The basic helix–loop–helix protein E2-2 is known to play a role in quiescence of endothelial cells (ECs). However, it is unclear how the activity of E2-2 is controlled in the cells. In this study, we identified FAM96B as an interaction partner of E2-2. FAM96B interfered with E2-2-mediated effects on luciferase reporter activities. Furthermore, the suppression of vascular endothelial growth factor receptor 2 promoter activity by E2-2 was rescued by the expression of FAM96B in a dose-dependent manner. Interestingly, FAM96B decreased the expression of ectopic and endogenous E2-2 proteins. Mutational analysis revealed that the middle region of FAM96B is required for the limited expression of E2-2 protein. When FAM96B was expressed in ECs, the EC migration, proliferation, and tube formation were potentiated. Taken together, these findings suggest that FAM96B acts as a regulator of E2-2 through the control of its protein expression. (Cancer Sci 2011; 102: 1808–1814)
The basic helix–loop–helix (bHLH) protein E2-2, alternatively termed ITF2, TCF4, SEF2, and SEF2-1B, was originally identified as a transcription factor that binds to the μE5 and κE2 motifs in the immunoglobulin heavy and light chain enhancers, respectively.(1) Together with E12/47 and HEB, E2-2 belongs to the E protein family. E proteins are widely expressed in tissues and bind to consensus DNA sequences containing (G/A)CAXXTG(G/A) as either homodimers or heterodimers with other HLH family molecules.(2,3) The E protein family is known to regulate lymphocyte development,(4) neural differentiation,(5) and myogenesis.(6) Recently, we and another group showed that E proteins also regulate blood vessel formation.(7–9)
The activity of E proteins is known to be controlled through their interaction with other HLH proteins,(10) coactivators,(11,12) or corepressors(13) as well as through their own phosphorylation.(14) The Id family, which lacks the basic region important for DNA binding activity, associates with E proteins to inhibit their DNA binding activity.(10) The coactivators CBP and p300 interact with E proteins through their AD1 domain to potentiate their transcriptional activity,(11,12) although the ETO family blocks the transcriptional activity of E proteins by its direct interaction.(13)
Angiogenesis, the formation of new blood vessels, is crucial for vascular development and homeostasis. Aberrant vascularization leads to a number of diseases including atherosclerosis, tumorigenicity, and retinopathy. Angiogenesis occurs through the sprouting of new vessels from pre-existing ones or by intussusceptive microvascular growth. Thus, angiogenesis is essential during embryonic development as well as in adulthood. In general, vascular formation is quiet in adulthood, although angiogenesis involved in wound healing, inflammation, ischemia, and the female reproductive cycle has been observed.(15) Angiogenesis is divided into two phases, the activation phase and the resolution phase. The angiogenic switch depends on a finely tuned balance between stimulators (e.g. vascular endothelial growth factor [VEGF], fibroblast growth factor [FGF]-2, angiopoietins, hypoxia) and inhibitors (e.g. angiostatin, endostatin, α-interferon). During the activation phase, proliferation of endothelial cells (ECs), increase of vascular permeability, and degradation of extracellular matrix components can be observed. Consequently, ECs make new capillary sprouts. During the resolution phase, the proliferation and migration of ECs stop, then the basement membrane is reconstituted and the vessels mature.(16)
In our recent studies, E2-2 as well as E2A inhibited the activity of the VEGF receptor 2 (VEGFR2) promoter to inhibit the activity of endothelium in vivo and in vitro. In contrast, Id1 and stem cell leukemia protein (SCL)/T cell acute lymphocytic leukemia 1 (TAL1), both of which possess the HLH domains, relieved E2-2-mediated inhibition of VEGFR2 promoter activity because of heterodimer formation with E2-2.(7,8) However, partner(s) other than HLH-containing proteins can still regulate the activity of E2-2.
To gain more insight into the molecular mechanisms by which E2-2 suppresses the activity of endothelium, we surveyed its interaction partner(s) and identified FAM96B, alternatively named CGI-128. However, its function has yet to be identified. In this study, we explored the regulatory role of FAM96B on the function of E2-2. In addition, we examined its function in EC activation. We found that FAM96B promoted decrease of E2-2 protein to rescue E2-2-mediated repression of VEGFR2 promoter activity. Consequently, FAM96B concomitantly enhanced EC migration, proliferation, and tube formation.
Materials and Methods
Plasmids and adenoviruses. The cDNA for human FAM96B was cloned by RT-PCR and sequenced to be verified prior to use. FAM96B (42-121) and FAM96BΔ (42-121) were generated by Pfx DNA polymerase (Invitrogen, Carlsbad, CA, USA) using human FAM96B as the template. Subsequently, each DNA was ligated into either Flag-pDEF3 or Myc-pDEF3.(17) Myc-E2-2 and Flag-E2-2 have been previously described.(8) MCKpfos-luc was generously provided by Dr. M. Sigvardsson (Lund University, Lund, Sweden),(18) and pGL2b-VEGFR2-luc (−166 bp/+267 bp) by Dr. C.C.W. Hughes (University of California Irvine, Irvine, CA, USA).(19) Adenovirus expressing Myc-FAM96B was generated using the pAdTrack-CMV vector.(20) After pAdTrack-CMV-Myc-FAM96B was recombined with pAdEasy-1,(20) the resulting plasmid was transfected into 293T cells, and the adenoviruses were amplified. LacZ- and constitutively active activin receptor-like kinase 6 (ALK6ca)/HA-expressing adenoviruses were previously described.(8)
Cell culture. COS7 cells, NIH3T3 cells, and mouse embryonic endothelial cells (MEECs)(21) were maintained in DMEM (Sigma, St. Louis, MO, USA) containing 10% FCS (Gibco, Carlsbad, CA, USA), 1× MEM non-essential amino acids (Invitrogen), and 100 U/mL penicillin/streptomycin. Calf pulmonary artery epithelial cells (CPAEs)(22) were cultured in DMEM with 10% FCS, 20 mM HEPES, and 100 U/mL penicillin/streptomycin. Primary HUVECs were cultured in endothelial cell basal medium (EBM) (Takara, Ohtsu, Japan) supplemented with 2% FCS. The MEECs and HUVECs were grown on 0.1% gelatin-coated dishes.
Adenoviral infections. Adenoviruses were incubated in DMEM containing polybrene (80 μg/mL) for 2 h and added to the dishes. Two hours after infection, the cells were washed and allowed to recover for 24 h prior to the experiments.
Immunoprecipitation and Western blot analysis. To detect interactions among proteins, plasmids were transfected into COS7 cells (5 × 105 cells/6-cm dish) using FuGENE6 (Roche, Indianapolis, IN, USA). Forty hours after transfection, the cells were lysed in 500 μL TNE buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM PMSF, 5 μg/mL leupeptin, 100 U/mL aprotinin, 2 mM sodium vanadate, 40 mM NaF, and 20 mM β-glycerophosphate). The cell lysates were precleared with protein G sepharose beads (GE Healthcare, Princeton, NJ, USA) for 30 min at 4°C then incubated with anti-Flag M5 (Sigma), anti-Myc9E10 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-E2-2 (M03) (Abnova, Taipei, Taiwan) antibody for 2 h at 4°C. Protein complexes were immunoprecipitated by incubation with protein G Sepharose beads for 30 min at 4°C, then washed three times with TNE buffer. The immunoprecipitated proteins and aliquots of the total cell lysates were boiled for 5 min in sample buffer, separated by SDS-PAGE, and transferred to Hybond-C Extra membranes (GE Healthcare). The membranes were probed with anti-Myc9E10 or anti-Flag M5 antibody. Primary antibodies were detected with HRP-conjugated goat anti-mouse IgG antibody (GE Healthcare) and chemiluminescent substrate (Pierce, Rockford, IL, USA). The protein expression in the total cell lysates was evaluated by Western blotting using anti-Flag M5, anti-Myc9E10, or anti-E2-2 (M03) antibody. As internal controls, the expressions of β-actin and GFP were detected using anti-β-actin (Sigma) and anti-GFP antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively.
Immunofluorescence. The MEECs were grown on glass coverslips coated with 0.1% gelatin 1 day prior to transfection. Cotransfection of Flag-E2-2 with Myc-FAM96B was carried out using Lipofectamine (Invitrogen) and Plus Reagent (Invitrogen). After 40 h, the slips were washed with PBS, fixed with 4% paraformaldehyde for 10 min, washed three times with PBS, permeabilized with 0.1% Triton X-100 (Sigma) for 10 min, and blocked with 5% normal swine serum in PBS for 1 h at 37°C. Mouse anti-E2-2 (M03) (1:250) and rabbit anti-Myc (1:250) (Upstate Biotechnology, Billerica, MA, USA) antibodies in 5% normal swine serum in PBS were added and incubated for 1 h at 37°C. The slips were washed three times with PBS then incubated with Alexa488-conjugated goat anti-mouse IgG (Molecular Probes, Carlsbad, CA, USA) or Alexa555-conjugated goat anti-rabbit IgG (Molecular Probes) at 1:250 for 1 h at room temperature. After the nuclei were stained with 2 μg/mL DAPI for 10 min, the slips were washed three times with distilled water, and the fluorescent signals visualized by microscopy (Zeiss, Göttingen, Germany).
Transcriptional reporter assay. The CPAEs were seeded at 5 × 104 cells/well in 12-well plates 1 day prior to transfection. The cells were transfected using Lipofectamine and Plus Reagent. After 40 h of transfection, the lysates were prepared and the luciferase activity was measured using a luciferase assay system (Promega, Madison, WI, USA). The results were corrected for β-galactosidase activity (pCH110; GE Healthcare). Each experiment was carried out in triplicate and repeated at least twice. Values are presented as the mean ± SD (n = 3).
Migration assay. Cell migration assays were carried out using a Boyden chamber. Costar nucleopore filters (8-μm pore) were coated with 10 μg/mL fibronectin (Sigma) overnight at 4°C. The chambers were washed three times with PBS. Adenovirus-infected HUVECs starved for 12 h without FCS were added to the top of each migration chamber at a density of 2.5 × 105 cells/chamber in 150 μL EBM without FCS. The cells were allowed to migrate to the underside of the chamber. After 24 h, the cells were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet (dissolved in 25% methanol). The upper surface was wiped with a cotton swab to remove non-migrating cells. Cells present on the lower surface were counted. Each experiment was carried out in triplicate and repeated at least twice. Values are presented as the mean ± SD (n = 3).
Network formation assay. The HUVECs (2 × 104 cells/well in an 8-well Lab-Tek chamber; ThermoFisher, Rockford, IL, USA) infected with adenoviruses were seeded on growth factor-reduced Matrigel (BD Biosciences, San Jose, CA, USA). Images were captured 6 and 12 h after ECs were seeded.(8) The total area of tube-like structures formed by HUVECs in each well was measured using ImageJ from the National Institutes of Health (http://rsbweb.nih.gov/ij/download.html).
Cell proliferation. Proliferation was measured by the incorporation of [3H]-thymidine to HUVECs. The HUVECs were seeded in 24-well plates 1 day before adenoviral infection. Values are presented as the mean ± SD (n = 3).
RNA isolation and RT-PCR. Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA, USA). Reverse transcription was carried out by using a First-Strand cDNA Synthesis kit (Takara). Polymerase chain reaction was carried out using ExTaq polymerase (Takara) as directed by the manufacturer. Primer sets used are shown in Table S1.
Identification of FAM96B as an interaction partner of E2-2. When we tried to identify HLH-interacting protein(s) by the yeast two-hybrid system using the HLH domain of Herp2 as a bait, FAM96B was isolated. Composed of 163 amino acids, FAM96B does not possess any known conserved domains. Among the HLH proteins examined, FAM96B clearly interacted with E2-2 in COS7 cells (Fig. 1a, and data not shown), although the expression of E2-2 was decreased in the presence of FAM96B (see below). This interaction between E2-2 and FAM96B led us to investigate whether E2-2 colocalizes with FAM96B. After MEECs were transfected with Flag-E2-2 and Myc-FAM96B, the subcellular localization was determined by fluorescence microscopy by staining E2-2 with Alexa488-conjugated goat anti-mouse IgG and FAM96B with Alexa555-conjugated goat anti-rabbit IgG. As seen in Figure 1(b), FAM96B could colocalize with E2-2 in the nucleus, which is consistent with an interaction between the two proteins. Id1 has been reported to interact with E2-2 to rescue E2-2-mediated inhibition of angiogenic reaction.(8) As Id1 induced by bone morphogenetic protein (BMP) or VEGF potentiates angiogenesis,(8,21,23,24) we tested the possibility that the expression of FAM96B is enhanced by BMP or VEGF stimulation. Instead of BMP stimulation, we infected ALK6ca/HA, which successively transduces BMP signaling into the cells without the ligands, into cells. As seen in Figure 1(c), FAM96B mRNA was augmented upon BMP signaling in a similar fashion to Id1. The expression of E2-2 mRNA was not changed upon ALK6 activation (Fig. S1). Furthermore, Id1 mRNA was induced in endothelial cells by VEGF as expected, whereas VEGF did not enhance the expression of FAM96B transcript (Fig. S2). It remains unknown why the stimulation of BMP, but not that of VEGF, accelerated the expression of FAM96B transcript in endothelial cells. This requires further exploration in future experiments.
Inhibitory effect of FAM96B on E2-2-mediated promoter activity. The MCKpfos-luc reporter construct consisting of four E-box elements has been used to investigate the function of E proteins.(18) Our previous studies reported that Id1 and SCL/TAL1, which are interaction partners of E2-2, could counteract E2-2-mediated MCKpfos-luc reporter activity.(7,8) To examine the possibility that FAM96B affects the MCKpfos-luc reporter activity induced by E2-2, we transfected a different amount of FAM96B expression vector into CPAE cells with or without E2-2. FAM96B inhibited the E2-2-mediated activity of this reporter in a dose-dependent manner (Fig. 2a). To further confirm that FAM96B influences the transcriptional activity of E2-2, we used the pGL2b-VEGFR2-luc (−166 bp/+267 bp) luciferase reporter construct, which E2-2 negatively controls.(7,8) Consistent with the results shown in Figure 2(a), FAM96B dose-dependently released the E2-2-mediated inhibition of pGL2b-VEGFR2-luc (−166 bp/+267 bp) luciferase activity (Fig. 2b). These results verified the possibility that FAM96B interferes with the transcriptional regulation of E2-2.
Activation of EC by FAM96B. E2-2 has been reported to hamper the expression of VEGFR2 in ECs to keep ECs quiescent, whereas Id1 and SCL/TAL1 rescue the E2-2-mediated suppression of ECs.(7,8) Migration, proliferation, survival, and differentiation in ECs are considerably altered during the process of EC activation. To investigate the effect of FAM96B on EC migration, we infected HUVECs with either GFP- or FAM96B-expressing adenoviruses prior to seeding the ECs on a fibronectin-coated filter (Fig. 3a). We then counted the number of ECs under the chamber. As seen in Figure 3(b), FAM96B potentiated the EC migration. Again, the overexpression of FAM96B in HUVECs decreased the E2-2 protein (Fig. 3a). We also tested whether FAM96B affected cell proliferation and network formation in ECs after ECs were infected with either GFP- or FAM96B-expressing adenoviruses. Overexpression of FAM96B in ECs potentiated both the cell growth (Fig. 3c) and the formation of cord-like structures (Fig. 3d,e). Thus, these observations indicate that, in contrast to E2-2, FAM96B has the ability to keep ECs active.
Effect of FAM96B on the expression of E2-2. Because ectopic FAM96B seems to influence the expression of E2-2 protein in COS7 cells (Fig. 1a) and HUVECs (Fig. 3a), we further investigated whether the expression of E2-2 protein is attenuated by the ectopic expression of FAM96B. When Myc-FAM96B was overexpressed with Flag-E2-2 in COS7 cells, the expression of Flag-tagged E2-2 was diminished with the increasing expression of Myc-FAM96B (Fig. 4a), and vice versa (Fig. 4b). Thus, the decrease of E2-2 expression mediated by FAM96B was not dependent on the tags. Furthermore, the ectopic expression of FAM96B decreased the endogenous E2-2 protein in NIH3T3 cells (Fig. 4c). As E12/47 belongs to the E protein family, we also tested whether FAM96B affected the expression of the E47 protein. As expected, the expression of E47 protein was also decreased by FAM96B to the same extent as that of E2-2. However, neither Smad2 (Fig. 4d) nor c-Myc (Fig. S3) expression was altered by FAM96B. Therefore, this finding strongly suggests that FAM96B is able to specifically affect the protein expression of the E protein family.
Exploration of the functional domain in FAM96B. To find the functional domain in FAM96B required for the decrease of E2-2 protein expression, we made two FAM96B mutants, termed FAM96B (42-121) and FAM96BΔ (42-121) (Fig. 5a). Then we explored which part(s) of FAM96B can affect the expression of E2-2 protein. As seen in Figure 5(b), FAM96B (42-121), but not FAM96BΔ (42-121), could decrease the expression of E2-2 even if the expression of FAM96B (42-121) was relatively lower than that of FAM96BΔ (42-121). Collectively, the domain from Asp42 to Ile121 in FAM96B is necessary for FAM96B to decrease the expression of E2-2.
Members of the E protein family, such as E2-2 and E12/47, are widely expressed and act as either transcriptional activators or transcriptional repressors, depending on the partner(s) with which the E protein family can interact.(13,25–27) E proteins are known to play key roles in the regulation of cell proliferation and differentiation. For example, E2-2 and E12/47 play critical roles in B-cell development and perturbed T-cell development.(28–30) In our previous studies, E2-2 kept ECs quiescent, whereas the HLH proteins Id1 and SCL/TAL1 were capable of overcoming the inhibitory action of E2-2 on EC functions.(7,8) In the present study, FAM96B, which is not a member of the HLH protein family, was found to decrease the expression of E2-2 protein in cells so as to perturb E2-2-mediated cellular responses such as transcriptional activities and EC quiescence. The experiments using FAM96B mutants predicted that the middle region of FAM96B is critical for it to show limited expression of E2-2 protein. Although FAM96B does not have any known conserved domains, the middle region of FAM96B shows similarity to the “domain of unknown function 59 (DUF59)” in prokaryotes. Thus, to the best of our knowledge, this is the first report to define the functional significance of DUF59.
A number of proteins in cells can be degraded through either the proteasomal or the lysosomal pathways,(31) so we investigated whether FAM96B can also decrease the expression of the E2-2 protein through these pathways. However, the decrease of E2-2 expression mediated by FAM96B was not rescued by inhibitors specific for either proteasomes or lysosomes (Figs S4,S5). As another possibility, we checked the transcript of E2-2 with or without FAM96B in the absence or presence of actinomycin D. However, the transcript of E2-2 was not altered by FAM96B (data not shown). These results indicate the possibility that FAM96B can activate a certain protease(s) that targets E2-2. Because we do not know the exact mechanism by which FAM96B can diminish the expression of the E2-2 protein, several possibilities still need to be explored. Furthermore, it is interesting to analyze whether FAM96B can be regulated at a transcriptional and/or a translational level by itself.
In a previous study, we found that E2-2 interfered with EC activation, whereas its interaction partners Id1 and SCL/TAL1 overcame the function of E2-2 and activated ECs.(7,8) FAM96B was also found to be a BMP-inducing gene, like Id1, in our present study. Therefore, we assumed that FAM96B could also potentiate the function of ECs. Indeed, we were convinced that FAM96B can promote EC activation. Importantly, the expression of endogenous E2-2 protein was diminished by FAM96B, indicating that FAM96B restricts the expression of E2-2 protein to control E2-2-mediated cellular responses in ECs. Our hypothesis can be further supported by the evidence that FAM96B could rescue the E2-2-mediated suppression of the VEGFR2 promoter (Fig. 2b and Ref. 8). Thus, it is highly possible that the protein expression of VEGFR2 is influenced by FAM96B. If we successfully produce knockout mice specific for the FAM96B gene, we will be able to validate the possibility. As E2-2 is ubiquitously expressed in cells, it would be interesting to know whether FAM96B regulates the function of E2-2 through the control of E2-2 expression in cell types other than ECs. Our preliminary study indicated that FAM96B could not compete with Id1 for interaction with E2-2, despite the fact that both Id1 and FAM96B can associate with E2-2. Therefore, the function of E2-2 might be fine-tuned at different steps within cells through, for example, the regulation of its protein expression, or its DNA binding ability.
In conclusion, we discovered a novel mechanism by which FAM96B impairs the expression of E2-2 protein in ECs to potentiate EC activation. The identification of compounds that bolster E2-2 activity through the activation of FAM96B in ECs may provide an exciting opportunity to modulate tumor angiogenesis for therapeutic intervention.
This research was supported by Grants-in-aid for Scientific Research (17390073, 20012007 and 21590328) (M.K. and S.I.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, a Grant-in-Aid for JSPS Fellows from the Japan Society for the Promotion of Science (W.Y. and F.I.), the Takeda Science Foundation (S.I.), and the Naito Foundation (S.I.). We thank Ms. F. Miyamasu for excellent English proofreading.