Dioxin Disrupts Thyroid Hormone and Glucocorticoid Induction of klf9, a Master Regulator of Frog Metamorphosis

Frog metamorphosis, the development of an air-breathing froglet from an aquatic tadpole, is under endocrine control by thyroid hormone (TH) and glucocorticoids (GC). Metamorphosis is susceptible to disruption by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AHR) agonist. Krüppel-Like Factor 9 (klf9), an immediate early gene in the endocrine-controlled cascade of expression changes that govern metamorphosis, can be synergistically induced by both hormones. This process is mediated by an upstream enhancer cluster, the klf9 synergy module (KSM). klf9 is also a target of the AHR. We measured klf9 mRNA expression following combined exposures to triiodothyronine (T3), corticosterone (CORT), and TCDD in the Xenopus laevis cell line XLK-WG. klf9 was induced 6-fold by 50 nM T3, 4-fold by 100 nM CORT, and 3-fold by 175 nM TCDD. Co-treatments of CORT and TCDD or T3 and TCDD induced klf9 mRNA 7- and 11-fold, respectively, while treatment with all 3 agents induced a 15-fold increase. Transactivation assays examined regulatory sequences from the Xenopus tropicalisklf9 upstream region. KSM-containing segments mediated a strong T3 response and a larger T3/CORT response, while induction by TCDD was mediated by a region ~1 kb farther upstream containing 5 AHR response elements. Unexpectedly, this region also supported a CORT response in the absence of readily- identifiable glucocorticoid responsive elements, suggesting mediation by protein-protein interactions. A similar AHRE cluster is positionally conserved in the human genome, and klf9 was induced by TCDD and TH in HepG2 cells. These results indicate that AHR binding to an upstream AHRE cluster represents an initiating event in TCDD disruption of klf9 expression and metamorphosis.

Role of FOXO1 in Glucocorticoid-Induced Somatotrope Maturation

Growth hormone (GH) is a well-known metabolic factor secreted by pituitary somatotropes. Transcription factors such as POU1F1 and NEUROD4 promote somatotrope differentiation, maturation, and function. The forkhead transcription factor, FOXO1, is necessary for the proper timing of somatotrope differentiation and function, but the underlying mechanisms behind it have yet to be unraveled. Pituitary gland development also depends on regulation by signaling factors and hormones. Glucocorticoids have mixed effects on growth hormone production. However, when the effects of glucocorticoid signaling on the hypothalamus and pituitary gland are uncoupled, the direct effects of glucocorticoid signaling on pituitary somatotropes are not only stimulatory, but necessary for initiation of somatotrope maturation and for maintenance of somatotrope function. We find that FOXO1 is necessary for glucocorticoid induction of important somatotrope genes. Activation of glucocorticoid signaling in the somatotrope-derived MtT/S cell line induces transient expression of the bZIP transcription factor, Crebl2 within 2 hours. Interestingly, glucocorticoid induction of Crebl2 as well as the somatotrope genes Ghrhr and Gh1, is impaired in the presence of the FOXO1 inhibitor (AS1842856). There are several possible mechanisms underlying the requirement of FOXO1 in glucocorticoid induction of somatotrope maturation. One possible mechanism is that glucocorticoid signaling upregulates expression of Foxo1 and ultimately FOXO1 targets. Consistent with this possibility, Foxo1 expression is induced 8 hours after activation of glucocorticoid signaling. This does not appear to be the only mechanism underlying the role for FOXO1 in mediating glucocorticoid-induced somatotrope maturation, however, because many FOXO1 target genes, such as Neurod4 and Fosl2 are not affected by glucocorticoid signaling. We are currently investigating whether cooperative binding between FOXO1 and the glucocorticoid receptor contributes to transcriptional regulation of common targets genes. Together these data demonstrate that FOXO1 is a key factor mediating glucocorticoid induction of somatotrope maturation.

LB004A RANDOMIZED, CONTROLLED TRIAL OF RITUXIMAB VERSUS AZATHIOPRINE AFTER INDUCTION OF REMISSION WITH RITUXIMAB FOR PATIENTS WITH ANCA-ASSOCIATED VASCULITIS AND RELAPSING DISEASE

Background and Aims Rituximab is an effective therapy for induction of remission in ANCA-associated vasculitis (AAV). However, the effect of rituximab is not sustained, and subsequent relapse rates are high, especially in patients with a history of relapse. The RITAZAREM trial (ClinicalTrials.gov identifier: NCT01697267) is an international, multi-center, open-labelled, randomized, controlled trial of patients with AAV with relapsing disease comparing the efficacy, after induction of remission with rituximab, of two relapse-prevention strategies: repeat dosing of rituximab or daily oral azathioprine. Method Patients with AAV were recruited at the time of relapse and received induction therapy with rituximab and glucocorticoids. If remission was achieved by month 4, patients were randomized in a 1:1 ratio to receive either rituximab (1000 mg every 4 months for 5 doses) or azathioprine (2 mg/kg/day) as maintenance therapy. Patients were followed for a minimum of 36 months, with the primary outcome being time to disease relapse. The formal hypothesis testing plan initially considers the hazard ratio for relapse across all time periods. If, and only if this global test is significant at a 5% level then the hazard ratios during the treatment period and the follow-up periods are considered separately. Results 190 patients were enrolled and 170 randomized at 4 months (85 to rituximab; 85 to azathioprine). The data are complete on all patients up to at least month 24. Median age was 59 years (range 19-89), with a prior disease duration of 5.3 years (0.4-38.5). 123/170 (72%) patients had a history of testing positive for anti-proteinase 3 ANCA; 47/170 (28%) for myeloperoxidase ANCA; 104/170 (61%) were enrolled having suffered a major relapse, and 48/170 (28%) received a pre-specified higher dose glucocorticoid induction regimen. Rituximab was superior to azathioprine in preventing disease relapse with a preliminary overall hazard ratio (HR) estimate of 0.36 (95% CI 0.23-0.57, p <0.001) and a during-treatment HR estimate of 0.30 (95% CI 0.15-0.60, p<0.001) (Figure 1). After adjustment, none of the randomization stratification covariates (ANCA type, glucocorticoid induction regimen, or relapse severity) had a significant differential effect on the primary outcome. By 24 months after entry, 20 months after randomization, 11/85 (13%) patients in the rituximab group had experienced a relapse compared to 32/85 (38%) patients in the azathioprine group. In the rituximab group 2/11 (18%) relapses were classified as major, compared to 12/32 (38%) in the azathioprine group. 19/85 (22%) patients in the rituximab group and 31/85 (36%) patients in the azathioprine group experienced at least one severe adverse event (SAE). 25/85 (29%) and 42/85 (49%) patients in the rituximab group developed hypogammaglobulinaemia (IgG <5g/l) and non-severe infections respectively, compared to 21/85 (25%) and 41/85 (48%) in the azathioprine group. Conclusion In the RITAZAREM trial, following induction of remission with rituximab, rituximab was superior to azathioprine for preventing disease relapse in patients with AAV with a prior history of relapse. There were no new major safety signals for use of these medications in this population.

Kynurenine Pathway of Tryptophan Metabolism: Regulatory and Functional Aspects

Regulatory and functional aspects of the kynurenine (K) pathway (KP) of tryptophan (Trp) degradation are reviewed. The KP accounts for ~95% of dietary Trp degradation, of which 90% is attributed to the hepatic KP. During immune activation, the minor extrahepatic KP plays a more active role. The KP is rate-limited by its first enzyme, Trp 2,3-dioxygenase (TDO), in liver and indoleamine 2,3-dioxygenase (IDO) elsewhere. TDO is regulated by glucocorticoid induction, substrate activation and stabilization by Trp, cofactor activation by heme, and end-product inhibition by reduced nicotinamide adenine dinucleotide (phosphate). IDO is regulated by IFN-γ and other cytokines and by nitric oxide. The KP disposes of excess Trp, controls hepatic heme synthesis and Trp availability for cerebral serotonin synthesis, and produces immunoregulatory and neuroactive metabolites, the B3 “vitamin” nicotinic acid, and oxidized nicotinamide adenine dinucleotide. Various KP enzymes are undermined in disease and are targeted for therapy of conditions ranging from immunological, neurological, and neurodegenerative conditions to cancer.