriate specificity; half-maximal displacement of tracer insulin binding occured at 33 ng/ml unlabeled insulin, but only at 2500 ng/ml unlabeled multiplication-stimulating activity. Specific multiplication-stimulating activity receptors also were demonstrated with which insulin did not interact even at 10 [mu]g/ml. Half-maximal displacement of tracer multiplication-stimulating activity occured at 200 ng/ml unlabeled multiplication-stimulating activity. We conclude that insulin cannot act via the multiplication-stimulating activity receptor and presumably acts via typical insulin receptors. The effects of multiplication-stimulating activity on enzyme induction and amino acid transport are probably mediated primarily via the multiplication-stimulating activity receptor.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23133/1/0000057.pd

Preclinical Targeting of the Type I Insulin-Like Growth Factor Receptor in Adrenocortical Carcinoma

Context: Drug therapy for adrenocortical carcinoma (ACC), a rare and lethal malignancy, is largely empirical and ineffective. New treatments directed at molecular targets critical to the pathophysiology of ACC may prove more efficacious

Progression to Adrenocortical Tumorigenesis in Mice and Humans through Insulin-Like Growth Factor 2 and beta-Catenin

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 beta-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 beta-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized beta-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 beta-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. (Ant J Pathol 2012, 181:1017-1033; http://dx.doi.org/10.1016/j.ajpath.2012.05.026)University of MichiganUniversity of MichiganNIH [DK062027, CA134606, T32 DK07245, T32 HD007505, T32-CA009676]NIHNational Council for Scientific and Technological Development of Brazil [CNPq 300209/2008-8]National Council for Scientific and Technological Development of BrazilCoordination for Improvement of Higher Education (CAPES), BrazilCoordination for Improvement of Higher Education (CAPES), Brazi