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VASCULAR BIOLOGY
1Department of Immunobiology and Vascular Biology and Transplantation Program, Yale University School of Medicine, New Haven, Connecticut; and Departments of 2Immunology and 3Cancer Biology, University of Texas and M. D. Anderson Cancer Center, Houston, Texas
Submitted 9 February 2007 ; accepted in final form 2 August 2007
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ABSTRACT |
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cardiovascular; mitogen-activated protein kinase; embryonic development
B kinase (IKK)/NF-B pathway (22, 36, 47).
Previously, we have reported that MEKK3 plays an important role in early embryonic cardiovascular development, especially in angiogenesis and heart development (46), by activating myocyte-specific enhancer factor (MEF)2c, a transcription factor crucial for early embryonic development (1, 29), through the p38 MAPK cascade (46). MEF2c has also been shown to be a downstream target of ERK5, one of the major MAPKs activated by MEKK3 and MEKK2 via MEK5 (3, 5, 21, 25). Disruption of ERK5 has been shown to cause multiple extraembryonic vascular and embryonic cardiovascular defects due to failure of endothelial cells (ECs) (21, 32, 44). Moreover, targeted deletion of ERK5 in adult mice led to lethality due to an EC failure (21). A study (31) on p38-
knockout mice showed that p38- has an essential role in placenta angiogenesis but not embryonic cardiovascular development. However, since there are different isoforms of p38 in mammalian cells (19, 23, 28, 43), other p38 isoforms other than p38- may function in embryonic cardiovascular development. In addition, MEKK3 has also been found to play a balancing role between pro- and antiapoptotic signals in ECs by regulating the cross-talk between the phosphatidylinositol 3-kinase (PI3K)/Akt and p38 signaling pathways (17). Both PI3K/Akt and p38 pathways are known to play important roles in ECs during vascular development (16, 18, 31). Thus, both biochemical and genetic studies have suggested that the two MAPK modules, MEKK3-ERK5-MEF2c and MEKK3-p38-MEF2c, may function in embryonic cardiovascular development.
Tie2 is an EC-specific receptor tyrosine kinase that plays an important role in embryonic vascular development (34). Disruption of Tie2 in mice resulted in embryonic lethality at approximately embryonic day 10.5 (E10.5) due to cardiac failure, hemorrhage, and other vascular defects (34). Tie2 is dimerized, autophosphorylated, and activated by binding to its ligand, angiopoietin-1 (Ang1), in ECs (30, 35, 41). Mice deficient for Ang1 have a similar phenotype as Tie2-deficient mice (38). Ang1 is an EC-specific mitogenic factor and is able to activate the MAPKs p38, ERK1/2, and JNK in cultured ECs (20, 24). However, the mechanism of MAPK activation by Ang1/Tie2 is still unknown. Since the phenotype of Mekk3-deficient mice resembles that of Tie2- and Ang1-deficient mice, it is likely that MEKK3 may be responsible for transducing Ang1/Tie2 signals to downstream MAPKs.
In this study, we further characterized the development of the vascular system in Mekk3-deficient embryos and determined the role of MEKK3 in ECs. We found that the proliferation and survival of Mekk3-deficient ECs were impaired. Interactions of Mekk3-deficient ECs with the myocardium were also affected in Mekk3-deficient embryos. Consistently, the dorsal aorta in mutant embryos was dilated or disrupted, suggesting a lack of support of ECs by smooth muscle cells (SMCs). At the biochemical level, we found that MEKK3 is required for Ang1-induced p38 and ERK5 activation.
Because angiogenesis plays a critical role in tumor development and growth (13), we also investigated if MEKK3 is involved in these processes. We found that Mekk3-deficient tumors grew at the same incidence and rate as Mekk3 wild-type tumors. Mekk3-deficient tumors produced similar levels of VEGF and induced angiogenesis in a manner indistinguishable from control wild-type tumors. Collectively, these results suggest that while MEKK3 is a key intracellular signaling molecule for ECs, it is not essential for tumor angiogenesis or growth of tumors.
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MATERIALS AND METHODS |
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All procedures in this study were performed in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996), and the protocol was approved by the Institutional Animal Care and Use Committee of the University of Texas and M. D. Anderson Cancer Center.
Immunohistochemistry. Embryos and yolk sacs for immunohistochemical staining were embedded in paraffin and cut into 6- to 7-µm-thick sections. Tissues for frozen sections were embedded in OCT (Tissue-Tek) and cut into 8-µm-thick sections. Frozen sections were fixed in acetone and acetone-chloroform. The hematoxylin and eosin staining procedure followed a standard protocol. Immunohistochemical staining with anti-smooth muscle actin (SMA; 1A4, Sigma), anti-PECAM (MEC13.3, Pharmingen), anti-VEGF (A20, Santa Cruz Biotechnology), and anti-proliferating cell nuclear antigen (PCNA) antibodies (BD Biosciences) was performed by basic immunoperoxidase procedures.
Bromodeoxyuridine incorporation assay. Mice carrying E10 embryos were injected with 250 µl of 250 µg/ml bromodeoxyuridine (BrdU; Sigma) through their tail veins. After 1.5–2 h, mice were killed, and embryos were collected. Embryos were fixed in 70% ethanol in PBS, embedded in paraffin, and cut into 6- to 7-µm sections. After deparaffination, tissues were treated with 1% Triton X-100 in PBS, 2 M HCl in PBS, and 0.1 M Tris·HCl (pH7.6) successively, followed by basic immunoperoxidase procedures with anti-BrdU antibody (B44, Becton Dickinson).
TUNEL assay. E9.5 embryos were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Apoptotic cells were detected by TUNEL assay using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions.
Cell culture. Wild-type and Mekk3-deficient mouse embryonic fibroblasts were prepared and cultured in 10% FBS in DMEM as previously described (47). Brain ECs (BECs) were maintained as previously described (14).
Plasmid construction. For construction of pHA-Fv-Tie2K plasmid, the intracellular kinase domain of Tie2 (amino acids 771–1122) was amplified by PCR and inserted into the vector pSH1/SN-E-Fv-Fvls-E (6, 15) at the SalI site to allow an in-frame fusion of the intracellular kinase domain of Tie2 (Tie2K) with the FKBP12V36 (Fv) domain from the vector.
Lentiviral infection and transient transfection. Production of the Mekk3 short interfering (si)RNA-producing lentivirus was performed as previously described (26). For lentiviral infection, 3 x 105 BECs were plated in a six-well plate and cultured at 37°C in 5% CO2 overnight. The overnight-cultured BECs were then centrifuged with lentivirus at a multiplicity of infection of 10 at 2,300 rpm for 90 min in the presence of 8 µg/ml polybrene. Viral supernatants were removed and replaced with 3 ml fresh DMEM plus 10% FBS containing antibiotics 6 h after centrifugation. Seventy-two hours later, MEKK3 protein levels in infected cells were determined by immunoblot analysis.
For transient transfection of mouse embryonic fibroblasts, 1 µg of pCMV-flag-p38 was cotransfected with 2 µg of pHA-Fv-Tie2K or empty vector pSH1/Sn-E-FV-Fvls-E using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Transfected cells were analyzed 36 h later.
Chemically induced dimerization. Synthetic dimerizer AP20187-mediated HA-Fv-Tie2K dimerization was carried out as previously described (6, 12).
Immunoblot analysis. Mouse embryonic fibroblasts cultured in a six-well plate were starved for 12 h before being stimulated with 100 nM AP20187 or vehicle control. BECs were also starved for 12 h before being stimulated with 100 ng/ml recombinant human Ang1 (R&D) for the indicated time periods. Cell lysates were prepared and analyzed by immunoblot analysis as previously described using antibodies against phosphorylated (p)-p38, p-JNK, and ERK5 (Cell Signaling Technology, Boston, MA); against p38 and JNK1 (Santa Cruz Biotechnology), against MEKK3 (BD Biosciences); and against pTyr (PY20; Transduction Laboratories). Anti-p-MEKK3 was as described in our previous study (48).
Growth of wild-type and Mekk3–/– tumors. Tumor-like wild-type and Mekk3–/– embryonic stem (ES) cells were prepared and characterized as described in Ref. 46. Tumor-like ES cells (1 x 106) were injected subcutaneously into nude mice, and tumor growth was monitored 3 times/wk using calipers. Tumor volume was calculated by the following formula: volume = W2 x L/2, where W is the shortest diameter and L is the longest diameter. For each experiment, 3 mice/group were used, and statistical analysis was performed.
Microvessel density of wild-type and Mekk3–/– tumors. To determine the extent of vascularization in ES cell-derived tumors, blood vessels were stained with anti-PECAM antibody. The microvessel density was counted in multiple sections of at least three subcutaneous tumors from each cell line, and SEs were calculated.
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RESULTS |
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Mekk3 deficiency causes decreases in cell proliferation. Because MEKK3 is a major intracellular signaling molecule involved in transducing many receptor-mediated signals, it is possible that in Mekk3-deficient embryos, the upstream receptor-mediated signals were blocked, which may lead to defective cell function. A previous study (11) in cell lines suggested that MEKK3 might be involved in regulating cell proliferation. To address this question, we determined cell proliferation in wild-type and Mekk3-deficient E9.5 yolk sacs by immunohistochemical staining (using anti-PCNA) and BrdU incorporation and staining assays. As shown in Fig. 2A, cell proliferation in Mekk3–/– yolk sacs was significantly decreased compared with that in wild-type yolk sacs. To determine the percentage of proliferating ECs, multiple sections (>10 blood islands each) of wild-type and Mekk3–/– yolk sacs were counted. The percentage of BrdU-positive cells in Mekk3–/– yolk sacs was 7% compared with 51% in wild-type yolk sacs (Fig. 2B).
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Mekk3 deficiency does not affect tumor-like cell growth. Since MEKK3 plays an essential role in embryonic angiogenesis, we then asked if MEKK3 is also required for tumor growth and tumor-induced angiogenesis. We established both wild-type and Mekk3–/– tumor-like cell lines from ES cells (46). ES cells are pluripotent cells derived from early mammalian embryos and possess tumor-like properties. If large numbers of ES cells are transplanted, they produce tumors (teratomas) (39).
Under normal growth condition, both wild-type and Mekk3–/– tumor-like cell lines grew similarly in vitro (data not shown). To further determine if MEKK3 is required for tumor growth in vivo, we implanted wild-type and Mekk3-deficient tumor-like ES cells subcutaneously into nude mice. Tumor growth was monitored from implanted mice initially at day 5 and continually through day 25, and tumor sizes were analyzed. As shown in Fig. 5A, the growth rates of wild-type and Mekk3-deficient tumors were almost identical, suggesting that Mekk3 is not required for tumor growth in vivo.
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Mekk3 deficiency does not affect VEGF production. Tumor cells are capable of producing VEGF, one of the most important angiogenic growth factors functioning in tumor angiogenesis (13). Therefore, we determined if the production of this angiogenic factor was affected in Mekk3-deficient tumors. As shown in Fig. 6A, we found that the tumors developed in nude mice implanted with either wide-type or Mekk3-deficient ES cells expressed similar levels of VEGF, as determined by immunohistochemical staing. To further confirm that the protein level of VEGF was not affected by Mekk3 disruption, we also determined VEGF protein levels using immunohistochemical staining in embryos (Fig. 6B). Again, we found similar levels of VEGF in E9.5 wild-type and Mekk3-deficient embryos, consistent with our previous finding showing that Mekk3 deficiency did not affect mRNA levels of VEGF and bFGF in embryos (46). Taken together, these results suggested that MEKK3 is not involved in regulating VEGF expression and may be also not required for other blood vessel growth factor productions.
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DISCUSSION |
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Although early EC development through vasculogenesis was not affected in the absence of MEKK3, our previous study (46) indicated that there is an intrinsic defect in Mekk3–/– ECs that affects their ability to generate new vasculature even in the presence of normal levels of vascular and angiogenic factors. MEKK3 has been suggested to play a role in regulating cell proliferation (11) and apoptosis (17). In this study, we found decreased cell proliferation in Mekk3–/– ECs in vivo (Fig. 2). Interestingly, this defect seems to be EC specific, since we found Mekk3-deficient tumor cells grew at a similar rate as wild-type tumors both in vitro and in vivo (Fig. 5). Furthermore, we found increased apoptosis in Mekk3-deficient ECs (Fig. 3). These results suggest that MEKK3 may play an intrinsic role in EC proliferation and survival.
To begin understanding the molecular mechanism of MEKK3 signaling in ECs, we used siRNA to knock down MEKK3 expression in EC lines. These experiments showed that MEKK3 is required for both p38 and ERK5 MAPK activation by Ang1 (Fig. 4). In control siRNA-infected BECs, p38 activation was nicely induced by Ang1, but this induction was clearly abolished in MEKK3 knockdown BECs (Fig. 4C). Importantly, we found that activation of ERK5, a major downstream target of MEKK3 during embryonic cardiovasulcar development (5, 21, 32), was severely impaired in Mekk3 knockdown BECs (Fig. 4C). This result suggests that the Ang1/Tie2-MEKK3-ERK5 axis may be a major functional module in angiogenesis. Interestingly, the basal phosphorylation level of p38 was slightly augmented in Mekk3 knockdown BECs. This might be due to MEKK2 compensation, since MEKK2 shares almost identical catalytic domains with MEKK3 (6, 48). We did not observe a significant defect in JNK activation in this assay, suggesting that other MAP3Ks might be involved in JNK activation during angiogenesis. Alternatively, JNK activation by Ang1/Tie2 may not be the dominant MAPK cascade.
To further determine the role of MEKK3 in Ang1/Tie2 signaling, we asked if MEKK3 is required for Tie2, a receptor tyrosine kinase for Ang1, signaling to the downstream targets. Tie2 consists of an extracellular ligand binding domain for angiopoietins (Ang1–4) and an intracellular kinase domain for signaling (35). Following ligand Ang1 binding, Tie2 becomes dimerized and autophosphorylated on its kinase domain, which leads to the activation of downstream effectors, including MAPK cascades (30, 35). We used a chemical-inducible system to artificially activate Tie2 in either wild-type or MEKK3-deficient cells. Our results showed that MEKK3 is critical for Ang1/Tie2 signaling to the p38 MAPK pathway (Fig. 4D). Taken together, the above experiments showed that at least part of the MEKK3 signaling is mediated by p38 and ERK5 MAPKs in response to angiogenic factor Ang1 stimulation. Since both p38 (4) and ERK5 (42) MAPK pathways are involved in controlling cell proliferation and survival in response to growth factors and developmental signals, the above results also suggest that the activation of these two MAPK pathways by MEKK3 during early angiogenesis may play a critical role in EC proliferation and survival, thus partially explain the phenotype of MEKK3-deficient embryos.
Precisely how MEKK3 is involved in regulating the early cardiovasculogenesis remains largely unknown. Since MEKK3 is ubiquitously expressed, disruption of the Mekk3 gene in mice may indirectly affect blood vessel development. Future investigation with EC-specific conditional Mekk3 knockout mice is needed to formerly confirm the crucial intrinsic role of MEKK3 in ECs.
Understanding the function of MEKK3 may provide new strategies for developing drugs to control angiogenesis and perhaps tumor growth. Thus, we also investigated the role of MEKK3 in tumor growth and angiogenesis in this study. We established both wild-type and Mekk3–/– tumor-like ES cell lines and implanted them into nude mice, where tumors developed due to the tumor-like properties of ES cells. Although we found that Mekk3-deficient mouse embryonic fibroblast cells grew slower than wild-type cells under normal culture conditions in vitro (data not shown), surprisingly we did not find any difference in the growth of tumors derived from both kinds of ES cells (Fig. 5A).
Tumor angiogenesis, the formation of new blood vessels in tumor tissues, provides enough nutrition for tumor growth. Since Mekk3 deficiency in tumors did not affect tumor growth, we further examined tumor angiogenesis in wild-type and Mekk3–/– ES cell-derived tumors. Consistent with the results in tumor growth, no differences were found in tumor angiogenesis of Mekk3–/– tumors (Fig. 5, B and C). These results suggest that MEKK3 is dispensable for both tumor growth and angiogenesis. Our study also suggests that MEKK3 is not a good target in tumor cells for therapeutic treatment of cancer, although it is an important regulator of angiogenesis.
Angiogenesis requires coordinated action of a variety of growth factors, and tumor cells produce many of these factors, including VEGF, one of the most important growth factors involved in both tumor and embryonic angiogenesis (13). We found that the production of VEGF protein was similar in wild-type and Mekk3–/– tumors (Fig. 6A). We also did not find any differences in VEGF protein levels in wild-type and Mekk3–/– embryos (Fig. 6B), consistent with our previous finding at the mRNA level (46). Since the newly formed blood vessels in tumors are derived from host tissues that have the wild-type Mekk3 gene, these data suggest that both tumor types are able to produce sufficient angiogenic growth factors for new blood vessel formation.
Together, our study showed that MEKK3 is required for EC proliferation, survival, and interactions with the myocardium during early embryonic development, but is not essential for tumor growth and angiogenesis. This study also suggests that MEKK3 might be a better target for antiangiogenic therapy in ECs rather than in tumor cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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