Down Syndrome Candidate Region 1 Isoform 1 Mediates Angiogenesis Through the Calcineurin-NFAT Pathway

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).

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). Ears were harvested at different stages of the angiogenic response and prepared for immunoblot assays. Using an antibody that recognized all mouse DSCR1 isoforms, we found that DSCR1 protein was FIGURE 1. Regulation of DSCR1 isoform expression in vitro. A. highly up-regulated as angiogenesis developed, reaching a Up-regulation of DSCR1 isoforms 1L and 4 in HUVEC and HMDVEC peak on day 3 (Fig. 2A). DSCR1 expression was not stimulated stimulated with VEGF-A165 as determined by semiquantitative RT-PCR by Ad-LacZ. DSCR1 protein was also up-regulated in the using isoform-specific primers. In the case of DSCR1-4, two bands are apparent; only the lower of these (arrow) is authentic, as was proved by angiogenic response that was induced in the mesenteries of nude DNA sequencing; the upper band reflects a lack of PCR primer specificity. mice bearing ascites mouse ovarian tumor (MOT; Fig. 2A). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as an internal loading control. B. Immunoblot of DSCR1 isoforms 1L and 4 in We next determined which DSCR1 isoforms were up- HUVEC that were serum-starved (0.1% fetal bovine serum) for 24 hours 164 regulated in Ad-mVEGF-A –induced angiogenesis, using and stimulated with 10 ng/mL of VEGF-A165 for 1, 2, 4, and 6 hours.

FIGURE 2. Regulation of DSCR1 and DSCR1 isoform expression in vivo. A. Immunoblot with a mDSCR1 antibody demonstrating time-dependent up-regulation of DSCR1 protein in mouse ears injected with 108 PFU of Ad-mVEGF-A164 and in mouse mesenteries at various times after i.p. injection of 106 MOT ascites tumor cells. A control adenovirus expressing LacZ did not induce DSCR1 protein expression; blots were stripped and probed with an anti – mitogen-activated protein kinase (MAPK) antibody to confirm equal protein loading (bottom). B. Time-dependent up- regulation of mRNAs of DSCR1 isoforms 1L and 4 in mouse tissues as determined by RT-PCR. Lanes 1 and 2, mouse ears following injection of 108 PFU of Ad-mVEGF-A164 or Ad-LacZ. Lane 3, mesenteries from mice injected i.p. with 106 MOT ascites tumor cells. No DSCR1 isoform 1L and only trace amounts of DSCR1-4 were detected in MOT tumor cells (lane 4). Glyceraldehyde-3- phosphate dehydrogenase served as an internal control for equal RNA loading. All experiments were repeated thrice. C. Immunohistochemical staining of normal human ovarian tissue (top) and a human ovarian adenocarcinoma (bottom) with antibodies specific for human CD31 (a and b), DSCR1-1L (c and d), and DSCR1-4 (e and f). Vessels (arrows) in normal and tumor tissue were CD31-positive, but only those in tumors stained with antibodies to DSCR1-1L and DSCR1-4. Tissue from one of five different patients, all of which exhibited similar staining. infection yields in HUVEC (23). Cell extracts from HUVEC thymidine incorporation was unaffected in FD4-transduced cells transduced with Flag-fused versions of DSCR1-1L (FD1L), (lane 7 versus lanes 1 or 3, P > 0.05) but the response to VEGF- DSCR1-3 (FD3), DSCR1-4 (FD4), and LacZ (FLacZ), were A165 was strikingly inhibited (lane 8 versus lanes 2 or 4, immunoprecipitated with an antibody against Flag and immu- P < 0.001). noblotted with an antibody reactive against all human DSCR1 Transduction with the DSCR1-1L-specific siRNA D1Si did isoforms. Figure 3A shows that FD1L, FD3, FD4, and Lac Z not affect baseline thymidine incorporation (lane 11 versus lanes proteins were all expressed in HUVEC. 1 or 3, P > 0.05) but significantly inhibited VEGF-A165-induced As a second approach, we designed siRNAs that specifically stimulation (lane 12 versus lanes 2 or 4, P < 0.001). targeted the NH2-terminal sequence of DSCR1-1L (D1Si) and On the other hand, transduction with the DSCR1-4-specific DSCR1-4 (D4Si), as well as a control siRNA (Neg-Si, a siRNA D4Si significantly enhanced thymidine incorporation scrambled version of the D4Si sequence). Cell extracts from both in the absence and presence of VEGF-A165 (lane 13 versus HUVEC that had been infected with retroviruses expressing lanes 1 or 3; lane 14 versus lanes 2 or 4, both P < 0.001). Control these siRNAs were immunoblotted with antibodies specific for Neg-Si had no effect on thymidine incorporation in HUVEC with DSCR1-1L and DSCR1-4. D1Si and D4Si strongly and or without VEGF-A165 stimulation (lane 15 versus lanes 1 or 3; specifically inhibited the expression of DSCR1-1 and DSCR1-4, lane 16 versus lanes 2 or 4, both P > 0.05). Transduction respectively, whereas Neg-Si had no effect on either (Fig. 3B). with FD3 also had no effect on thymidine incorporation without VEGF-A165 (lane 9 versus lanes 1 or 3, P > 0.05) or Effects of DSCR1 Isoforms and Isoform-Specific siRNAs following its addition (lane 10 versus lanes 2 or 4, P > 0.05). on HUVEC Proliferation We next tested whether DSCR1 isoform-specific siRNAs As expected, VEGF-A165 stimulated (24) thymidine incorpo- had effects on KDR (VEGF receptor 2) expression or on ration in control HUVEC and in HUVEC transduced with LacZ VEGF-A165-induced KDR phosphorylation. Serum-starved (Fig. 3C, lane 2 versus lane 1 and lane 4 versus lane 3, both HUVEC that had been transduced with D1Si, D4Si, or with P < 0.001). HUVEC transduced with FD1L showed significantly control Neg-Si were stimulated with 10 ng/mL of VEGF-A165 increased thymidine incorporation in the absence of for 2 minutes. Cell extracts were immunoprecipitated with an VEGF-A165 (lane 5 versus lane 3, P < 0.001), and further antibody against KDR and immunoblotted with an antibody enhanced thymidine incorporation following VEGF-A165 stim- against phosphorylated tyrosine (PY20). Neither D1Si nor D4Si ulation (lane 6 versus lanes 5 and 4, P < 0.01). Baseline had any effect on VEGF-A165-stimulated KDR phosphorylation

cells transfected to overexpress VEGF-A165 (SK-MEL/VEGF cells; ref. 27) were mixed with PT67 cells packaging retroviruses that expressed full-length DSCR1 isomer cDNAs or their respective siRNAs. The cell mixtures were incorporated into Matrigels that were implanted in the s.c. space of nude mice. As previously reported (25-27), VEGF-A165 secreted by SK- MEL/VEGF cells induces nearby vascular endothelial cells to divide and therefore to become susceptible to infection with retroviruses secreted by PT67 packaging cells. The angiogenic response that developed after the implantation of Matrigel plugs containing various cell mixtures was evaluated on day 3 (Fig. 4A). Angiogenesis was assessed by macroscopy (top) and by histology and immunohistochemistry for the endothelial cell marker CD31 (bottom). Matrigel plugs containing only PT67 cells packaging LacZ-expressing retroviruses (PT67/LacZ cells) induced minimal angiogenesis (lane 1). However, strong angiogenesis with typical ‘‘mother’’ vessels was induced in plugs containing SK-MEL/VEGF cells, whether alone or combined with PT67/LacZ cells (lanes 2 and 3). Mother vessels are enlarged, thin-walled, pericyte-poor vessels that are the first new vessel type induced by VEGF-A164 in vivo (22). The angiogenic response and mother vessel formation were strikingly depressed by the inclusion of PT67/ D1Si cells (lane 4) but were not affected by the inclusion of either PT67/D4Si or PT67/Neg-Si cells (lanes 5 and 6). However, the inclusion of PT67/FD4 cells completely inhibited VEGF-A- induced angiogenesis and mother vessel formation (lane 7), whereas overexpression of FD1L was associated with strong FIGURE 3. Roles of DSCR1-1L and DSCR1-4 in HUVEC proliferation. angiogenesis (lane 8). The inclusion of PT67/FD3 cells had no A. Cell extracts from HUVEC transduced with Flag-DSCR1-4 (FD4 cells), Flag-DSCR1-1L (FD1L cells), Flag DSCR1-3 (FD3 cells), and Flag-LacZ effect on the angiogenic response induced by SKMEL/VEGF (FLacZ cells) were immunoprecipitated with an antibody against Flag and cells (lane 9). Immunostaining with antibody against DSCR1 the precipitates immunoblotted with an antibody reactive with all human indicated that DSCR1 was induced in the vessel structure DSCR1 isoforms (top). Expression of mitogen-activated protein kinase a, arrows (MAPK) indicates equal protein loading (bottom). B. Cell extracts from (Supplementary Fig. S2, ), which was almost completely HUVEC transduced with isoform-specific siRNAs or with a scrambled inhibited by D1Si or D4Si (Supplementary Fig. S2, b and c, DSCR1-4 siRNA (Neg-Si) were immunoblotted with antibodies specific for arrows), but was not affected by SiNeg (Supplementary Fig. S2, hDSCR1-1L and hDSCR1-4. D1Si specifically knocked down the expression of DSCR1-1L but had no effect on DSCR1-4. D4Si specifically d, arrow). In situ hybridization also confirmed the knockdown knocked down the expression of DSCR1-4 but had no effect on DSCR1- of DSCR1 expression by D1Si and D4Si (data not shown). 1L. Neg-Si had no effect on either DSCR1-1L or DSCR1-4 expression. C. in situ HUVEC were transduced with retroviruses as indicated, then serum- Furthermore, hybridization indicated that the expression 165 starved and cultured with or without VEGF-A165 for 24 hours before levels of VEGF-A were similar in Matrigels containing 3 assessing H-thymidine incorporation as a measure of cell proliferation these different cell mixtures (see ref. 25); also, transfection of (see Materials and Methods). See text for statistical comparisons (n = 4). SKMEL and PT67 cells with FD1L, FD4, and etc., has no effect on these cells’ proliferation. Therefore, the results obtained top (Supplementary Fig. S1, ). The blot was then stripped and cannot be attributed to the effects of VEGF-A expression. reprobed with an antibody against KDR. The expression of We next tested whether the overexpression of DSCR1-1L KDR was also not affected by D1Si or D4Si transduction could induce angiogenesis in the absence of VEGF-A165 bottom (Supplementary Fig. S1, ). These data indicate that, (Fig. 4B). Matrigels containing only PT67/FD1L cells were 165 as anticipated, the effects of D1Si and D4Si on VEGF-A - able to induce angiogenesis and mother vessel formation stimulated HUVEC proliferation are not due to the inhibition of (lane 2) to an extent similar to that induced by VEGF-A165- KDR expression or phosphorylation. expressing cells (lane 1). However, Matrigels containing only PT67/FD3 did not induce angiogenesis (lane 3). When Effects of DSCR1 Isoforms on VEGF-A-Induced DSCR1-4 expressing FD4 cells were included in Matrigels Angiogenesis In vivo along with DSCR1-1L expressing FD1L cells in the absence of In order to elucidate the mechanisms of angiogenesis, it SKMEL/VEGF cells, the angiogenic response expected from would be desirable to modulate the expression of individual FD1L cells was abrogated (lane 4). Immunostaining and in situ vascular genes in vivo using the gene overexpression and hybridization confirmed the similar expression of DSCR1-1, silencing approaches that have proved to be so powerful DSCR1-3, and DSCR1-4 with an antibody against Flag in vitro. Recently, a novel system has been developed that (Supplementary Fig. S3; data not shown). allowed us to introduce DSCR1 isoform–specific cDNAs or To confirm that the angiogenesis induced by PT67/FD1L siRNAs into endothelial cells in vivo (25-27). SK-MEL-2 tumor was not due to the induction of VEGF-A expression, we made

use of SU1498, an inhibitor of VEGF receptor 2/KDR kinase dye was injected i.v. into mice 3 days after implanting Matrigel activity (KDR is the VEGF-A receptor responsible for plugs containing various cell mixtures. Evan’s blue dye binds to mediating the angiogenic response; ref. 28). When incorporated plasma proteins, and therefore, the amount of plasma within the into Matrigel plugs and administered systemically, SU1498 Matrigel-associated vasculature can be calculated from simul- strongly inhibited angiogenesis in Matrigel plugs containing taneous measurements of dye concentration in peripheral blood SKMEL/VEGF cells, but had no inhibitory effect on the plasma. Matrigel plugs were harvested 5 minutes after i.v. dye angiogenesis induced by PT67 cells expressing FD1L (Supple- injection, when blood vessels were filled with dye-plasma mentary Fig. S4). Taken together, these data provide strong protein complexes, but before there was time for significant evidence that DSCR1-1 can induce angiogenesis and mother extravasation. vessel formation even in the absence of exogenous VEGF-A165 In Matrigel plugs containing VEGF-A165-expressing and that DSCR1-4 inhibits that response. SKMEL/VEGF cells (alone or with PT67/LacZ cells), intravascular plasma volume as measured by Evan’s blue dye Quantification of the Effects of DSCR1 Isoforms and accumulation increased >2-fold above baseline levels (Fig. 4C, siRNAs on Angiogenesis lanes 2 or 3 versus lane 1, P < 0.001). The presence of We next used the intravascular plasma volume of Matrigel PT67/D1Si cells expressing DSCR1-1 siRNA strikingly plug–associated blood vessels as a novel measure to quantitate blocked the angiogenic response expected from SKMEL/VEGF the angiogenic response induced by VEGF-A165 and DSCR1 cells (Fig. 4C, lane 4 versus lanes 2 and 3, P < 0.001). isoforms (25). Intravascular plasma volume is an appropriate However, cells expressing DSCR1-4 siRNA (D4Si) or control measure as enlarged mother vessels are a signature property of siRNA (Neg-Si) had no effect on the angiogenic response the early angiogenic response to VEGF-A (22, 29). Evan’s blue induced by SKMEL/VEGF cells (Fig. 4C, lanes 5 and 6 versus

FIGURE 4. DSCR1-1 stimulates, whereas DSCR1-4 inhibits, VEGF-A165-induced angiogenesis in the in vivo Matrigel assay. A and B. Macroscopic (top) and CD31-stained microscopic (bottom) images of the angiogenic response induced in Matrigels 3 days after implantation with indicated contents of VEGF- A165-secreting SKMEL/VEGF cells and PT67 cells generating LacZ, D1Si, D4Si, Neg-Si, FD1L, FD3, and FD4 retroviruses; all experiments were repeated thrice. C. Intravascular plasma volumes (AL/g) in Matrigels from (A and B) as a quantitative measure of the angiogenic response. Intravascular plasma volumes were determined by the accumulation of Evan’s blue dye administered i.v. 5 minutes prior to euthanasia (n = 4). D. Effect of cyclosporin A, a calcineurin inhibitor, on DSCR1-1L-induced angiogenesis in the Matrigel assay. Left, typical macroscopic and microscopic data as in (A) and Supplementary Fig. S2. Right, intravascular plasma volumes measured by the Evan’s blue dye assay as in (B; n = 4). All experiments were repeated twice.

lanes 2 or 3, P > 0.05). The inclusion of PT67/FD1L cells VEGF-A165-stimulated NFATc1 activity (lane 10 versus lane 2, expressing DSCR1-1L or PT67/FD3 cells expressing DSCR1-3 P < 0.001), without affecting baseline activity (lane 9 versus lane also had no effect (Fig. 4C, lanes 7 and 8 versus lanes 2 or 3, 1, P > 0.05). D4Si, the DSCR1-4-specific siRNA, had an P > 0.05). However, when PT67/FD4 cells expressing opposite effect, up-regulating NFATc1 activity both in the DSCR1-4 were incorporated in Matrigel plugs, the increased presence and absence of VEGF-A165 (lanes 11 and 12 versus dye accumulation expected from SKMEL/VEGF cells was lanes 1 and 2, respectively, although the effects were not strongly inhibited (Fig. 4C, lane 9 versus lanes 2 or 3, statistically significant, P > 0.05). Control siRNA (Neg-Si) had P < 0.001). In the absence of SKMEL/VEGF cells, dye no effect on NFATc1 activity in the presence or absence of accumulation in Matrigel plugs containing PT67/FD1L cells VEGF-A165 (lane 13 versus lane 1 and lane 14 versus lane 2, was similar to that of Matrigels containing SKMEL/VEGF cells both P > 0.05). We further tested whether the overexpression of (Fig. 4C, lane 10 versus lane 2, P > 0.05). However, dye DSCR1-1L induced DSCR1-4 expression. Cellular extracts from accumulation in Matrigels containing PT67/FD3 or PT67/FD4 cells transduced with FD4, FLacZ, and FD1L were immuno- cells was similar to that of Matrigels containing PT67/LacZ blotted with an antibody against DSCR1. Figure 5B clearly cells (Fig. 4C, lanes 11 or 12, versus lane 1, P > 0.05). indicated that overexpression of DSCR1-1L induced the The quantitative measurements of vascular plasma volumes expression of DSCR1-4 (Fig. 5B). presented in Fig. 4C therefore confirm the qualitative measures of angiogenesis presented in Fig. 4A and B. Discussion Recent studies from several laboratories have shown that VEGF-A165 up-regulates DSCR1-4, one of four proposed Effects of DSCR1-1L and DSCR1-4 on the Calcineurin- DSCR1 isoforms, in vascular endothelial cells. DSCR1-4 then NFAT Pathway triggers a negative feedback loop that quenches VEGF-A165- The COOH-terminal domain of DSCR1 has been shown to induced endothelial cell proliferation and tube formation in vitro bind to calcineurin and to inhibit its activity, preventing the and angiogenesis in vivo (13-16). We have confirmed and activation (dephosphorylation) of NFAT and its translocation to extended these findings but have also shown for the first time the nucleus (13-16, 30). However, a recent report has shown that another DSCR1 isoform, DSCR1-1L, plays an important that phosphorylation of RCN1, the Drosophila orthologue of proangiogenic role that is opposite to and opposes that MCIP1/DSCR1, has an opposite effect, enhancing calcineurin of DSCR1-4. On the other hand, expression of a third DSCR1 activity (31). These results suggested to us that different isomer, DSCR1-3, apparently does not have a significant role in DSCR1 isoforms might be responsible for these opposite effects VEGF-A165-mediated angiogenesis (Figs. 3C, 4, and 5). of DSCR1 on calcineurin binding and activation. To test this VEGF-A165 up-regulated DSCR1-1L in HUVEC at both the possibility, we investigated the effects of DSCR1-1L on the mRNA and protein levels, although to a lesser extent than calcineurin pathway. We included cyclosporin A, an inhibitor of DSCR1-4 (Fig. 1A and B). In addition, both DSCR1-1L and calcineurin, in Matrigel plugs containing FD1L cells that DSCR1-4 were up-regulated in the angiogenic responses expressed DSCR1-1L; cyclosporin A was also injected i.p. induced in vivo by a VEGF-A164-expressing adenovirus and daily. As shown in Fig. 4D, cyclosporin A strongly inhibited by VEGF-A164-expressing MOT tumors (Fig. 2B). Of impor- DSCR1-1L-induced angiogenesis, suggesting that DSCR1-1L tance, the MOT tumor cells did not themselves express regulated angiogenesis through calcineurin activation. DSCR1-1L detectably, and expressed only trace amounts of We next tested the effects of DSCR1 isoforms and their DSCR1-4. Because antibodies reactive with mouse DSCR1 in specific siRNAs on NFATc1-dependent reporter expression. tissue sections are not available, we could not further localize NFATc1 is the only NFAT protein that is activated by VEGF in DSCR1 in these models. However, both DSCR1-1L and endothelial cells (32). HUVEC transduced with DSCR1 isoform DSCR1-4 were selectively localized to the microvessels cDNAs or their isoform-specific siRNAs were transfected with a supplying human ovarian cancers (Fig. 2C). NFATc1-targeted promoter-luciferase plasmid and an internal Of considerable interest, DSCR1-1L and DSCR1-4 were control luciferase plasmid, and then stimulated with VEGF-A165 found to have antithetical effects on the angiogenic response, for 6 hours. Luciferase activity was measured and normalized to both in vitro and in vivo. Consistent with earlier reports the control luciferase activity for equal transfection efficiency. As (13-16), overexpression of DSCR1-4 strikingly inhibited shown in Fig. 5A, VEGF-A165 stimulated a f2-fold increase in VEGF-A165-mediated 3H-thymidine incorporation in cultured NFATc1 activity in HUVEC transduced with LacZ (lane 2 versus HUVEC, whereas a DSCR1-4-specific siRNA (D4Si) stimu- lane 1, P < 0.001). In HUVEC transduced with DSCR1-1L, lated such incorporation in both the presence and absence of NFATactivity was strongly up-regulated both in the absence and added VEGF-A165 (Fig. 3C). Similarly, PT67/FD4 cells presence of VEGF-A165 (lane 3 versus lane 1 and lane 4 versus strongly inhibited angiogenesis in Matrigel assays in vivo, lane 2, both P < 0.001). By contrast, in FD4 cells that whereas PT67/D4Si cells had an opposite, proangiogenic effect overexpressed DSCR1-4, baseline NFATc1 activity was strik- (Fig. 4). In contrast, the overexpression of DSCR1-1L ingly reduced (lane 7 versus lane 1, P < 0.01) and the response to significantly increased 3H-thymidine incorporation above VEGF-A165 was strongly inhibited (lane 8 versus lane 2, control levels both in the presence and absence of added P < 0.001). Overexpression of DSCR1-3 did not show a VEGF-A165, whereas VEGF-A165-mediated stimulation of significant effect (lane 5 versus lane 1 and lane 6 versus lane 2, thymidine incorporation was strikingly inhibited in HUVEC both P > 0.05). On the other hand, knocking down DSCR1-1L transduced with a DSCR1-1L-specific siRNA (Fig. 3C). Also, expression by its specific siRNA (D1Si) completely inhibited PT67/FD1L cells overexpressing DSCR1-1L induced strong

FD1L cells from inducing angiogenesis in Matrigel plugs (Fig. 4D). Also, using a luciferase reporter assay, NFATc1 activity was increased in FD1L cells (HUVEC transfected with DSCR1-1L), either in the presence or absence of VEGF-A165, and VEGF-A165-mediated activity of NFATc1 was depressed when HUVEC were transfected with D1Si (Fig. 5A). Furthermore, NFAT activation was strongly inhibited in FD4 cells that overexpressed DSCR1-4 (Fig. 5A). In addition, experiments from Drosophila indicate that phosphorylation of RCN1, an orthologue of MCIP1/DSCR1, enhances calcineurin activity (34); these data support our findings that the DSCR1 gene can both inhibit and activate calcineurin. Remaining to be elucidated is the signaling pathway by which VEGF-A165 induces DSCR1-1L expression. Unlike DSCR1-4, DSCR1-1L is not likely to be induced through the calcineurin-NFAT pathway (13-16). Whereas the promoter regulating DSCR1-4 is found in the intron preceding exon 4 and contains NFAT binding sites (34), the promoter regulating DSCR1-1L lies upstream of exon 1 and lacks NFAT binding sites (our unpublished data). Also remaining to be investigated FIGURE 5. Differential regulation of calcineurin-NFATc1 pathway by are the mechanisms regulating the interplay between DSCR1-1L DSCR1 isoforms. A. FD1L, FD3, FD4, LacZ, D1Si, D4Si, and Neg-Si cells and DSCR1-4 signaling that determine whether angiogenesis is were transfected with a NFATc1-regulated reporter gene. After 24 hours of serum starvation, cells were cultured with or without 10 ng/mL of VEGF for induced or inhibited. In the only experiment in which both of 6 hours. NFATc1 activity was expressed as the level of transcription these isoforms were introduced into Matrigel (Fig. 4B, lane 4), activity of the promoter after normalization with standard (n =6).B. the net effect was inhibition of angiogenesis. Cellular extracts from HUVEC transduced with FD4 (lane 1), LacZ (lane 2), and FD1L (lane 3) were subjected to immunoblotting with an antibody DSCR1-1L was overlooked in earlier studies of VEGF- against DSCR1. All experiments were repeated thrice. A-mediated regulation of DSCR1 function, likely for technical reasons that may have included the source of angiogenesis in the in vivo Matrigel assay in the presence or cultured cells, differences in serum starvation conditions absence of a source of VEGF-A165, whereas the inclusion of (0.5% versus 0.1% serum), plate coatings (fibronectin versus PT67/D1Si cells expressing an DSCR1-1L-specific siRNA collagen), etc. This would not be the first time that apparently strongly inhibited VEGF-A165-induced angiogenesis (Fig. 4). minor differences in tissue culture technique have profoundly Taken together, these data indicate that, whereas DSCR1-4 affected VEGF-A165 signaling. For example, plating HUVEC provides a negative feedback loop, DSCR1-1L promotes and is on collagen or fibronectin determined whether PLCg or actually required for VEGF-A165-mediated angiogenesis. Fur- phosphatidylinositol-3-kinase was required for VEGF-A165- thermore, mechanistic studies determined that isoform-specific stimulated HUVEC proliferation (23). Also, DSCR1-1L is DSCR1 siRNAs did not affect KDR expression or phosphory- expressed at much lower levels than DSCR1-4 in HUVEC lation (Supplementary Fig. S1) and that the proangiogenic effect and HMDVEC and its expression at both mRNA and protein of DSCR1-1L was not inhibited by SU1496, an inhibitor of levels was less stimulated by VEGF-A165 in these cultured VEGF receptor 2 (KDR), the receptor through which VEGF- cells (Fig. 1A and B). Nonetheless, although undetectable in A165 mediates endothelial cell proliferation and angiogenesis the microvasculature of several normal tissues, DSCR1-1L (Supplementary Fig. S4). Therefore, DSCR1-1L must be acting was strongly expressed in vivo in the angiogenic responses downstream of VEGF-A and its receptor. induced by Ad-mVEGF-A164 and by mouse and human The antiangiogenic activity of DSCR1-4 has been attributed ovarian cancers (Fig. 2). to its suppression of the calcineurin-NFAT pathway (refs. 14, In summary, DSCR1-1L and DSCR1-4 represent distinct 16; Fig. 6). Calcineurin is a calcium-regulated, serine/threonine isoforms of the same gene that are regulated by different phosphatase that is activated by VEGF-A and other growth promoters and that have opposite effects on VEGF-A164/5- factors to dephosphorylate and thereby activate the transcription induced angiogenesis. How these conflicting activities are factor, NFAT (14-16, 33). Activated NFAT translocates to the balanced in tumor angiogenesis in vivo remains to be cell nucleus in which it acts cooperatively with other determined. The possibilities include differential promoter transcription factors to induce the expression of a large number regulation, expression levels, timing of expression, interacting of genes, including many that have roles in angiogenesis and proteins, etc., questions we are now addressing. The inflammation. By binding to and inactivating calcineurin, expression of different DSCR1 isoforms by different types DSCR1-4 reverses NFAT activation and shuts down the of tumors could explain the puzzling findings that, in patients transcription of proangiogenic genes such as GM-CSF, with Down syndrome, the incidence of certain types of Cox 2, IL-8, E-selectin, and tissue factor (13-16). malignancy is increased, whereas that of others is decreased How do the proangiogenic effects of DSCR1-1L fit into this (9-12). In any event, DSCR1-1L should be considered as a model? The calcineurin-NFAT axis is apparently involved possible target for the therapeutic regulation of pathologic because cyclosporin A, a calcineurin inhibitor, prevented PT67/ angiogenesis.

In vivo Angiogenesis Models Female, 4- to 5-week-old nu/nu mice (NIH) were injected i.d. in ear skin with 1 Â 108 plaque-forming units of adenoviral vectors expressing either mouse VEGF-A164 (Ad- mVEGF-A164) or, as a control, Ad-LacZ (22). MOT cells were maintained by i.p. passage in C3H/HeJ mice and 106 cells were injected i.p. for growth in ascites form as previously described (36). Ears and mesenteries were homogenized in T-PER tissue protein extraction reagent (Pierce Biotechnology, Inc. Rockford, IL) or in RNA extraction buffer (Qiagen, Inc., Valencia, CA). Equal amounts of protein were subjected to immunoblotting using the mDSCR1 antibody. RNA was isolated and subjected to RT- PCR with DSCR1-1L- or DSCR1-4–specific primers. Experi- ments were repeated thrice.

Human Ovarian Carcinomas Tumors were obtained at the time of surgery and were fixed in 4% paraformaldehyde, embedded in OCT compound and prepared for immunohistochemistry (see below). These experi- FIGURE 6. Schematic diagram of proposed mechanisms for DSCR1- 1L and DSCR1-4 regulation of angiogenesis. ments were carried out according to a protocol approved by the hospital’s Committee on Clinical Investigation.

Materials and Methods Immunohistochemistry Materials Implanted Matrigel plugs were dissected free, fixed in 4% Rat anti-mCD31 antibody and mouse anti-hCD31 antibody paraformaldehyde for 4 hours, changed to 30% sucrose were obtained from BD Biosciences (BD Biosciences, overnight, and embedded in OCT compound. Frozen sections PharMingen, San Diego, CA) and Dako (Carpinteria, CA), were then blocked with 5% goat serum and stained with the respectively. Anti-KDR antibody and anti-phosphorylated following primary antibodies at room temperature for 1 hour: rat tyrosine (p-PY20) were purchased from Santa Cruz Biotech- anti-mCD31 antibody (1:50 dilution; BD Biosciences, PharMin- nology, Inc. (Santa Cruz, CA). Mouse monoclonal antibodies gen), mouse anti-hCD31 (1:100 dilution, Dako), or mouse anti- against human DSCR1 (all isoforms, hDSCR1), against specific hDSCR1-1L, mouse anti-hDSCR1-4, mouse anti-mDSCR1, all human DSCR1 isoforms (hDSCR1-1, hDSCR1-3, and at 1:100 dilution (Center for Biomedical Inventions, University hDSCR1-4), and against mouse DSCR1 (all isoforms, of Texas Southwestern Medical School). Sections were then mDSCR1) were obtained from the Center for Biomedical washed thrice with PBSand incubated for 1 hour with Inventions, University of Texas Southwestern Medical School appropriate secondary antibodies: biotinylated polyclonal anti- and from Abnova Corporation (Taipei, Taiwan, ROC). Flag rat IgG antibody (1:500 dilution) or biotinylated polyclonal anti- antibody was from Sigma-Aldrich (St. Louis, MO). mouse IgG antibody (1:200 dilution; Vector Laboratories, Inc. Burlingame, CA). Sections were then washed thrice with PBS, Cell Culture and Assays reacted with the ABC peroxidase kit (Vector Laboratories) at HUVECs (Clonetics, Biowhittaker, Inc., Walkersville, MD) room temperature for 45 minutes, and washed twice with PBS were cultured and transduced with retroviruses carrying various prior to mounting for light microscopy and photography. constructs as previously described (23). HMDVECs were isolated from newborn foreskins (35) and were cultured in Cloning and Expression of DSCR1 Isoforms and siRNAs EBM medium. At 80% confluence, HUVEC and HMDVEC DSCR1 isoforms were cloned by RT-PCR using RNA were incubated in 0.1% fetal bovine serum–containing isolated from HUVEC cultured for 1 hour with 10 ng/mL of 165 EBM medium for 24 hours and then treated with VEGF-A VEGF-A165.The5¶ primers are listed below: DSCR1-1, (10 ng/mL) for different times and subjected to proliferation ATGGAGGAGG TGGACCTGCAGG; DSCR1-1L, CTGATG- assays as previously described (23). GAGGACGGCGTGGCCGG; DSCR1-3, ATGGTGTATGC CAAATTTGAGTCC; DSCR1-4, ATGCATTTTAGAAACTT- Reverse Transcription-PCR Analyses TAACTAC. The 3¶ primer was TCAGCTG AGGTGGATCG- RNA was isolated from HUVEC that had been serum- GCGTGTAC. The identities of the PCR fragments were starved for 24 hours and stimulated with 10 ng/mL of VEGF-A confirmed by DNA sequencing. The cDNAs were fused for the times indicated. RT-PCR with isoform-specific primers in-frame to the Flag sequence, cloned into the retroviral was carried out as described (17). Glyceraldehyde-3-phosphate vector pMMP, and overexpressed in HUVEC as described dehydrogenase served as a control for equal RNA loading. RT- previously (23). PCR products were analyzed on 4% agarose gels. Experiments Recently, a new vector system, pSUPER-retro, was used were repeated thrice. to direct the synthesis of siRNAs in mammalian cells.

Sequence-specific siRNAs destroy the endogenous mRNAs PBS. Two microliters of Fugene were added directly to 50 AL that match the siRNA sequence, thus inhibiting the expression of OPTI-MEM1 medium and incubated at room temperature of their cognate protein (37). siRNAs were designed with the for 5 minutes. NFAT-luc plasmid (0.75 Ag) and pRL-Tk software from OligoEngine, Co. (Seattle, WA), cloned into (0.15 Ag) were added to the mixture and incubated at room pSUPER-retro vector (OligoEngine), and expressed in temperature for 15 minutes, and then added to cells with HUVEC. The D1Si and D4Si sequences were GCTTCATT- 300 AL of medium. Twenty-four hours later, cells were changed GACTGCGAGA and CCAGGGCCAAATTTGAGTC, respec- to EBM medium with 0.1% fetal bovine serum. After 24 hours, tively. Neg-Si is the scrambled sequence of D4Si. The Neg-Si cells were stimulated with 10 ng/mL of VEGF-A165 for 6 hours. is GAACAAATACGCGTGTGTC. Cells were washed twice with PBSand incubated with 120 AL of passive buffer from a dual-luciferase reporter Assay system (Promega, Madison, WI) at room temperature until cells were Matrigel Angiogenesis Assays dissolved. Luciferase activity was assayed according to the Matrigel angiogenesis assays were carried out as described manufacturer’s protocol. (25). SKMEL/VEGF cells (1 Â 107), alone or mixed with 1 Â 7 10 of PT67 cells infected with retroviruses expressing various Animal Welfare full-length DSCR1 or LacZ cDNAs or DSCR1 isoform– All animal experiments were done in compliance with the specific siRNAs were suspended in 0.5 mL of growth factor– Beth Israel Deaconess Medical Center’s Animal Care and Use reduced Matrigel (BD Biosciences, Bedford, MA) and injected Committee. s.c. into nu/nu mice. Tissues were harvested, photographed, and fixed with 4% paraformaldehyde for immunohistochemis- Statistics try. 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Mol Cancer Res 2006;4(11). November 2006 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Down Syndrome Candidate Region 1 Isoform 1 Mediates Angiogenesis through the Calcineurin-NFAT Pathway

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