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Evidence for Orphan Nuclear Receptor TR4 in the Etiology of Cushing Disease

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Evidence for Orphan Nuclear Receptor TR4 in the Etiology of Cushing Disease Evidence for orphan nuclear receptor TR4 in the etiology of Cushing disease Li Dua,1, Marvin Bergsneidera, Leili Mirsadraeia,2, Steven H. Younga, Johan W. Jonkerb, Michael Downesc, William H. Yonga, Ronald M. Evansc,d,3, and Anthony P. Heaneya,3 aDepartments of Medicine and Neurosurgery, David Geffen School of Medicine, University of California, Los Angeles, CA 90095; bCenter for Liver, Digestive, and Metabolic Diseases, Department of Pediatrics, University Medical Center Groningen, University of Groningen, 9713 GZ, Groningen, The Netherlands; and cGene Expression Laboratory and dHoward Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, CA 92037 Contributed by Ronald M. Evans, April 9, 2013 (sent for review March 5, 2013) Cushing disease (CD) is a life-threatening disorder attributed to Here, we demonstrate that in the normal human pituitary excess pituitary tumor-derived adrenocorticotrophic hormone gland, TR4 is almost exclusively expressed in the cytosol of cor- (ACTH) and adrenal steroid secretion caused by pituitary tumors. ticotroph cells, and that TR4 expression is markedly increased in Whereas CD was first described in 1932, the underlying genetic corticotroph tumors from both humans and mice. We further basis driving tumor growth and ACTH secretion remains unsolved. demonstrate that TR4 is a potent regulator of POMC transcrip- Here, we show that testicular orphan nuclear receptor 4 (TR4, tion, ACTH secretion and corticotroph tumor growth in vitro and nuclear receptor subfamily 2, group C, member 2) is overex- in vivo thereby identifying TR4 as a potential unique therapeutic pressed in human corticotroph tumors as well as in human and target in Cushing disease. mouse corticotroph tumor cell lines. Forced overexpression of TR4 Results in both human and murine tumor cells increased proopiomelano- cortin transcription, ACTH secretion, cellular proliferation, and tumor ACTH-Secreting Corticotroph Tumors Exhibit Higher TR4 Expression. invasion rates in vitro. Conversely, knockdown of TR4 expression In normal human pituitary tissue, immunocytochemistry colocalized reversed all phenotypes. Mechanistically, we show that TR4 tran- TR4 and ACTH in the cytoplasm of corticotroph cells (autopsy- derived tissue, n = 5, Fig. 1 A–D). Minimal TR4 immunoreactivity scriptionally activates proopiomelanocortin through binding of was observed in other pituitary cell subtypes and no TR4 expression a direct repeat 1 response element in the promoter, and that this was observed in the nucleus of normal pituitary cells. is enhanced by MAPK-mediated TR4 phosphorylation. In vivo, In contrast, analysis of paraffin-embedded pituitary tumors TR4 overexpression promotes murine corticotroph tumor growth demonstrated abundant TR4 expression in corticotroph tumors as well as enhances ACTH and corticosterone production, whereas (++ to +++ expression in 10/12 tumors (84%), Table 1) which, TR4 knockdown decreases circulating ACTH and corticosterone unlike the normal pituitary corticotrophs, was clearly evident in levels in mice harboring ACTH-secreting tumors. Our findings the nucleus (Fig. 1E). The increased TR4 levels in tumors from directly link TR4 to the etiology of corticotroph tumors, hormone CD patients were confirmed by Western blot analyses (Fig. 1F). secretion, and cell growth as well as identify it as a potential tar- In comparison with corticotroph tumors, lower TR4 expression get in the treatment of CD. was noted in the nucleus of somatotroph (3/5 tumors, 60%), lactotroph (2/5 tumors, 40%), and clinically nonfunctioning pi- ituitary tumors are common, with a reported overall preva- tuitary adenomas (3/5 tumors, 60%, Table S1). Plence of 15% in the general population (1). Although almost always benign, these tumors cause significant morbidity and TR4 Overexpression Induces POMC and ACTH in Murine Corticotroph mortality through mass effects and/or excess pituitary hormone Tumor Cells. The marked increase in expression of TR4 in corti- secretion (2). Cushing disease (CD), due to a pituitary cortico- cotroph tumors compared with normal pituitary corticotrophs, troph tumor, results in excessive adrenocorticotrophic hormone combined with its redistribution from the cytosol to the nucleus (ACTH)-directed adrenal-derived steroid hypersecretion (3, 4). suggested an altered functional role for TR4 in tumor cells. As It results in various disabling symptoms including diabetes, hy- human pituitary tumors do not survive in long-term culture, we pertension, osteoporosis, obesity, and psychological disturbances used murine corticotroph AtT20 cells to examine the role of TR4 and has a 3.8-fold increased mortality. Surgical removal of cor- in corticotroph tumor function in vitro. Like human CD, these ticotroph adenomas is first-line therapy and although initial re- cells express POMC, secrete ACTH, and produce a Cushing mission rates approximate 80% in expert centers, the disease phenotype when inoculated s.c. in mice (16). Consistent with recurs in up to 25% of cases where careful long-term follow-up is previous findings, dexamethasone (Dex, 100 nM for 8 h) treatment used (5). Additional therapies that directly target corticotroph of murine pituitary corticotroph cell-line AtT20 cells transfected tumor growth and/or ACTH production are still needed. Im- with a POMC-luciferase reporter construct led to a 50% decrease CELL BIOLOGY proved understanding of the mechanisms regulating expression of in POMC luciferase (POMC-Luc) activity, whereas treatment the ACTH precursor polypeptide proopiomelanocortin (POMC) with corticotropin-releasing hormone (CRH, 100 nM for 8 h) may lead to unique therapies for Cushing disease. increased POMC-Luc approximately threefold (Fig. 2A). In this Testicular orphan receptor 4 (TR4, nuclear receptor subfamily 2, group C, member 2) belongs to the nuclear receptor superfamily and encodes a 67-kDa protein (6). TR4 acts as a homodimer or Author contributions: L.D. and A.P.H. designed research; L.D., M.B., L.M., S.H.Y., J.W.J., heterodimer with TR2 and is a master transcriptional regula- and W.H.Y. performed research; M.D. contributed new reagents/analytic tools; L.D., tor in various processes including spermatogenesis, lipoprotein M.D., and A.P.H. analyzed data; and L.D., J.W.J., M.D., R.M.E., and A.P.H. wrote regulation, and CNS development. TR4 binds to AGGTCA the paper. DNA sequence motifs in direct repeat (DR) orientation with The authors declare no conflict of interest. a variable number of spacer nucleotides to regulate target genes Freely available online through the PNAS open access option. such as CD36, phosphoenolpyruvate carboxykinase, apolipo- 1Present address: Beckman Research Institute, City of Hope, Duarte, CA 91010. protein E (ApoE), and beta-globin (7–14). TR4 expression has 2Present address: University of California, San Diego, La Jolla, CA 92103. been demonstrated in rat and mouse pituitary gland and a po- 3To whom correspondence may be addressed. E-mail: [email protected] or AHeaney@mednet. tential TR4-binding site has been identified in the POMC ucla.edu. promoter (15), leading us to examine potential actions of TR4 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. on POMC regulation. 1073/pnas.1306182110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1306182110 PNAS | May 21, 2013 | vol. 110 | no. 21 | 8555–8560 Downloaded by guest on September 26, 2021 A B C and stable TR4 knockdown. Transient and lentiviral-mediated stable TR4 knockdown resulted in ∼50% and ∼25% decreased corticotroph tumor proliferation, respectively, compared with siCtrl and nonsilencing shRNA transfectants (NS shRNA) (Fig. 3 A and B). Furthermore, transient and stable TR4 overexpression led to 1.5-fold and 1.7-fold increases in corticotroph tumor pro- liferation rates, respectively, compared with control transfectants D EFNF CD (Fig. 3 C and D). Importantly, we also found that murine corti- TR4 cotroph tumor cell invasion rates correlated with TR4 expression, actin with a 75% reduction and threefold increase in invasion rates observed after TR4 knockdown and overexpression, respectively *** (Fig. 3 E and F). 4 2 (~ fold) TR4 Knockdown Inhibits Corticotroph Tumor ACTH Secretion and Growth in Vivo. To examine the effects of TR4 expression on TR4 Expression NF CD pituitary corticotroph tumor growth in vivo, AtT20 cells stably Fig. 1. Human ACTH-secreting pituitary tumor tissue showed higher TR4 transfected with TR4 shRNA (shTR4-621) or nonsilencing expression compared with normal pituitary tissue. (A–C) Immunofluores- shRNA (NS shRNA) were inoculated s.c. into 6-wk-old male cence images of normal corticotroph cells using fluorescently tagged nude mice. Mice inoculated with AtT20 cells with reduced TR4 antibodies to ACTH (green, A)andTR4(red,B) colocalized TR4 and ACTH expression (shTR4-621) developed tumors later than mice in- exclusively in the cytoplasm (C). Magnification (200×). Immunocytochemistry oculated with control cells (Fig. 4A). At 3 wk postinoculation, and hematoxylin & eosin staining showing (D) cytoplasmic TR4 expression tumors were detected in both groups and a similar rate of tumor (brown, arrowed) in a representative autopsy-derived normal pituitary tissue growth was observed. Whereas lower tumor weights were ob- in contrast to strong nuclear TR4 expression in (E) human corticotroph tumor served in the mice inoculated with AtT20 cells with reduced TR4 fi from patients with CD. (F) Western blot and quanti cation for TR4 ex- expression (124.0 ± 29.3 vs. 94.5 ± 32.7 mg, n =
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