Down Syndrome Candidate Region 1 Isoform 1 Mediates Angiogenesis through the Calcineurin-NFAT Pathway Liuliang Qin,1 Dezheng Zhao,2 Xin Liu,1 Janice A. Nagy,1 Mien Van Hoang,1 Lawrence F. Brown,1 Harold F. Dvorak,1 and Huiyan Zeng1 Departments of 1Pathology and 2Medicine, Gastroenterology Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts Abstract Introduction Down syndrome candidate region 1 (DSCR1) is one of Down syndrome candidate region 1 (DSCR1) is one of more more than 50 genes located in a region of chromosome than 50 genes present in that portion of chromosome 21 that is 21 that has been implicated in Down syndrome. DSCR1 duplicated in trisomy 21, the chromosomal abnormality can be expressed as four isoforms, one of which, responsible for Down syndrome (1-3). DSCR1 is identical to isoform 4 (DSCR1-4), has recently been found to be MCIP1 (modulatory calcineurin–interacting protein; ref. 4). It strongly induced by vascular endothelial growth is homologous to Adapt78, an oxidant stress-inducible gene in factor A (VEGF-A165) and to provide a negative feedback hamsters (5-7) and also to Rcn1/nebula in Drosophila (8). loop that inhibits VEGF-A165-induced endothelial cell Down syndrome is a major cause of mental retardation and is proliferation in vitro and angiogenesis in vivo. We report also associated with various cardiac and gastrointestinal here that another DSCR1 isoform, DSCR1-1L, is also anomalies, immune system defects, and Alzheimer’s disease. up-regulated by VEGF-A165 in cultured endothelial cells Of relevance to cancer, patients with Down syndrome have an and is strongly expressed in several types of pathologic increased risk of certain malignancies (leukemia, germ cell angiogenesis in vivo. In contrast to DSCR1-4, the tumors) but an extremely low incidence of many solid tumors, overexpression of DSCR1-1Linduced the proliferation particularly breast cancer (9, 10). On the other hand, clinically and activation of the transcription factor NFAT in aggressive ovarian carcinomas often have trisomy of chromo- cultured endothelial cells and promoted angiogenesis some 21, whereas certain other solid tumors have deletions of in Matrigel assays in vivo, even in the absence of this chromosome. Together, these and other data suggest that VEGF-A. Similarly, small interfering RNAs specific for genes such as DSCR1 that are duplicated in Down syndrome DSCR1-1Land DSCR1-4 had opposing inhibitory and may play important roles in tumorigenesis (11, 12). stimulatory effects, respectively, on these same Interest in DSCR1 has recently been sparked by several functions. DSCR1-4 is thought to inhibit angiogenesis by reports indicating that it is up-regulated in cultured vascular inactivating calcineurin, thereby preventing activation endothelial cells by vascular endothelial growth factor (VEGF- and nuclear translocation of NFAT, a key transcription A165), and furthermore, that it provides a negative feedback 165 factor. In contrast, DSCR1-1L, regulated by a different loop that inhibits VEGF-A -induced angiogenesis (13-16). promoter than DSCR1-4, activates NFAT and its However, these conclusions were based on the study of only proangiogenic activity is inhibited by cyclosporin, an one DSCR1 isoform, isoform 4 (DSCR1-4). The DSCR1 gene inhibitor of calcineurin. In sum, DSCR1-1L, unlike is comprised of seven exons, the first four of which can serve DSCR1-4, potently activates angiogenesis and could be as start sites that then combine with exons 5 to 7 to produce an attractive target for antiangiogenesis therapy. four different mRNA transcripts (2, 17). Exon 1 was originally (Mol Cancer Res 2006;4(11):811–20) thought to encode a 29–amino acid peptide (DSCR1-1; refs. 2, 17), but later studies by Genesca et al. (18) revealed a start site further upstream that encoded an 84–amino acid peptide (DSCR1-1L). Exon 2 is probably not translated into protein because it lacks a methionine start site. Exon 3 encodes only three amino acids (2, 17). Exon 4, under the control of a Received 5/8/06; revised 8/9/06; accepted 8/21/06. different promoter from that regulating isoforms 1 to 3 (2, 17), Grant support: NIH grant K01 CA098581 and ACS grant RSG-05-118-01-CSM encodes a 29–amino acid peptide that initiates a fourth (H. Zeng); K01 DK064920-01A1 (D. Zhao), and HL-59316 and P01 CA-92644 (H.F. Dvorak). DSCR1 isoform. Several DSCR1 isoforms have different The costs of publication of this article were defrayed in part by the payment of expression patterns and likely different functions and page charges. This article must therefore be hereby marked advertisement in regulatory mechanisms (2, 17). All four isoforms are accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Molecular Cancer expressed in heart and skeletal muscle (2, 17). Isoform 1 Research Online (http://mcr.aacrjournals.org/). has also been detected in brain, whereas isoform 4 has been L. Qin and D. Zhao contributed equally to this work. detected in placenta and kidney (2, 17). DSCR1-1L has been Requests for reprints: Huiyan Zeng, Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Avenue RN found to play a protective role against cell stress (5-7). In 270A, Boston, MA 02215. Phone: 617-667-2329; Fax: 617-667-3591. E-mail: addition to inhibiting angiogenesis (13-16), DSCR1-4 plays an [email protected]. Copyright D 2006 American Association for Cancer Research. inhibitory role in cardiac and skeletal muscle hypertrophy doi:10.1158/1541-7786.MCR-06-0126 (19-21). Mol Cancer Res 2006;4(11). November 2006 811 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. 812 Qin et al. The present study was undertaken to determine whether other semiquantitative RT-PCR (Fig. 2B). We found that both DSCR1 isoforms had roles in angiogenesis analogous to that of DSCR1-1L and DSCR1-4 RNAs were induced by Ad- DSCR1-4. We report here that DSCR1-1L, like DSCR1-4, is up- mVEGF-A164, peaking at 12 hours after ear injection (lane 1); regulated by VEGF-A165 in cultured endothelial cells Ad-LacZ did not stimulate expression of either isoform (lane 2). and also in several types of pathologic angiogenesis in vivo. DSCR1-1L and DSCR1-4 were also both up-regulated in the DSCR1-1L is also expressed in the microvasculature of at mesenteries of mice bearing MOT ascites tumors (lane 3). least some human ovarian cancers, but not in the tumor cells However, MOT cells themselves did not express detectable themselves nor in the microvessels of normal ovary. Of particular amounts of DSCR1-1L and only trace amounts of DSCR1-4 interest, the effects of DSCR1-1L in angiogenesis are antithetical (lane 4). to those of DSCR1-4. Using a novel modification of the standard Specific antibodies are available against human DSCR1 Matrigel assay, we found that overexpression of DSCR1-1L isoforms 1 and 4, and we used these to perform immunohis- promoted endothelial cell proliferation in vitro and angiogenesis tochemistry on five cases of human ovarian adenocarcinoma. in vivo, whereas a DSCR1-1L–specific small interfering Both DSCR1-1L and DSCR1-4 were selectively expressed in RNA (siRNA) had opposite effects. Furthermore, whereas the tumor vascular endothelium but not in the tumor cells nor DSCR1-4 binds to calcineurin and prevents it from activating in the vessels of adjacent normal ovarian tissue (Fig. 2C). the transcription factor NFAT, DSCR1-1L activates the calci- Together, these data indicate that both DSCR1-1L and DSCR1- neurin-NFAT pathway. Taken together, our data indicate that 4 are highly expressed in the newly formed vessels of several DSCR1 isoforms 1 and 4 have opposing stimulatory and types of pathologic angiogenesis in mice and in at least some inhibitory effects on VEGF-A164/5-induced angiogenesis. human ovarian cancers. Transfection of HUVEC with Full-Length DSCR1 Isoforms Results or with Isoform-Specific siRNAs Up-Regulation of DSCR1-1L In vitro and In vivo We used two approaches to investigate the effects of DSCR1 In agreement with recent reports (13-15), we found that isoforms on vascular endothelium. First, we used RT-PCR to 165 VEGF-A strongly up-regulated DSCR1 expression in human clone full-length DSCR1-1L, DSCR1-3, and DSCR1-4 cDNAs umbilical vein endothelial cells (HUVEC) as determined by from total RNA that we isolated from HUVEC that had been DNA microarrays and quantitative real-time reverse transcrip- treated for 1 hour with VEGF-A165. After confirming their tion-PCR (RT-PCR). Maximum DSCR1 mRNA expression identity by DNA sequencing, we fused the Flag sequence in- appeared 1 hour after serum-starved HUVEC were stimulated frame to the NH2 terminus of each and subcloned the products 165 with 10 ng/mL of VEGF-A (data not shown). We then set out into a retroviral expression vector that gave nearly 100% to determine whether DSCR1 isoforms 1 and 4 were regulated differentially by VEGF-A165 in vascular endothelium. Because isoform-specific internal probes could not be developed for quantitative real-time reverse transcription-PCR, we used semiquantitative RT-PCR to evaluate the expression of DSCR1 isoforms. Using NH2-terminal-specific primers (17), we found that VEGF-A165 up-regulated both DSCR1-1L and DSCR1-4 mRNAs in serum-starved HUVEC and human microdermal vascular endothelial cells (HMDVEC; Fig. 1A). Immunoblots revealed that DSCR1-1L protein was up-regulated in HUVEC in a time-dependent fashion by VEGF-A165, but to a lesser extent than DSCR1-4 (Fig. 1B). Similar results were obtained with HMDVEC cells (data not shown). To evaluate the role of DSCR1 in vascular endothelium in vivo, we made use of an established angiogenesis assay in which an adenoviral vector expressing mVEGF-A164 (mouse equivalent of human VEGF-A165) was injected into nude mouse ear skin; an adenoviral vector expressing LacZ, that did not induce angiogenesis, served as a control (22).