ELAVL1 regulates of eIF4E transporter to promote postnatal angiogenesis

Sung-Hee Changa,1, Olivier Elementob, Jiasheng Zhangc, Zhen W. Zhuangc, Michael Simonsc, and Timothy Hlaa,1

aCenter for Vascular Biology, Department of Pathology and Laboratory Medicine, and bInstitute for Computational Biomedicine, Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, New York, NY 10065; and cYale Cardiovascular Research Center, Yale University School of Medicine, New Haven, CT 06520

Edited by Napoleone Ferrara, University of California, San Diego, La Jolla, CA, and approved October 28, 2014 (received for review June 27, 2014) Posttranscriptional RNA regulation is important in determining the angiogenic factor induced by hypoxia-inducible factor 1α (HIF-1α) plasticity of cellular phenotypes. However, mechanisms of how RNA (20). We and others recently showed that macrophage ELAVL1 is binding (RBPs) influence cellular behavior are poorly under- important in the angiogenic expression program (9, 21). stood. We show here that the RBP embryonic lethal abnormal vision In this report, we investigated how posttranscriptional gene like 1 (ELAVL1, also know as HuR) regulates the alternative splicing of regulations via ELAVL1 control postnatal angiogenesis. This eukaryotic initiation factor 4E nuclear import factor 1 work shows that ELAVL1 regulates alternative splicing of the (Eif4enif1), which encodes an initiation factor eukaryotic translation initiation factor 4E nuclear import factor 1 4E transporter (4E-T) and suppresses the expression of capped (Eif4enif1), which encodes an eIF4E transporter (4E-T) protein. mRNAs. In the absence of ELAVL1, skipping of 11 of Eif4enif1 The 4E-T is required for cytoplasmic RNA processing body (PB) forms the stable, short isoform, 4E-Ts. This alternative splicing event formation and functions in mRNA translational suppression and results in the formation of RNA processing bodies (PBs), enhanced mRNA degradation (22, 23). We hypothesize that ELAVL1- turnover of angiogenic mRNAs, and suppressed sprouting behavior regulated alternative splicing of Eif4enif1 controls mRNA turn- of vascular endothelial cells. Further, endothelial-specific Elavl1 knock- over, which regulates postnatal pathological angiogenesis. out mice exhibited reduced revascularization after hind limb ischemia and tumor angiogenesis in -induced mammary cancer, Results and Discussion resulting in attenuated blood flow and tumor growth, respectively. ELAVL1 Regulates Alternative Splicing of Eif4enif1. To examine the ELAVL1-regulated alternative splicing of Eif4enif1 leading to en- mechanisms by which ELAVL1 regulates angiogenesis, we con- hanced formation of PB and mRNA turnover constitutes a novel post- ducted exon-microarray analysis using mouse lung endothelial transcriptional mechanism critical for pathological angiogenesis. cells (MLECs) isolated from endothelial cell-specific Elavl1 knockout mice (Elavl1 ECKO) (Fig. S1) as well as bone-marrow– angiogenesis | RNA binding protein | eIF4e transporter | derived macrophages (BMDMs) isolated from myeloid-specific alternative splicing | tumor angiogenesis Elavl1 knockout mice (Elavl1 MøKO) (9) and compared them with the wild-type (WT, Elavl1f/f) counterparts. Alternative ngiogenesis, also known as new vessel formation, is a funda- splicing (AS) analysis by GeneSpring (Agilent Technologies) and Amental process in embryonic development, tissue growth, and AltAnalyze (24) identified four (Eif4enif1, Dlst, Usp1, and recovery from tissue injury (1). In addition, dysregulated angiogen- BC005537) to be alternatively spliced in an ELAVL1-dependent esis is important in many conditions such as cancer growth, metas- manner in both cell types (Fig. 1 and Figs. S2 and S3). Among tasis, age-related macular degeneration, and chronic inflammatory these, the coding exon 11 of Eif4enif1 gene is spliced out in the disease (2). Both developmental and postnatal angiogenesis are absence of ELAVL1. This 72-nt exon encodes a 24-amino-acid initiated by paracrine factors acting on endothelial cells to induce domain, which is positioned between the two nuclear export the formation of angiogenic sprouts, their fusion to form the pri- signal motifs of the eukaryotic initiation factor 4E transporter mary vascular plexus and maturation processes that stabilize the newly formed blood vessels (3). However, programs Significance in endothelial cells that drive the angiogenic process are poorly understood. Hypoxia- and flow-regulated transcriptional events have Angiogenesis, or new blood vessel formation, is critical not only been characterized as major mechanisms that regulate gene ex- for normal processes such as embryonic development but also for pression during angiogenesis (4, 5). Recently, posttranscriptional progression of diseases such as tumor growth, metastasis, and gene regulation by RNA binding proteins (RBPs) and miRNAs is chronic inflammatory disease. This work elucidated a molecular recognized to play important roles in the regulation of fundamental mechanism that is important in postnatal angiogenesis in tumor biological processes (6, 7). Indeed, miRNAs were shown to regulate growth and ischemia–reperfusion injury in the hind limb. of angiogenesis and expression of key regulators (8–12). Specifically, we identified a posttranscriptional gene regulatory ELAVL1 (also known as Hu antigen R, HuR) is an AU-rich mechanism that controls the activity of a potent suppressor of element (ARE) and U-rich element (URE) RBP that stabilizes gene expression, named eIF4e transporter (4E-T). Alternative mRNAs and promotes gene expression (13). Although this RBP is splicing of 4E-T controls the level of the active form of 4E-T, which located primarily in the nucleus, it is translocated into the cytoplasm suppresses gene expression in endothelial cells. This mechanism after cellular activation to promote gene expression. ELAVL1 binds may be targeted to control angiogenesis-dependent diseases.

to the 3′ UTRs of many mRNAs, often at or near miRNA bind- MEDICAL SCIENCES Author contributions: S.-H.C. and T.H. designed research; S.-H.C., J.Z., and Z.W.Z. per- ing sites (14, 15). Indeed, ELAVL1 functions in part to modulate formed research; S.-H.C., O.E., J.Z., Z.W.Z., M.S., and T.H. analyzed data; O.E. performed miRNA-dependent gene regulation (9, 16). Mice deficient for bioinformatic analysis; and S.-H.C., M.S., and T.H. wrote the paper. Elavl1 are embryonic lethal due to defects in placental development The authors declare no conflict of interest. (17). Inducible postnatal deletion of Elavl1 leads to stem/progenitor This article is a PNAS Direct Submission. cell leading to intestinal and hematopoietic failure 1To whom correspondence may be addressed. Email: [email protected] or and death within 10 d (18), and zebrafish elavl1 is important for [email protected]. regulation of expression and embryonic erythropoiesis (19). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ELAVL1 stabilizes the mRNA for VEGF-A, which encodes a key 1073/pnas.1412172111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1412172111 PNAS | December 23, 2014 | vol. 111 | no. 51 | 18309–18314 Downloaded by guest on October 1, 2021 (4E-T) protein (25). In contrast, the three other ELAVL1- were shown to be involved in posttranscriptional gene regulation regulated, alternatively spliced genes contain affected in in germ cells (27). However, the role of 4E-T in angiogenesis has the 5′ UTR (Usp1)or3′ UTR (Dlst and BC005537). not been determined. RT-PCR analysis indicated that Eif4enif1 We further studied ELAVL1 regulation of alternative splicing exon 11 is skipped in cells that lack ELAVL1, resulting in the re- of Eif4enif1, which encodes an important factor that is critical for duced expression of Eif4enif1-L (4E-TL) isoform and increased cytoplasmic RNA PB formation, suppression of mRNA trans- expression of Eif4enif1-S (4E-Ts) isoform (Fig. 1B). ELAVL1- lation, and mRNA degradation (22, 23). Loss-of-function muta- dependent alternative splicing of Eif4enif1 was observed in primary tions of EIF4ENIF1 gene are associated with primary ovarian BMDMs, MLECs, immortalized mouse embryonic endothelial cells Elavl1 insufficiency (26) and both mouse and Drosophila homologs of 4E-T (IMECs) transfected with siRNA targeting ,andIMECstably expressing the shRNA for Elavl1 (shElavl1), indicating that this event is not cell-type specific. Eif4enif1 genomic sequence shows that 10 contains 18 U-rich sites including a long, U-rich poly- pyrimidine tract (∼71 bp) immediately preceding the 3′-splice ac- ceptor site (Fig. S4). Because ELAVL1 interaction with binding sites located close to 3′-splice junctions have been observed in the pho- toactivatable ribonucleoside enhanced crosslinking and immuno- precipitation analysis of human cells (14, 15), and because it was shown to regulate splicing of the FAS in HeLa cells (28), our data suggest that nuclear function of ELAVL1 may involve the regulation of splicing of Eif4enif1 primary transcript. This event results in the predominant expression of the 4E-TL isoform. Eif4enif1 mRNA expression was similar between WT and Elavl1 ECKO MLECs. When MLECs were treated with acti- nomycin D, Eif4enif1 mRNA decayed with a half- of ∼2h, which was similar between WT and Elavl1 ECKO cells (Fig. 1C), suggesting that ELAVL1 does not regulate the turnover of this transcript. However, immunoblot analysis using the 4E-T detected a polypeptide band of ∼140 kDa, which was markedly increased in Elavl1 ECKO cells (Fig. 1D). Further, siRNA- mediated knockdown of Elavl1 in IMECs resulted in dose- dependent increase in the expression of 4E-T immunoreactive band (Fig. 1D). Because the SDS/PAGE cannot distinguish the 4E-TL and 4E-TS isoforms, we hypothesized that protein stability may be different between the two isoforms. Indeed, in shElavl1 IMEC cells, which primarily express the 4E-TS isoform, immuno- reactive 4E-T is much more prominent than the control shRNA- treated counterparts and exhibits a longer half-life (Fig. 1E). To confirm the differential stability between 4E-TL and 4E-TS isoforms, we expressed each isoform in IMECs in which endogenous 4E-T was down-regulatedbyshRNA.AsshowninFig.1F, the half-life of 4E-TL is much shorter than the 4E-TS isoform, suggesting that ELAVL1- induced inclusion of exon 11 destabilizes the 4E-T polypeptide. Thus, in the presence of ELAVL1, alternative splicing of Eif4enif1 gene results in the expression of the short-lived 4E-T isoform. Fig. 1. ELAVL1 regulates alternative splicing of Eif4enif1.(A) Venn diagram L depicting alternative splicing events in BMDMs and MLECs as determined by 4E-T Protein in ELAVL1 Depleted Cells Induces PB Potently. 4E-T, the microarray expression results of WT, Elavl1 ECKO MLECs, and Elavl1 S originally identified as a nucleocytoplasmic shuttling protein, MøKO BMDMs by GeneSpring (Agilent) and Altanalyze (open source) pro- ′ grams. Alternative exon analysis parameters are described in Fig. S2. Four binds to eIF4E, the mRNA 5 cap-binding protein (25). We genes (Eif4enif1, Dlst, Usp1, and Bc005537) were identified in both BMDMs observed that both 4E-TL and 4E-TS isoforms bind to eIF4E in a and ECs. Scheme of the differentially expressed exons (blue box) are shown. GST pull-down assay (Fig. S5). Interestingly, recent work revealed (B) RT-PCR analysis of alternative splicing of Eif4enif1. Scheme of the alter- that 4E-T is required for the formation of PBs and transports the native splicing of Eif4enif1. Primers (F1 forward; R1 reverse) are indicated in eIF4E/mRNA complex to PBs for translational repression and flanking exons 10 and 12. RT-PCR was done using RNA isolated from primary mRNA decay (22, 23). Thus, we examined the effect of alterna- BMDMs, MLECs, siRNA targeting Elavl1 (siElavl1) transfected IMECs, and tively spliced 4E-T protein isoforms in PB formation in endothelial stable knockdown of Elavl1 in IMECs (shElavl1). (C) Eif4enif1 transcript ex- cells. Immunofluorescence staining confirmed that 4E-T localizes pression and decay. The expression of Eif4enif1 transcripts was quantified using primers (F2 and R2) located in exon 10 in a qRT-PCR assay. Actinomycin predominantly within discrete foci in the cytoplasm and is colo- D(5μg/mL) was used to treat WT and Elavl1 ECKO for various times. Data calized with the mRNA decapping factor Dcp1a, a marker of PBs present mean ± SEM, n = 3. (D) The 4E-T polypeptide expression in Elavl1 (22, 23) (Fig. 2A). Transfection of siElavl1 in IMECs to reduce depleted cells. Protein extracts from WT and Elavl1 ECKO MLECs, and IMECs ELAVL1 protein increased both the number and the area of 4E-T treated with siElavl1, were prepared and immunoblotted with anti 4E-T, positive PBs (Fig. 2A). Similarly, primary endothelial cells that lack ELAVL1, or β- . (E) IMECs expressing control shRNA (shCtl) or Elavl1 (Elavl1 ECKO) and Elavl1 knockdown IMECs (shElavl1) shElavl1 were treated with cycloheximde (100 μg/mL) for indicated times and showed a similar phenotype (Fig. 2 B–D). These data suggest that immunoblot analysis of lysates was performed with antibodies specific to 4E-T, expression of stable 4E-TS isoform in Elavl1 depleted cells induces ELAVL1, β-actin, or cyclin D1. (F) Differential stability of 4E-TL and 4E-TS poly- . Endogenous Eif4enif1 was knocked down in IMECs using lentiviral the formation of abundant, large PBs. To further examine this possibility, we overexpressed 4E-T or 4E-T in IMECs by lenti- shEif4enif1 construct. These cells were transduced with cDNAs encoding 4E-TL L S and 4E-TS polypeptides, treated with cycloheximde (100 μg/mL) for indicated virus transduction (Fig. S6). Dcp1a immunostaining revealed that times and analyzed by immunoblot analysis. Results from D–F were from overexpression of 4E-TL enhanced PB formation approximately a representative experiment that was repeated two to three times. twofold compared with control lentiviral transduction (Fig. 2E).

18310 | www.pnas.org/cgi/doi/10.1073/pnas.1412172111 Chang et al. Downloaded by guest on October 1, 2021 Fig. 2. The 4E-TS protein in ELAVL1 depleted cells induces exaggerated PB formation. (A) The 4E-T colocalizes with Dcp1a, a marker for PBs. IMECs transfected with siCtl or siElavl1 were stained with anti–4E-T antibody (green), anti-Dcp1a (red), and DAPI (blue). Higher magnification views of boxed areas are shown in Bottom Right Insets.(B and C) MLECs from WT or Elavl1 KO mice and stable knock- down of Elavl1 in IMECs (shElavl1) were stained with anti-ELAVL1 antibody (green) and anti–4E-T antibody (red). (D) Quantitative analysis of the number and the area of PBs. The number of 4E-T positive PBs per cell are as follows: shCtl vs. shElavl1: 4.19 ± 0.56 vs. 38.06 ± 4.0. The area of 4E-T positive PBs per cell is as follows: shCtl vs. shElavl1: 67.90 ± 9.79 vs. 939.9 ± 131.6. The number of pixels per PB is as follows: shCtl vs. shElavl1: 16.20 ± 0.72 vs. 23.65 ± 1.29. n = 21 or 17, ***P < 0.0001. (E) IMECs were transfected with con- trol siRNA or Elavl1siRNA, subsequently infected with

lentivirus expressing control vector, 4E-TL, or 4E-TS and stained for Dcp1a. (F) Quantitative analysis of the

number and the areas of PBs in control, 4E-TL, or 4E-TS overexpressed cells. *P < 0.05, ***P < 0.005, **P < 0.0001. (Scale bars, 10 μm.)

In sharp contrast, 4E-TS expression was much more potent in the (Fig. 3C). Knockdown of Elavl1 and Eif4enif1 did not alter the induction of PBs (approximately sixfold) (Fig. 2F). In Elavl1- loading of Fos, Hif1a,andVegfa mRNAs on light or heavy poly- depleted IMECs, the number and size of PBs were maximal and somes (Fig. 3D). In contrast, turnover of angiogenic regulatory overexpression of 4E-TL or 4E-TS did not have an additive effect. mRNAs was affected by ELAVL1 and 4E-T. Fos and Hif-1a These data indicate that formation of 4E-TS isoform, which is mRNA, which contain several ARE−/URE− motifs, revealed inhibited by ELAVL1-induced alternative splicing, is a potent a longer half-life in Eif4enif1 knockdown cells compared with inducer of PB formation in IMECs. control cells (Fos: 103.2 min vs. 20.4 min and Hif1a:186minvs. 55.8 min), whereas a shorter half-life in Elavl1 knockdown cells 4E-TS Protein in ELAVL1-Depleted Endothelial Cells Promotes mRNA (Fos: 15.6 min vs. 20.4 min and Hif1a: 49.2 min vs. 55.8 min) (Fig. Turnover. PBs are the sites at which translationally inactive 3E). These data strongly suggest that ELAVL1-regulated alterna- mRNAs, , RBPs, and mRNA decay machinery ach- tive splicing of Eif4enif1 and the formation of 4E-TS isoform con- ieve translational repression and mRNA degradation (29). In trol, at least in part, the delivery of specific ARE/URE-containing particular, ARE-bearing mRNAs are targeted to PBs for trans- mRNAs to PBs and facilitate mRNA degradation. Even though lational silencing and rapid degradation (30). To better un- Vegf-a mRNA half-life was similar in control, Elavl1 knockdown and derstand the ELAVL1-mediated gene regulation in endothelial Eif4enif1 knockdown cells (25.2 min, 28.2 min, and 32.4 min), cells, we analyzed global gene expression profiles in MLECs steady-state Vegf-a mRNA expression was significantly reduced in isolated from WT and Elavl1 ECKO mice. Among 1,473 dif- Elavl1 knockdown cells and enhanced in Eif4enif1 knockdown cells ferentially expressed genes, the majority (69%, 1,014 genes) were (Fig. 3F). In addition, the secretion of VEGF is significantly reduced down-regulated in Elavl1 ECKO MLECs (fold change >1.4, n = 4/ in the supernatant of Elavl1 knockdown cells and enhanced in the group) (Fig. 3A). In addition, down-regulated transcripts con- supernatant of Eif4enif1 knockdown cells under hypoxia (Fig. 3G). tained higher density of both ARE and URE (14, 15) than the Accordingly, in the Boyden chamber chemotaxis assay, stimulation transcripts that are stable or up-regulated in 3′ UTR, 5′ UTR, of IMEC migration in response to sphingosine 1-phosphate (S1P) MEDICAL SCIENCES exons, and (Fig. 3B and Fig. S7). These data suggest that was attenuated in Elavl1 knockdown cells but is greatly exaggerated ELAVL1 stabilizes ARE- and URE-containing transcripts (14, in Eif4enif1 knockdown cells (Fig. 3H). These data suggest that the 15) in vascular endothelial cells. impaired angiogenic phenotype in Elavl1 knockout cells is the result To test the possible role of 4E-TS in translational repression, of the function of 4E-TS protein. we measured the polysome loading of posttranscriptionally reg- ulated angiogenesis-regulatory mRNAs, Fos, Hif1a, and Vegfa by Endothelial ELAVL1 Regulates Postnatal Pathological Angiogenesis. fractionating cytoplasmic components in 15–45% sucrose gra- To examine the angiogenic functions of ELAVL1-mediated gene dients followed by quantitative RT-PCR (qRT-PCR) analysis regulation in primary endothelial cells, we analyzed in vitro

Chang et al. PNAS | December 23, 2014 | vol. 111 | no. 51 | 18311 Downloaded by guest on October 1, 2021 Fig. 3. The 4E-TS protein in ELAVL1-depleted en- dothelial cells promotes mRNA turnover. (A) Micro- array analysis in MLECs from WT or Elavl1 KO mice. A total of 1,014 genes were down-regulated in Elavl1 KO MLECs. Fold change is >1.4, n = 4 per group. (B) Average density of AUUUA (the number of AUUUA per kilobase of transcript) in each region (3′ UTRs, 5′ UTRs, exons, and introns) of genes analyzed by microarray. (C) Polysome profiles of IMECs stably knocked down with shRNA targeted against control (Ctl), Elavl1,orEif4enif1 mRNAs. Cell extracts were size fractionated by centrifugation through sucrose density gradients (15–45%). Arrows indicate the di- rection of sedimentation. Below each profile, 18S and 28S rRNA were visualized by nanogel. (D) RNA extracted from each of the 12 fractions, followed by qRT-PCR to assess the relative distribution of Fos, Hif1a, Vegfa, and β-actin mRNAs. Data represent mean ± SEM from three independent experiments. (E) Percentage of Fos, Hif1a, and Vegfa mRNA remaining in shcontrol, shElavl1, and shEif4enif1 cells upon addition of actinomycin D (5 μg/mL) for indicated times. The half-life (t1/2) of mRNA was determined from the slope and Y intercept of a best fit value in the semilogarithmic plot of mRNA

abundance versus time; log10 of 50% = 10^(slope × t1/2 + Y intercept). Data represent mean ± SEM from four independent experiments. (F) Vegfa mRNA ex- pression in shcontrol, shElavl1, and shEif4enif1 cells. Data represent mean ± SEM from five independent experiments. *P < 0.05, **P = 0.01. (G) VEGF-A se- cretion in the supernatant of normoxia- or hypoxia- treated shcontrol, shElavl1, and shEif4enif1 cells. Data represent mean ± SEM from three independent experiments. *P < 0.05, **P = 0.01. (H) Chemotaxis of IMECs. Migration of IMECs stably knocked down with shRNA control, Elavl1,orEif4enif1 in response to 100 nM S1P was analyzed using a modified Boy- den chamber. Data represent the mean ± SEM from three independent experiments. *P < 0.05.

angiogenic phenotypes of MLECs from WT and Elavl1 ECKO femoral artery ligation (32), significantly attenuated blood flow mice. Loss of ELAVL1 did not affect endothelial cell pro- recovery was seen in Elavl1 ECKO mice (Fig. 5 A and B). Micro- liferation in vitro (Fig. S8). However, migratory and sprouting CT scan of the affected limb shows attenuated angiogenesis and responses of endothelial cells were greatly affected. MLECs collateral formation (Fig. 5C) and quantitative analysis of micro- from Elavl1 ECKO mice did not migrate as much as the WT CT data confirmed that the relative number of arteries with counterparts in the scratch-induced migration assay (Fig. 4A). <144-μm diameter in Elavl1 ECKO mice is significantly reduced Similarly, 3D spheroid sprouting assay showed significantly re- compared with WT mice, supporting the significant reduction in duced number and length of sprouts in MLECs from Elavl1 blood flow recovery after femoral artery ligation in Elavl1 ECKO ECKO mice compared with the WT controls (Fig. 4 B and C). mice. Tissue sections were immunostained with CD31 to high- Next, the vascular phenotypes of Elavl1 ECKO mice were light microvessels in the ischemic gastrocnemius muscle (Fig. examined. Crosses between Elavl1f/f mice with or without VE- 5D). Quantitative analysis revealed reduced number of capil- cadherin-Cre (31) resulted in the birth of WT and Elavl1 ECKO laries per field and per muscle fiber in Elavl1ECKO mice. These mice at 1:1 ratio (Fig. S9A). Both WT and Elavl1 ECKO mice data indicate that the endothelial ELAVL1 is critical for optimal appeared normal, suggesting that ELAVL1 is not required for revascularization response after ischemic injury of the hind limb. embryonic vascular development. In addition, postnatal angio- To examine the role of endothelial ELAVL1 in tumor an- genesis in the ear, retina, and trachea of WT and Elavl1 ECKO giogenesis, we implanted Lewis lung carcinoma (LLC) cells s.c. mice looks similar, suggesting the undisturbed angiogenesis in and examined tumor growth and associated tumor microvessels. adult mice (Fig. S9B). However, when these mice were subjected LLC tumor implant in Elavl1ECKO mice showed significantly to a model of ischemic angiogenesis in the hind limb following attenuated tumor volume and weight compared with WT mice

18312 | www.pnas.org/cgi/doi/10.1073/pnas.1412172111 Chang et al. Downloaded by guest on October 1, 2021 Fig. 4. Endothelial ELAVL1 regulates postnatal path- ological angiogenesis. (A) Scratch wound assay and (B and C) spheroid sprouting assay using MLECs isolated from WT and Elavl1 ECKO mice. Represen- tative images of endothelial cells in scratched area at 0 h (Top) and 16 h (Bottom). (A) Quantitative analysis of scratch closed area in WT ECs (91.18 ± 3.39, n = 6) vs. Elavl1 KO ECs (48.43 ± 5.66, n = 5). ***P < 0.0001. Data are presented as the percent of scratch covered area to the initial cell-free area. (B and C) Representative images and quantitative analysis of sprouts from spheroids at 3 d and 5 d. Number of sprouts per spheroid at 3 d (WT: 13.80 ± 1.24 vs. KO:1.33 ± 0.56) and 5 d (WT 22.56 ± 1.15 vs. 8.53 ± 0.54). Mean length of sprout per spheroid at 3 d (WT: 55.34 ± 7.40 vs. KO: 14.18 ± 4.81) and 5 d (WT: 56.86 ± 2.22 vs. KO: 39.88 ± 1.86). Cumulative sprout length of spheroids at 3 d (WT: 768.6 ± 135.5 vs. KO: 28.95 ± 13.40) and 5 d (WT: 1283 ± 79.85 vs. KO: 345 ± 29.50). Three days: n = 5 or 6 per group; 5 d: n = 16 or 17 per group, **P < 0.005, ***P < 0.0005.

(Fig. 6A). CD31 immunostained tumor images and quantitative were removed at 16 wk and the total tumor burden was eval- analysis of CD31 positive vessels indicate that Elavl1 ECKO uated. PyMT:Elavl1 ECKO mice had significantly reduced tu- mice had significantly reduced vascular density and branching mor burden than their WT counterparts (Fig. 6B). Histological patterns compared with the WT counterparts (Fig. 6A). We sections were stained for tissuearchitectureaswellasimmu- also examined the role of endothelial ELAVL1 in a sponta- nohistochemistry to analyze the microvessels (Fig. 6B). Data neous model of mammary cancer. Elavl1 ECKO mice were revealed that mammary tumors in PyMT:Elavl1 ECKO mice crossed with transgenic mice expressing polyoma virus middle T showed the reduced vascular density and fewer malignant oncogene (PyMT) under the transcriptional control of mouse lesions compared with the WT counterparts. These data sug- mammary tumor virus (MMTV-PyMT)(33).Tumors gest that posttranscriptional gene regulation by ELAVL1 in MEDICAL SCIENCES

Fig. 5. Endothelial ELAVL1 regulates postnatal pathological angiogenesis. Revascularization of the mouse ischemic hind limb was conducted as described in WT and Elavl1 ECKO mice. (A) Representative laser Doppler flow images of hind limb perfusion before (pre) and at different time points (post 3, 7, 10, and 14 d) after femoral artery ligation (R, control; L, ligated). (B) Changes in perfusion are shown as ratio of ischemic to control limb flow perfusion (n = 7 per group, *P < 0.05, ***P < 0.001). (C) Micro-CT reconstruction of a representative mouse hind limb 14 d after femoral artery ligation. Quantitative analysis of micro-CT angiograms in the calf is presented as total number of vascular structures of specified diameter. (n = 7 per group, *P < 0.05) (D) Representative images of CD31 immunostained sections of ligated limb muscle and quantification of CD31 positive vessels. The number of capillaries per field (20× WT: 91.1 ± 10.9 vs. Elavl1 ECKO: 35.5 ± 4.4, P = 0.0002, n = 8 images from four mice per group). The number of capillaries per fiber (WT: 3.57 ± 0.11 vs. Elavl1 ECKO 1.55 ± 0.06, P < 0.0001, n = 101 fibers from eight images per group). (Scale bar, 50 μm.)

Chang et al. PNAS | December 23, 2014 | vol. 111 | no. 51 | 18313 Downloaded by guest on October 1, 2021 suppresses angiogenic gene expression in endothelial cells. Our work also revealed a novel mode of regulation of angiogenic gene expression, which is to control mRNA turnover by limiting the expression of highly stable isoform of a posttranscriptional re- pressor. The 4E-TS promotes turnover of mRNAs for endo- thelial factors (Fos and Hif-1a) and reduces the expression of a key growth factor (VEGF-A), suppressing chemotactic migration and sprouting behavior. This mode of gene regulation may be highly relevant in postnatal angiogenesis in the revascularization of ischemic muscle of the limb and tumor angiogenesis. Indeed, regulation of eIF4E by the mTOR/4EBP1 pathway is therapeutically targeted in oncology (34). We propose that the ELAVL1/Eif4enif1 posttranscriptional RNA regulon de- scribed here may be relevant in many pathological processes and therefore therapeutically actionable. Materials and Methods Fig. 6. Endothelial ELAVL1 regulates postnatal pathological angiogenesis. (A) LLC tumor isograft model in WT and Elavl1 ECKO mice. Representative and . Tumor isograft, spontaneous mammary tumor images of CD31 immunostaining of LLC tumors. Quantitative analysis of model, and hind limb ischemic model and laser Doppler blood flow analysis vascular density (CD31 positive pixels per field, 20×). LLC tumor volume and were performed with institutional review board approval as described in SI LLC tumor weight of WT (0.525 ± 0.056 n = 34) and Elavl1 ECKO mice (0.285 ± Materials and Methods (Weill Cornell Medical College for tumor study, Yale 0.031, n = 32). *P < 0.05, **P < 0.005, ***P < 0.0001. (B) PyMT-induced University School of Medicine for hind limb ischemic mouse model). Primary mammary tumor model in WT and Elavl1 ECKO mice. Representative images mouse lung endothelial cells were isolated, cultured, and scratch wound of CD31 immunostaining of mammary tumors. Total mammary mass of assay and spheroid sprouting assay were performed as described in SI PyMT:WT (3.311 ± 0.202, n = 26) and PyMT: Elavl1 ECKO mice (2.082 ± 0.219, Materials and Methods. n = 23) at the age of 16 wk. ***P < 0.0001. (Scale bar, 50 μm.) RNA and Protein Analysis. RNA isolation, mouse exon chip array, alternative splicing analysis, polysomal mRNA profiling, RT-qPCR analysis, Western endothelial cells is critical for tumor angiogenesis and optimal blotting, and immunofluorescence experiments are described in SI Mate- tumor growth. rials and Methods. Our results elucidate previously unidentified function of ACKNOWLEDGMENTS. We thank Professor Thomas Tuschl (The Rockefeller ELAVL1, which is to regulate the alternative splicing of Eif4enif1 University) for critical comments and Dr. Jerry Pelletier (McGill University) primary transcript. In the absence of ELAVL1, exon 11 exclusion for the gift of reagents. This work was supported by National Institutes produces 4E-TS, a stable isoform that strongly induces PBs and of Health Grants HL49094, HL117798, and CA77839 (to T.H.).

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