0888-8809/05/$15.00/0 Molecular Endocrinology 19(10):2610–2623 Printed in U.S.A. Copyright © 2005 by The Endocrine Society doi: 10.1210/me.2005-0047 Activin Regulates Luteinizing Hormone ␤-Subunit Gene Expression through Smad-Binding and Homeobox Elements Djurdjica Coss, Varykina G. Thackray, Chu-Xia Deng, and Pamela L. Mellon Departments of Reproductive Medicine and Neuroscience (D.C., V.G.T., P.L.M.), Center for Reproductive Science and Medicine, University of California, San Diego, La Jolla, California 92093- 0674; and Genetics of Development and Disease Branch (C.-X.D.), National Institute of Diabetes and Downloaded from https://academic.oup.com/mend/article/19/10/2610/2738010 by guest on 23 September 2021 Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 LH ␤-subunit (LH␤), which is essential for ovulation site found in this region of the promoter. Juxta- and reproductive fitness, is synthesized specifi- posed to the HB are three Smad-binding elements cally in pituitary gonadotropes. In this study, we (SBEs), which are essential for LH␤ induction. In- show that LH␤ gene expression is induced by ac- terestingly, two of the SBEs are also critical for tivin in mouse primary pituitary cells if the cells are basal expression of the LH␤ gene. We demonstrate treated within 24 h after dispersion in culture. Fur- that Smad proteins are necessary and sufficient for thermore, male mice deficient in Smad3, and there- activin induction of the LH␤ gene. Furthermore, fore in activin signaling, have lower expression of Smad proteins can bind one of the identified SBEs. both LH␤ and FSH␤ mRNAs compared with their In addition to binding this SBE, Smad proteins in- wild-type littermates. Using the L␤T2 immortalized teract with pituitary homeobox 1 (Ptx-1) and ortho- mouse gonadotrope cell line that endogenously denticle homeobox 1 (Otx-1), which can bind the expresses LH, we identify specific elements in the HB located close to the Smad-binding site. Thus, regulatory region of the rat LH␤ gene necessary for activin induction of LH␤ gene expression requires its induction by activin. Activin responsiveness is a combination of several transcription factors, conferred by a promoter-proximal region located both basal and activin induced, as well as cooper- ؊121/؊86 from the transcriptional start site. Max- ation between multiple DNA elements. (Molecular imal LH␤ induction by activin requires a homeobox Endocrinology 19: 2610–2623, 2005) (element (HB) and a 5؅-early growth response (Egr H IS ESSENTIAL for steroidogenesis and repro- initially described as functioning in gonadal feedback Lductive function in both males and females, be- on gonadotropin synthesis. Activin increases release cause a lack of this hormone leads to hypogonadism of FSH (6) from the pituitary and induces FSH␤ ex- and infertility in both sexes (1). LH synthesis is re- pression in gonadotrope cells (7), whereas inhibin an- stricted exclusively to the anterior pituitary gonado- tagonizes activin action. Follistatin, a potent activin- tropes (2). It is a heterodimeric glycoprotein, com- binding protein (8), can inhibit the biosynthesis and posed of an ␣-subunit, in common with FSH and TSH, secretion of FSH (9). Interestingly, activin, follistatin, and a unique ␤-subunit, which confers biological and inhibin are expressed in the mature pituitary go- specificity. Transcription of the ␤-subunit is the rate- nadotrope and can function in an autocrine manner limiting step for LH production (3, 4). LH ␤-subunit (10–12). Follistatin is also synthesized by folliculostel- (LH␤) gene transcription is induced by GnRH and re- late cells in the pituitary and regulates activin availabil- pressed by gonadal steroids (5). ity in a paracrine manner (13, 14). Activin and inhibin, members of the TGF␤ family of TGF␤ family members, including activin, activate growth factors, also play important roles in the mod- signaling molecules known as receptor-associated ulation of gonadotropins. These glycoproteins were Smads, which, in the case of activin, are Smad2 and/or Smad3 (15). Smad2 or Smad3 then associate First Published Online June 16, 2005 with a common Smad, Smad4 (DPC4). The activated Abbreviations: BSA, Bovine serum albumin; Egr, early growth response; GAPDH, glyceraldehyde-3-phosphate de- heteromeric Smad complex translocates into the nu- hydrogenase; GFP, green fluorescent protein; GST, glutathi- cleus, where it binds a Smad-binding element GTCTA- one-S-transferase; HB, homeobox element; LH␤,LH␤-sub- GAC, or either half of this palindrome, within DNA to unit; NP-40, Nonidet P-40; Otx, orthodenticle homeobox; regulate the expression of target genes (16). Ptx, pituitary homeobox; SBE, Smad-binding element; SF-1, As mentioned above, there is considerable evidence steroidogenic factor 1. that activin regulates FSH synthesis and secretion. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the However, the initial reports regarding the role of activin foremost professional society serving the endocrine in gonadotropes failed to detect an effect of activin on community. LH secretion (6, 17). In contrast, a number of subse- 2610 Coss et al. • Activin Regulation of LH␤ Mol Endocrinol, October 2005, 19(10):2610–2623 2611 quent studies suggest that activin can influence LH basal expression. In this region, we also identified synthesis, both in vivo and in cell culture. Stouffer et al. three Smad-binding elements (SBEs), and mutation of (18) have shown an acute and sustained increase in LH any one of these sites completely abolished activin secretion in response to activin in female rhesus mon- response. Interestingly, two of these SBEs are also keys. McLachlan et al. (19) reported that 2-d activin important for basal expression of LH␤. Finally, we infusion in adult male monkeys significantly increased demonstrate that overexpressed Smad proteins acti- both LH and FSH release in response to GnRH injec- vate LH␤, bind a SBE in gel-shift assay, and interact tion. Attardi and Miklos (20) demonstrated that treat- with orthodenticle homeobox (Otx) and pituitary ho- ment of primary rat pituitary cell cultures with activin meobox (Ptx) proteins, which can bind the HB. There- results in significant increases in both LH secretion fore, activin regulation of LH␤ gene expression, similar ␤ ␤ and protein content in the cells, as well as LH mRNA to LH regulation by GnRH, requires multiple, com- Downloaded from https://academic.oup.com/mend/article/19/10/2610/2738010 by guest on 23 September 2021 levels. More recently, this observation was confirmed posite DNA elements and involves a combinatorial in human fetal primary cell culture, where recombinant action of several DNA binding proteins. human activin caused a significant increase in LH se- cretion into the medium. In addition, inhibin decreased FSH and LH secretion, but the LH response to inhibin RESULTS was less prominent than that of FSH (21). Furthermore, our laboratory has previously shown that an LH␤ re- Activin Induces LH␤ in Primary Mouse Pituitary porter gene is induced by activin in transiently trans- Cell Culture fected L␤T2 cells (22), and, recently, this finding was extended in a study in which the endogenous LH␤ There is little doubt that activin is a major regulator of gene was induced by activin in this mature gonado- FSH synthesis; however, examination of the effects of trope cell model (23). Despite these reports, activin is activin on LH has resulted in conflicting reports. Some still generally regarded as a selective regulator of FSH. studies have shown that activin regulates LH␤ induction In this report, we used primary mouse pituitary cells in animals and cell culture, both in rat primary pituitary enzymatically dispersed in culture to address this dis- cells and a gonadotrope cell model derived from the crepancy in the literature by showing that the LH␤ mouse; however, others did not detect this response. In gene can be induced by activin treatment. Moreover, this study, we sought to provide a possible explanation using Smad3-deficient mice, we confirmed a role for for these differing results regarding activin regulation of activin in regulation of LH␤ expression in vivo, when LH synthesis as well as determine whether activin regu- we found that these mice have lower expression of lates LH␤ induction in mouse primary cells. Using mouse LH␤ mRNA, in addition to lower levels of FSH␤, com- cells instead of rat primary cells allowed us to directly pared with their wild-type littermates. We then sought correlate our results with experiments in genetically to identify the molecular mechanism of activin induc- modified mice and more detailed mechanistic studies tion of LH␤ gene expression using L␤T2 cells, the using the mouse-derived gonadotrope cell line, L␤T2. mature gonadotrope cell model derived from a trans- We enzymatically dispersed mouse pituitary cells and genic mouse pituitary tumor, which endogenously ex- treated them with activin or GnRH for 5 h, either1dor3d presses LH␤. We found that in L␤T2 cells, LH␤ re- after the dispersion. When cells were treated within 24 h sponds to activin in a time- and dose-dependent after dispersion, both GnRH and activin induced robust manner, and the response maps between Ϫ86 and expression of LH␤ compared with the vehicle-treated Ϫ121 bp from the transcriptional start site. This is an cells (Fig. 1). Therefore, activin, as well as GnRH, induces active region of the promoter that contains a ho- LH␤ gene expression. However, when cells were al- meobox element (HB) at Ϫ100 that is crucial for basal lowed to recover for 66 h before treatment with GnRH or and cell-specific expression of the LH␤ gene (24). In activin, GnRH treatment still caused a significant induc- close proximity, on either side of the HB, are tandem tion of LH␤, although to a lesser extent than at 24 h, steroidogenic factor 1 (SF-1) and early growth re- whereas activin induction failed to reach a significant sponse (Egr) sites (25).