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Peroxisome Proliferator-Activated Receptor ; Is a Zac Target Gene Mediating Zac Antiproliferation

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Peroxisome Proliferator-Activated Receptor ; Is a Zac Target Gene Mediating Zac Antiproliferation Research Article Peroxisome Proliferator-Activated Receptor ; Is a Zac Target Gene Mediating Zac Antiproliferation Thomas Barz, Anke Hoffmann, Markus Panhuysen, and Dietmar Spengler Molecular Neuroendocrinology, Max-Planck-Institute of Psychiatry, Munich, Germany Abstract Zac also potently coactivates or corepresses the hormone- dependent activity of nuclear receptors, including androgen, Zac is a C2H2 zinc finger protein, which regulates apoptosis estrogen, glucocorticoid, and thyroid hormone receptors (13); all and cell cycle arrest through DNA binding and transactivation. of these are key regulators of development, homeostasis, differen- During tumorigenesis and in response to mitogenic activation, tiation, and cell growth. Recent data, showing tightly controlled Zac gene expression is down-regulated in a methylation- spatio-temporal Zac expression during embryogenesis in mesen- sensitive manner. As yet, no target genes have been identified chymal and neural stem/progenitor cells, have suggested additional that could explain the potent antiproliferative function of Zac. roles related to differentiation and development (14, 15). Consis- Here, applying genome-wide expression analysis, we identify tent with this concept, other studies have disclosed that the Zac peroxisome proliferator-activated receptor g (PPARg) as a new gene is maternally imprinted (16, 17) and that defects of its bona fide Zac target gene, which is induced by direct Zac imprinting status underlie the etiology of transient neonatal binding to the proximal PPAR;1 promoter. We show that in diabetes mellitus (TNDM), an uncommon form of childhood human colon carcinoma cells, ZAC activates expression of diabetes (OMIM *601410), which probably results from a delayed PPAR; target genes in a PPAR;-dependent manner. More- maturation of pancreatic h-cells (18, 19). over, we show that treatment of pituitary tumor cells with Our earlier work revealed that Zac can act as a transcription octreotide, a somatostatin analogue, leads to Zac induction factor through its monomeric or dimeric binding to either a GC-rich and subsequent Zac-dependent up-regulation of PPAR;, palindromic DNA element or to GC-rich direct and reverse repeat which thereupon mediates part of the antiproliferative activity elements, respectively (2, 20, 21). Zac confers transactivation in a of Zac. Our work provides a first step toward elucidating a strictly HAT-dependent manner via recruitment of p300 and the functional relationship between Zac and PPAR; that could be coordinated regulation of p300’s substrate affinities and catalytic relevant to the understanding of tumorigenesis and diabetes activity by zinc fingers 6and 7 and its COOH terminus (22). In this as well. (Cancer Res 2006; 66(24): 11975-82) way, Zac DNA binding is directly coupled to p300-HAT regulation, indicating that modification of the chromatin status may play an Introduction important role in target gene recognition and activation. However, it Zac is a zinc finger protein, which potently induces apoptosis is far from clear how Zac exerts its antiproliferative functions and cell cycle arrest and prevents tumor formation in nude mice because only a few potential Zac target genes involved in (1, 2). Expression of Lot1, the rat orthologue of Zac, is lost during differentiation (PAC1 and KRT14) have been proposed until now spontaneous transformation of ovary surface epithelial cells in vitro (21, 23). Therefore, a genome-wide screen for Zac target genes (3), whereas human ZAC, which is widely expressed in normal should reveal the transcriptional activities responsible for biological tissues, is frequently down-regulated in a methylation-sensitive effects of Zac and the gene networks addressed. manner in various tumors (3–7). Additionally, Zac/Lot1 expression The transcription factor peroxisome proliferator-activated is repressed by epidermal growth factor and Ras/jun oncogenes receptor g (PPARg) is a member of the nuclear hormone receptor (8, 9), suggesting that it might be integrated in a negative feedback family. Besides its key role in adipogenesis, PPARg has increasingly loop controlling cell proliferation in response to mitogen been recognized to participate in lipid metabolism, glucose activation. Consistent with the antiproliferative role of Zac, its homeostasis, inflammatory responses, differentiation, anti- down-regulation by small interfering RNA (siRNA) enhances proliferation, and apoptosis (24), raising intense clinical interest pituitary tumor cell proliferation (10). Conversely, octreotide, a on regulation of its expression and pharmacologic control of its somatostatin analogue, inhibits tumor cell growth by inducing Zac activity. PPARg exists as two protein isoforms, expressed from expression via activated glycogen synthase kinase-3h (GSK3h) and different promoters and alternatively spliced at the 5¶-end of the possibly through p53 (10, 11). In addition, Zac coactivates p53 (12), gene, resulting in 30 additional amino acids at the NH2 terminus of for example, to enhance transactivation of the proapoptotic gene PPARg2 compared with PPARg1 (25, 26). Whereas expression of Apaf-1 (11). These findings suggest that Zac and p53 cooperate at PPARg2 is mainly restricted to adipose tissue, PPARg1 has also different levels in antiproliferation. been detected in other tissues, including heart, skeletal muscle, liver, colon, kidney, spleen, pancreas, pituitary, and brain. Studies on regulation of PPARg expression have been focused primarily on the PPARg2 promoter in adipocyte precursor cells in terms of adipogenesis, whereas control of PPARg1 transcription is still Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). poorly understood. Requests for reprints: Dietmar Spengler, Molecular Neuroendocrinology, Max- Here, using genome-wide expression analysis, we identify PPARc Planck-Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany. Phone: as a new bona fide Zac target gene that mediates antiprolifera- 49-89-30622-559; Fax: 49-89-30622-605; E-mail: [email protected]. I2006American Association for Cancer Research. tion in cancer cells. This functional link between Zac and PPARg doi:10.1158/0008-5472.CAN-06-1529 may also apply to other common diseases, in particular diabetes. www.aacrjournals.org 11975 Cancer Res 2006; 66: (24). December 15, 2006 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Cancer Research Materials and Methods Cell culture and transfection. Cells were cultivated in DMEM with 10% FCS and penicillin/streptidin. Transfections were carried out by electro- poration or Metafectene (Biontex, Munich, Germany). Inducible Zac clones of the hippocampal cell line HW3-5 (27) were generated as described previously (1) using the pCMVtetr vector (28). Proliferation rate was measured using a Coulter Counter (Beckman, Krefeld, Germany) following seeding of 2 Â 103 cells in 24-well plates and cultivation in the absence or presence of tetracycline (100 ng/mL) for 10 days. 3-(4,5-Dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assays were done as described (1). For transfection assays, luciferase values were normalized on h- galactosidase activity by cotransfecting a pRK7-h-Gal vector. All chemicals were purchased from Sigma (Taufkirchen, Germany). PSG5mPPARg1 plasmid was generously provided by L. Michalik (University of Lausanne, Lausanne, Switzerland). See ref. 21 for Zac expression constructs. RNA, Northern blot, microarray analysis, and reverse transcrip- tion-PCR. RNA was isolated using Trizol (Invitrogen, Karlsruhe, Germany). For Northern blot, 20 Ag total RNA was separated by denaturating gel electrophoresis, blotted overnight, blocked, and hybridized in Rapid-hyb buffer (Amersham, Piscataway, NJ) to P32- random-primed cDNA. Blot was exposed to phosphoimage plate overnight and plate was scanned using a BAS reader (Fuji, Gru¨nwald, Germany). For microarray analysis, 40 AgtotalRNApersamplewasdye coupled using indirect labeling. To exclude dye bias, one half of each Figure 1. Characterization of inducible Zac clones. A, Northern blot and sample was coupled to Cy3 and to Cy5, respectively. The samples were RT-PCR showing endogenous Zac expression in HW3-5cells. The corticotroph pituitary cell line AtT-20 and the neuroblastoma cell line Neuro-2A (N-2A) hybridized on four 24k mouse cDNA arrays (Max-Planck-Institute of served as positive and negative controls, respectively. BglII digestion confirmed Psychiatry,Munich,Germany;ref.29)foreachdyecouplingcombination specificity of the HW3-5PCR product. M, size marker. B, immunoblot and scanned on a Perkin-Elmer Life Sciences (Rodgau-Ju¨gesheim, [40 Ag whole-cell extract (WCE)] showing increased Zac expression in inducible Germany) ScanArray 4000 laser scanner. Reverse transcription was HW3-5Zac clones 24 hours after tetracycline ( Tc) removal. C, induced Zac expression in HW3-5Zac clones inhibits proliferation. Cells were cultivated in the carried out with Omniscript reverse transcription kit (Qiagen, Hilden, presence or absence of tetracycline and cell numbers were measured daily with Germany). One microliter of reverse transcription reaction was used as a cell counter. Medium was renewed every 3rd day. Points, mean of three template for PCRs of 30 cycles using Taq DNA Polymerase (Fermentas, independent experiments for each clone; bars, SD. D, immunoblot (40 Ag WCE) St. Leon-Rot, Germany). Quantitative PCR was done
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