Histol Histopathol (2006) 21: 167-178 Histology and http://www.hh.um.es Histopathology Cellular and Molecular Biology Identification of gene networks modulated by activin in LßT2 cells using DNA microarray analysis W. Mazhawidza1, S.J. Winters2, U.B. Kaiser3 and S.S. Kakar4 1Department of Biochemistry and Molecular Biology, 2Division of Endocrinology and Metabolism, 4James Graham Brown Cancer Center, University of Louisville, and 3Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, USA Summary. Activins, members of the TGFß family of Introduction proteins, are widely expressed in a variety of tissues. First identified based on their ability to regulate Activins and inhibins are structurally related biosynthesis and secretion of follicle-stimulating members of the transforming growth factor-ß (TGFß) hormone (FSH), activins have also been shown to family of growth factors, and were initially isolated modulate development, cell growth, apoptosis, and based on their activity in regulating follicle-stimulating inflammation. Despite their many known functions, the hormone (FSH) release from the anterior pituitary. precise mechanisms and downstream signaling pathways Activins stimulate whereas inhibins inhibit FSH by which activins mediate their diverse effects remain secretion (Woodruff and Mather, 1995; Woodruff, 1998). unknown. We have used a DNA microarray assay to Activins are secreted by the gonads and within the identify genes that are regulated by activin, alone or in pituitary gland, where they act as autocrine or paracrine combination with gonadotropin-releasing hormone regulators of the reproductive axis (Ying, 1988; Corrigan (GnRH), another major regulator of FSH, in a murine et al., 1991). In addition, activins are found to be gonadotrope-derived cell line (LßT2). We used mRNA expressed in many other tissues, including testis from these cells to screen Affymetrix Mu74av2 mouse (Vliegen et al., 1993), ovary (Gurusinghe et al., 1995), Gene Chip oligonucleotide microarrays, representing endometrium (Otani et al., 1998), prostate (Mellor et al., approximately 12,400 mouse genes. Treatment of LßT2 2000), breast (Di Loreto et al., 1999), adrenal gland cells with activin A, a gonadotropin-releasing hormone (Spencer et al., 1992), retinal epithelium (Coulombe and agonist (GnRHA) or activin A plus GnRHA resulted in Kos, 1997 ), placenta (Petraglia et al., 1992), bone alterations in levels of gene expression that ranged in marrow stroma (Yamashita et al., 1992), oocyte magnitude from 15 to 67-fold. Data analysis identified (Uchiyama et al., 1994) and pancreas (Ogawa et al., 268 transcripts that were up- or down-regulated by two- 1993). Activins are also expressed in several cancers, fold or more. Distinct sets of genes were affected by including ovarian prostate, testicular, breast, pituitary, treatment with activin, GnRHA and activin plus endometrial, placental and liver cancers (Risbridger et GnRHA, suggesting interactions between activin and al., 2001). In recent years, activins have been shown to GnRHA. Changes in expression of seven randomly regulate a number of additional biological functions, selected representative genes identified by the including erythroid differentiation, neural induction and microarray technique were confirmed by real-time nerve cell survival, Xenopus laevis embryonic quantitative PCR and semi-quantitative reverse mesoderm induction, bone growth promotion, transcription/PCR (RT/PCR). Modulation of expression somatostatin induction, cell proliferation, differentiation, of genes by activin suggests that activin may mediate its and apoptosis (Chen et al., 2002; Welt et al., 2002). effects through a variety of signaling pathways. The mature form of activin is a homodimer held together by a single disulfide bond. Five isoforms of the Key words: Activin, GnRH, FSH, LH, Microarray Abbreviations: GnRH, Gonadotropin releasing hromone; FSH, Follicle- stimulating hormone; and TGFß, Transforming gorowth factor, Aebp1, AE binding protein 1, WNT5A, WNT-1 related protein 5A, Ptrj, protein tyrosine-like phosphatase receptor j, SDF-1, stormal cell derived factor, Offprint requests to: Sham S. Kakar, Department of Medicine, 570 S Myh8, perinatal skeletal myosin heavy chain polypetide 8, Ephx2, Preston Street, Baxter Building, Room 204 C, University of Louisville, epoxide hydrolase 2, Tyrp1, tyosine-related protein, Erg1, early growth Louisville, KY 40202, USA. Email: [email protected] response1 and Egr2, early growth response 2. 168 Genes regulated by activin activin monomeric subunits have been identified to date activin-dependent, and that follistatin attenuates the in different species. These include: ßA, ßB, ßC, ßD, and GnRH stimulation of FSHß gene expression (Weiss et ßE (Woodruff, 1998; Welt et al., 2002). ßA and ßB being al., 1992; Meriggiola et al., 1994; Pernasetti et al., 2001). the most common and best-characterized). Much as We have therefore studied the effects of activin both activins share structural homology with TGFß, the alone and in combination with GnRH. activin receptor and signaling system similarly resemble, and share components with the TGFß receptor system Materials and methods (Nakao et al., 1997; Whitman, 1998; Yue and Mulder, 2000; Risbridger et al., 2001). Activation of the activin Cell culture, treatment of cells, and purification of mRNA signaling pathway is initiated by direct binding of activin to a transmembrane type II serine-threonine kinase LßT2 cells were obtained from Dr. Pamela Mellon receptor, ActRII or ActRIIB. Upon ligand binding, the (University of California, San Diego). Cells were plated type II receptor associates with and phosphorylates a in 15 cm culture flasks and maintained at 37°C in 5% type I receptor, ActRIB (ALK4), which in turn leads to CO2 in high glucose DMEM supplemented with 10% phosphorylation and hence activation of members of the fetal bovine serum (FBS) (Hyclone). For experiments, 4- SMAD transcription factor family. The activation of 5x106 cells were seeded in each flask. After 24 h, media SMADs, SMAD2 and SMAD3, leads to their association were replaced with fresh media containing human with the common mediator SMAD4, and translocation to recombinant activin A (30 ng/ml; R&D System, the nucleus. Once in the nucleus, the SMAD complex Minneapolis, MN). LßT2 cells were treated with activin participates in binding to specific DNA sequences, the A for 48 h because early studies revealed that the FSHß consensus element having been defined as 5'- mRNA expression by activin is delayed (Graham et al., GTCTAG[N]C-3' (SMAD binding element or SBE), to 1999) although more recent studies using PCR revealed regulate gene transcription (Welt et al., 2002). a much earlier response (Bernard, 2004). After 48 h of Several mechanisms exist to achieve diversity in treatment, media were again replaced with fresh media, responses to activin. Cellular specificity of the activin this time containing 0.1% FBS and activin A (30 ng/ml). signal is believed to be achieved in part by interaction of One hour later, 100 nM GnRH agonist, ([D-Ala6]GnRH, SMADs with cell-specific transcriptional coregulators at GnRHA), or vehicle was added for an additional 1 h. the level of DNA binding and transcriptional activation. Cells were treated with GnRHA for 1 h because SMADs have also been reported to interact with prolonged treatment down regulates GnRH receptor and additional DNA binding partners such as AP-1, ATF2, post receptor signaling (Junoy et al., 2002; Liu et al., and the vitamin D receptor, which in turn are activated 2003). The cells were then rinsed with PBS and lysed by other signaling pathways, providing a mechanism for with Trizol reagent (Invitrogen). Total RNA was integrating a variety of signals to regulate cellular prepared from the three samples in each treatment group, response. Extracellularly, binding of activin to its using manufacturer’s instructions. The quality of the receptor is inhibited by follistatin, an activin binding RNA from each sample was examined on a denaturing protein (Schneyer et al., 1994; Chapman et al., 2002), agarose gel. Equal amounts of total RNA from each and inhibin antagonizes the actions of activin by binding sample were used to purify poly (A)+ RNA using an to betaglycan on the cell surface, sequestering ActRII Oligotex mRNA purification kit (Qiagen, Valencia, CA). into nonfunctional complexes (Lewis et al., 2000; Chapman et al., 2002; Gray et al., 2002). In addition, Affymetrix genechip experiments there is accumulating evidence for a role of other intracellular signaling pathways, such as activation of Poly (A)+ RNA was processed according to the MAP kinase pathways, in mediating at least some of the Affymetrix Expression Analysis Technical Manual and actions of activin (Murase et al., 2001; Funaba et al., as described previously (Kakar et al., 2003). Briefly, 2 2002). The MAP kinase pathway, in turn, may modify µg of poly(A)+ RNA was used from each sample in each SMAD activity. The precise mechanisms by which experiment. cDNA was generated using SuperScript activin mediates its diverse biological effects remain Reverse Transcriptase (Invitrogen) and an oligo-dT incompletely understood. A greater and more primer linked to a T7 RNA polymerase-binding site sophisticated knowledge of the molecular pathways and sequence. The resulting cDNA was used to synthesize mediators of the actions of activins will lead to a better biotin-labeled cRNA via an in vitro transcription understanding of the diverse physiologic