|Home | About | Journals | Submit | Contact Us | Français|
Nuclear Factor of Activated T-cells cytoplasmic 1 (NFATc1) activity in endocardial cushion (ECC) endothelial cells is required for normal ECC growth and extracellular matrix (ECM) remodeling during heart valve development.
The mechanisms of NFATc1 activation and downstream effects on cell proliferation and ECM remodeling enzyme gene expression were examined in NFATc1 mutant mice and chick ECC explants.
NFATc1−/− mice display reduced proliferation of ECC endothelial and mesenchymal cells at embryonic day 10.5, while myocardial cells are unaffected. Vascular Endothelial Growth Factor A (VEGF) activates NFATc1 and promotes ECC cell proliferation via the regulatory phosphatase, Calcineurin (Cn), and MAPK-ERK Kinase 1 - Extracellular signal-Related Kinase 1/2 (MEK1-ERK1/2)-dependent signaling. As ECCs mature, Receptor Activator of NFκB Ligand (RANKL) and the ECM remodeling enzyme, Cathepsin K (CtsK), are expressed by ECC endothelial cells. RANKL inhibits VEGF-induced cell proliferation while causing increased expression of CtsK via Cn/NFATc1 and c-Jun N-terminal Kinase 1/2 (JNK1/2)-dependent signaling.
These data support a novel mechanism for the transition from ECC growth to remodeling in which NFATc1 promotes a sequential pattern of gene expression via cooperation with ligand-specific cofactors such as MEK1-ERK1/2 or JNK1/2.
Congenital malformations of cardiac valves affect 1–2% of the population; however, the molecular mechanisms that govern valve development are still not completely understood. 1 Cardiac valvulogenesis is a complex process that begins with the formation of endocardial cushions (ECCs) in the atrioventricular canal and outflow tract regions of the looping heart. The ECCs are populated as overlying endothelial cells delaminate, undergo epithelial to mesenchymal transformation (EMT), and invade the extracellular matrix (ECM). 2 During EMT, ECC endothelial cells are highly proliferative, and increased mitotic index is a feature of both endothelial and mesenchymal cells of the developing valves. 2,3 After outgrowth, the ECM of the valves is remodeled into a highly organized, trilaminar architecture characteristic of mature cardiac valves. During ECM remodeling, valve interstitial cell proliferation is decreased and endothelial cells express ECM remodeling enzymes. 4,5 It is clear that precise regulation of valve cell cycle, growth, and remodeling is required for normal valve development, as alterations in these processes are linked to valve defects. 2,6,7 While much is known about signaling mechanisms regulating ECC formation and EMT, mechanisms governing the transition from growth to remodeling in the developing valves are relatively uncharacterized.
Nuclear Factor of Activated T-cells cytoplasmic 1 (NFATc1/NFAT/NFATc/NFAT2), a transcription factor of the NFAT family, functions in development and homeostasis of the brain, skeleton, immune system, and heart. 8 In both mouse and chick embryos, cardiac NFATc1 expression is specific to ECC endothelial cells and overlaps with expression of its regulatory phosphatase Calcineurin (Cn). 7,9,10 NFATc1 null mice, or those lacking NFATc1 expression specifically in endothelial cells, have normal ECC formation and EMT, however, these ECCs fail to grow and remodel, resulting in lethality at embryonic day (E)12.0–E14.5. 9,11 Mice lacking expression of Cnb1 specifically in endothelial cells or chick embryos treated with the Cn inhibitor Cyclosporin A (CsA), just prior to cushion growth, phenocopy NFATc1−/− mouse models, thereby illustrating the importance of the Cn-NFATc1 interaction for valve growth and remodeling. 7,10 A necessary spatiotemporal window for Cn-NFATc1 activation in ECC endothelial cells has been defined, however, upstream effectors and intersecting pathways of NFATc1 in ECC endothelial cells during this critical period were not previously identified.
Vascular endothelial growth factor A (VEGF/VEGFA/VEGF165) is critical for development and maintenance of heart, lung, and vascular tissues. 12–14 VEGF levels must be tightly controlled during cardiac valve morphogenesis, as VEGF signaling maintains the ECC endothelial cell layer during ECC formation, but is also a potent inhibitor of EMT. 6,15 VEGF is expressed by myocardium and ECC endothelial cells during ECC formation and growth, however, expression is extinguished during ECC remodeling. 14 While it is known that under or overexpression of VEGF in ECC endothelial cells disrupts ECC morphogenesis, the role of VEGF in post-EMT ECC growth is not known. 15 VEGF/NFATc1 signaling has been implicated in homeostasis of valve endothelial tissue, as VEGF treatment of adult human pulmonary valve endothelial cells (HPVECs) increases cell proliferation. 16 However, VEGF regulation of NFATc1 in ECC cells during development has not been demonstrated.
Receptor Activator of NFκB Ligand (RANKL/TRANCE/TNFSF11/OPGL/ODF) is a member of the TNF family of signaling molecules that is best known for its role in promoting osteoclast differentiation and production of ECM remodeling enzymes, such as Cathepsin K (CtsK). 17,18 In myxomatous and diseased human mitral valves containing high levels of fragmented collagen and elastin, CtsK expression is upregulated by interstitial cells. 19 Consistent with RANKL/NFATc1 pathway activity during valve remodeling, CtsK is normally expressed by murine valve endothelial cells at E13.5. 5 However, CtsK is not expressed in NFATc1 null embryos. 5 Conserved spatiotemporal expression of RANKL/NFATc1-related genes among vertebrates has not been previously demonstrated. Likewise, the ability of RANKL to promote CtsK transcription in a Cn-dependent manner in ECC cells has not been tested.
The present study examines the relationship of VEGF and RANKL signaling mechanisms in the regulation of NFATc1 in cardiac valve growth and remodeling. In isolated avian ECC cultures, VEGF signaling promotes cell proliferation via activation of Cn/NFATc1 together with MAPK-ERK Kinase1-Extracellular signal-Related Kinase1/2 (MEK1-ERK1/2) copathways. In vivo, NFATc1 is necessary for normal ECC growth, as mice lacking NFATc1 expression have decreased ECC endothelial cell proliferation at E10.5. In addition, RANKL/NFATc1 signaling is conserved among avian and mammalian embryos and RANKL acts during valve remodeling to promote CtsK expression via activation of Cn/NFATc1 and Jun N-terminal Kinases1/2 (JNK1/2) signaling. These data suggest NFATc1 plays a central role in the transition from ECC cell proliferation, in response to VEGF signaling, to ECM remodeling enzyme production, in response to RANKL signaling. VEGF/NFATc1 signaling promotes cell proliferation and not CtsK expression via cooperation with MEK1-ERK1/2-dependent cofactors, while RANKL/NFAT signaling inhibits cell proliferation and increases CtsK expression via cooperation with JNK1/2-dependent cofactors.
E10.5 NFATc1 mouse embryos were collected and genotyped as previously described. 11 Proliferative indices of ECC cells were determined via quantification of bromodeoxyuridine (BrdU) labeled nuclei versus total nuclei in paraffin embedded mouse heart sections. Explanted superior and inferior atrioventricular ECCs, isolated from Hamburger and Hamilton stage 25 (HH25; E4.5) chicken embryos,20 were maintained in culture for 2–7 days with one or more of the following treatments added to the culture media: VEGF (R&D Systems) 50ng/mL, soluble Flt receptor chimera (sFlt1) (R&D Systems) 50ng/mL, RANKL (R&D Systems) 800ng/mL, Osteoprotegerin (OPG) (R&D Systems) 1ug/mL, Cyclosporin A (CsA) (Novartis) 1ug/mL, Bovine Serum Albumin (BSA) (Sigma) 100–800ng/mL, U0126 (Promega) 10uM, SP600125 (Calbiochem) 2.5uM, and/or DMSO (Sigma) 0.005–0.01%. Real Time RT-PCR for CtsK expression and in situ hybridization for NFATc1, RANKL, and CtsK mRNA were performed as previously described. 21–23 An expanded materials and methods section is included as an online supplement at http://circres.ahajournals.org/.
During ECC formation, endothelial cells populate the ECC mesenchyme by undergoing EMT and migrating into the cardiac jelly. ECCs then enter a growth period (mouse E10.5–E13.5) during which endothelial and mesenchymal cells of the ECC are highly proliferative. 3,4 In order to determine if NFATc1 is necessary for ECC endothelial cell proliferation, the proliferative index of ECC endothelial cells was examined in NFATc1+/+, +/− and −/− E10.5 mouse embryos. Pregnant NFATc1+/− females were injected intraperitoneally with BrdU labeling solution. The proliferative index of endothelial cells overlying ECCs along with ECC mesenchymal cells and ventricular myocytes was assessed for at least six embryos of each genotype isolated from separate litters. Adjacent sections were labeled with MF20 antibody in order to visualize the myocardial boundaries of atrioventricular ECCs (Figure 1A–C). Wild type (NFATc1+/+) and heterozygous (+/−) mice have 32% and 30% of ECC endothelial cell nuclei labeled with BrdU respectively, while NFATc1 null (−/−) littermates have a significantly lower (21%) percentage of BrdU labeled nuclei (Figure 1D). Decreased proliferation also was observed in ECC mesenchymal cells that arise from ECC endothelial cells via EMT (Figure 1D). 4 The proliferative defect observed is not due to general embryo failure, as no differences were detected in the mitotic index of ventricular myocytes among genotypes (Figure 1D). It is important to note that cardiac myocytes do not express NFATc1. This suggests that Cn/NFATc1 signaling is necessary during ECC formation and growth for normal ECC cell proliferation. No difference in the number of apoptotic cells were observed in ECCs of NFATc1+/+, +/−, or −/− E10.5 mice, as determined by anti-Cleaved Caspase3 labeling (data not shown). These results indicate that the hypoplastic ECC phenotype observed in NFATc1−/− embryos at E13.5 is likely due to decreased proliferation of ECC endothelial and endothelial-derived mesenchymal cells as early as E10.5, when these embryos are grossly indistinguishable from NFATc1+/+ and +/− littermates.
VEGF regulates endothelial cell adhesion, cell cycle, and inflammatory cell recruitment during development and throughout postnatal life. 13 The ability of VEGF to increase NFATc1 nuclear localization and cell proliferation was examined in ECC cells isolated from HH25 (E4.5) chicken embryos and placed in cell culture. The avian ECC culture system was used because HH25 chick embryos have much larger ECCs than murine embryos at the same stage in development, large numbers of synchronously staged ECCs can be collected at one time, and cultured ECC explants can be treated with cytokines or inhibitors to manipulate developmental pathways without the confounding effects of myocardial or systemic interactions. 3,21,23 These explants contain valve progenitor cells as well as precursors of septum intermedium. 2 NFATc1-positive ECC cells are endothelial as indicated by co-expression with endothelial markers such as Sox17 and VEGFR2 (Online Figure IA-A″; data not shown). 12,24 NFATc1-positive cells from non-dissociated ECC explants exist as clusters of Sox17 or VEGFR2 positive endothelial cells surrounded by Smooth Muscle α-Actin positive, MF20, and Sox17 negative mesenchymal cells. Myocyte contamination of cultures was not detected by immunofluorescence and confocal laser scanning microscopy (ICLSM) or Real Time RT-PCR. Isolated atrioventricular ECCs were treated with VEGF, soluble Flt1 receptor chimera (sFlt1, a VEGF inhibitor), Cyclosporin A (CsA, a Cn inhibitor), VEGF+sFlt1, VEGF+CsA, or BSA (as a vehicle control). Nuclear localization NFATc1 of was evaluated by ICLSM.
Cultures treated with VEGF contained significantly more cells with nuclear NFATc1 labeling than control cultures (76% vs 16%), indicating that VEGF promotes NFATc1 nuclear localization (Figure 2A–B, G). In contrast, cultures treated with VEGF+sFlt1 or sFlt1 alone had a comparable number of cells containing nuclear NFATc1 as control cultures, indicating that increased NFATc1 nuclear localization was a specific effect of VEGF treatment (Figure 2C, E, G). ECC cells treated with VEGF+CsA or CsA alone also showed low level NFATc1 nuclear localization comparable to controls, demonstrating that NFATc1 nuclear accumulation following VEGF treatment is Cn-dependent (Figure 2D, F–G). It is important to note that nuclear size and morphology in all cultures of this study were comparable and apparently normal with no evidence of toxicity. Since NFATc1 is predominantly expressed by endothelial cells of the ECC in vivo and in culture, these results demonstrate that VEGF treatment promotes NFATc1 nuclear localization in ECC endothelial cells.
To determine if VEGF induces proliferation of ECC cells, HH25 chick ECCs were cultured and treated with VEGF, VEGF+sFlt1, VEGF+CsA, sFlt1, CsA, or BSA. The proliferative index was calculated as the percent of total nuclei labeled with the M-phase marker anti-phosphohistone H3 (pHH3) antibody. Cultures treated with VEGF had a significantly higher percentage of pHH3 positive cells (4.4%) than BSA treated controls (2.3%) (Figure 3A–B, G). Proliferation indices from cultures treated with a combination of VEGF+sFlt, VEGF+CsA or sFlt1 alone were comparable to BSA treated controls, indicating that the increased proliferation is a specific effect of VEGF treatment and is Cn-dependent (Figure 3C–E, G). Cultures treated with CsA alone had a significantly lower percentage of pHH3 positive nuclei, which could be due to the ability of CsA treatment to block endogenous Cn/NFAT signaling and affect cell proliferation in the absence of added VEGF (Figure 3F–G). Endogenous expression of VEGFR2, the main receptor for VEGF signaling, is specific to ECC endothelial cells and it was determined that VEGR2 is localized to clusters of endothelial cells in culture (data not shown). 14,25 Similarly, the majority of pHH3 positive cells co-express endothelial markers such as Sox17 and NFATc1 in VEGF-treated cultures (Online Figure IB-C). Together, these data indicate that VEGF increases proliferation of ECC endothelial cells in a Cn-dependent manner.
In osteoclasts, RANKL/NFATc1 signaling promotes bone ECM remodeling by inducing expression of NFATc1 target genes, including the ECM remodeling enzyme CtsK. 17 RANKL/NFATc1 pathway components are expressed by murine ECC endothelial cells during physiological valve remodeling and by human ECC cells during pathological remodeling, suggesting that this pathway has a role in normal valve development and human disease mechanisms. 5,19,26 Since species-specific differences between mouse and chicken in TGFβ signaling molecules have been noted during ECC formation, we sought to determine that the spatiotemporal expression patterns of NFATc1, RANKL, and CtsK during valve growth and remodeling are conserved in chicken embryos. 2 In situ hybridization (ISH) revealed that in chicken embryos, NFATc1 is expressed by ECC endothelial cells throughout growth and remodeling (chicken E4.5–E14) (Figure 4A–B; data not shown). In contrast, RANKL and CtsK are absent in ECC but are expressed later during valve remodeling (E7), as detected by ISH (Figure 4C–F), or Real Time RT-PCR (data not shown), which correlates with data from mouse models.5 Previous studies indicate that, in mouse embryos, VEGF expression is upregulated in atrioventricular canal cells at E10.5, during ECC growth, and is depleted by E14.5. 14,15 Similarly in avian embryos, VEGF expression is upregulated in the atrioventricular canal during ECC growth. 27 Therefore, VEGF and NFATc1 are expressed during ECC growth, while RANKL/NFATc1 pathway components are not expressed until ECC remodeling in both chicken and mouse model systems. These findings suggest that VEGF/NFATc1 signaling during ECC growth and RANKL/NFATc1 signaling during valve remodeling are conserved mechanisms controlling valve development among vertebrates.
To examine the ability of RANKL to induce NFATc1 nuclear localization in ECC cells, avian ECCs were cultured and treated with RANKL, Osteoprotegerin (OPG, a soluble RANKL inhibitor), CsA, RANKL+OPG, RANKL+CsA, or BSA. The percentage of cells with nuclear NFATc1 was determined by ICLSM. Cells treated with RANKL had a significantly higher percentage of cells containing nuclear NFATc1 (37%) than BSA treated controls (16%), indicating that RANKL promotes NFATc1 nuclear localization in ECC cells (Figure 5A–B, G). Cells treated with RANKL+OPG or OPG alone had NFATc1 nuclear localization comparable to controls, indicating that increased nuclear NFATc1 is a specific effect of RANKL treatment (Figure 5C, E, G). ECC cells treated with RANKL+CsA or CsA alone also showed NFATc1 nuclear localization comparable to controls, demonstrating that NFATc1 nuclear accumulation induced by RANKL is Cn-dependent (Figure 5D, F–G). Together, these results indicate that RANKL treatment promotes NFATc1 nuclear localization in cultured ECC cells.
To determine if RANKL induces expression of the ECM remodeling enzyme CtsK in ECC cells, avian ECCs were explanted and cultured for 7 days with the aforementioned treatments added to media. In these experiments, addition of RANKL to cultured ECCs resulted in 7.1 fold higher CtsK mRNA expression than BSA treated controls, as detected by Real Time RT-PCR (Figure 5H). Treatment with RANKL+OPG or OPG alone yielded CtsK expression levels comparable to controls, indicating that increased CtsK expression is a specific effect of RANKL treatment (Figure 5H). Treatment with RANKL+CsA significantly inhibited the induction of CtsK expression, while CsA treatment alone had expression comparable to BSA (Figure 5H). For all Real Time RT-PCR experiments, GAPDH and β-actin mRNA levels prior to normalization were comparable among culture groups, indicating that cultures contained a similar number of live cells at collection. Taken together, these results indicate that RANKL promotes expression of CtsK via Cn signaling in ECC cells.
The results obtained for 7 day cultures are comparable to those obtained when cells were maintained in culture for 4 days. However, no induction of CtsK mRNA expression was observed at 48 hours, even though RANKL-induced NFATc1 nuclear localization at this time point (Figure 5B, G; data not shown). Expression of markers associated with ECC maturation were assessed for ECC explants maintained in culture for 2 days versus those cultured for 7 or 10 days. While NFATc1 expression remained comparable among all cultures, CtsK expression increased over the culture period. This increase in CtsK mimics gene expression of ECCs and valves in vivo (Online Figure IIA–B). Similarly, expression of Periostin and Versican in ECC cultures increased over time, while Scleraxis mRNA levels remain relatively unchanged. This pattern of gene expression closely resembles gene transcription in vivo (Online Figure IIA–B). Together, these data suggest that cultured ECC cells have a pattern of gene expression consistent with maturing valves in vivo and that RANKL-induced CtsK expression in ECC cells is not only ligand-dependent, but is time-dependent as well.
The specificity of VEGF and RANKL induction of cell proliferation and ECM remodeling enzyme expression was examined. In order to determine if VEGF/NFATc1 signaling can induce CtsK transcription, HH25 avian ECC cells were cultured in the presence of VEGF, VEGF+sFlt1, VEGF+CsA, sFlt1, or CsA. None of these treatment groups expressed increased Ctsk mRNA compared to BSA treated controls (Figure 6A). To determine if RANKL/NFATc1 signaling promotes ECC cell proliferation, ECCs were cultured in the presence of RANKL, RANKL+OPG, RANKL+CsA, OPG, CsA, or BSA. In these experiments, none of the treatment groups exhibited increased proliferation compared to BSA treated controls (Figure 6C). These results show that downstream effects of NFATc1 activation in ECC cells are ligand-dependent.
To examine the signaling hierarchy and crosstalk at the level of VEGF and RANKL receptors upstream of NFATc1, ECC explants were cultured and treated with VEGF+OPG (RANKL inhibitor) or RANKL+sFlt (VEGF inhibitor). These data demonstrated that VEGF-induced ECC cell proliferation does not require RANK receptor function (Online Figure IIIA), and likewise, RANKL-induced CtsK mRNA expression does not require VEGF receptor function (Online Figure IIIB). Therefore, VEGF and RANKL signaling act independently with separable downstream effects in ECCs, however, both VEGF-induced cell proliferation and RANKL-induced CtsK expression are Cn/NFATc1-dependent.
The above results are consistent with a mechanism whereby VEGF activation of NFATc1 promotes ECC proliferation, followed by RANKL activation of NFATc1 to induce ECM remodeling enzyme expression in maturing valves. Therefore, experiments were performed to examine the response of ECC cells in the presence of both VEGF and RANKL signals concurrently. Cells treated with VEGF+RANKL together had a proliferative index comparable to BSA treated controls (2.6% vs 2.3%) (Figure 6D). This is in contrast to VEGF-treated cultures that had a significantly higher proliferative index than BSA treated controls (Figure 6D). These results indicate that RANKL treatment of ECC cells inhibits VEGF-induced ECC cell proliferation. In addition, treatment of ECC cells with RANKL alone significantly inhibits cell proliferation compared to control cultures (Figure 6C–D), consistent with RANKL inhibition of endogenous ECC cell proliferation mechanisms. Similarly, ECC cells were cultured in the presence of VEGF+RANKL to determine the effects on CtsK transcription. Addition of VEGF with RANKL to cultures does not significantly inhibit RANKL-induced CtsK expression (Figure 6B) and VEGF alone does not affect CtsK expression. Taken together, these results indicate there is crosstalk in the signaling pathways that regulate NFATc1, whereby RANKL inhibits VEGF-induced proliferation of ECC cells while activating CtsK transcription.
RANKL stimulates ECM remodeling enzyme production via co-activation of Cn/NFATc1 and JNK1/2 pathways in osteoclasts. 17 JNK1/2 activation in cardiac valves was examined in vivo via immunohistochemistry on E12.5 mouse heart sections with anti-phosphorylated JNK(Thr183/Tyr185) antibody. JNK1/2 activation was detected in mitral and tricuspid valve endothelial cells consistent with RANKL and JNK1/2 activity during valve ECM remodeling in vivo (Figure 7). To determine if RANKL-induced CtsK expression requires JNK1/2 signaling in ECC cells, avian ECC explants were cultured for 7 days in the presence of RANKL+DMSO, RANKL+SP600125 (a JNK1/2 inhibitor), SP600125, or DMSO (vehicle control). ICLSM was used to determine that SP600125 treatment of cultured ECC cells significantly decreased phosphorylated JNK(Thr183/Tyr185) expressing ECC cells, while RANKL-induced NFATc1 nuclear localization was not significantly altered (Online Figure IVE-H and data not shown). Real Time RT-PCR demonstrated that SP600125 treatment significantly decreased RANKL-induced CtsK expression (Figure 8C). Therefore, RANKL-induced CtsK expression in ECC cells is JNK1/2-dependent.
In vascular endothelial cells, VEGF stimulation of VEGFR2 activates Cn/NFAT and ERK1/2 copathways together to promote gene transciption. 13,28 In order to determine if ERK activation is specifically required for VEGF-mediated effects downstream of NFATc1 in ECC cells, ECCs were cultured in the presence of VEGF+DMSO, VEGF+U0126 (a MEK1-ERK1/2 inhibitor), U0126, or DMSO (vehicle control). ICLSM was used to determine that U0126 treatment of cultured ECC cells significantly decreased diphosphorylated ERK1/2 expressing ECC cells, while VEGF-induced NFATc1 nuclear localization was not significantly altered (Online Figure IVA–D and data not shown). VEGF and DMSO control treated cells had mitotic indices of 4.0% and 2.6% respectively, as determined by pHH3 immunoreactivity (Figure 8A). Addition of U0126 to cultures either alone or in combination with VEGF blocked the effects of VEGF treatment, and presumably endogenous VEGF signaling on cell proliferation, resulting in a significantly decreased mitotic index of 1.3% for both, compared to control cultures (Figure 7A). In contrast, MEK1-ERK1/2 inhibition had no effect on RANKL-induced CtsK expression in cultured ECC cells. As a specificity control, treatment with Phosphoinositide 3-Kinase (PI3K) inhibitor LY294002 had no effect on either VEGF-induced proliferation or RANKL-induced CtsK expression in ECC cells (Figure 7D; data not shown). These data demonstrate that VEGF-induced ECC cell proliferation is MEK1-ERK1/2-dependent, however, RANKL-induced CtsK expression does not require MEK1-ERK1/2 activity.
Conversely, JNK1/2 inhibition with SP600125 did not significantly alter VEGF-induced ECC cell proliferation, demonstrating that VEGF-induced ECC cell proliferation is JNK1/2-independent (Figure 8B). To determine if RANKL inhibition of VEGF-induced ECC cell proliferation requires JNK1/2 activation, ECC cells were treated with VEGF, RANKL, and SP600125. These experiments showed that RANKL-mediated inhibition of VEGF-induced ECC cell proliferation is JNK1/2-dependent (Figure 8E). Interestingly, JNK1/2-dependent signaling has not previously been associated with maturation of ECC cells. Overall, these results show that, in conjunction with Cn/NFATc1, MEK1-ERK1/2 activation is necessary to achieve VEGF-induced ECC cell proliferation, while JNK1/2 activation is necessary for RANKL-induced CtsK expression and for RANKL-mediated inhibition of ECC cell proliferation.
During heart valve morphogenesis, ECCs transition from growth, characterized by high cell proliferation, to remodeling, during which the ECM is stratified and mature valve leaflets become apparent. 3 Investigation into the role of NFATc1 in valve maturation supports a model whereby VEGF/NFATc1/ERK1/2 signaling promotes ECC cell proliferation during ECC growth, and RANKL/NFATc1/JNK1/2 signaling inhibits VEGF-induced cell proliferation, while promoting CtsK expression, during valve remodeling. These data also support a novel mechanism for the transition from ECC growth to remodeling in which NFATc1 promotes a sequential pattern of gene expression via cooperation with ligand-specific cofactors MEK1-ERK1/2 and JNK1/2 (Online Figure V).
NFATc1−/− mouse embryos exhibit decreased proliferation of ECC endothelial and mesenchymal cells at E10.5. Prior to this time, ECCs are apparently normal, demonstrating that NFATc1 is not required for ECC formation and EMT. 7,9,11 VEGF is an upstream activator of Cn/NFATc1 and requires MEK1-ERK1/2 activation in promoting proliferation of cultured ECC cells. In vivo ECC growth is characterized by nuclear localization of NFATc1 in endothelial cells in addition to expression of VEGF and VEGFR2. 7,15,27 VEGF signaling must be tightly regulated for normal valvulogenesis to occur, as VEGF is necessary for endothelial proliferation and maintenance, as well as being a potent inhibitor of EMT during initial formation of the ECCs.15,27 In the atrioventricular canal myocardium, VEGF expression is negatively regulated by NFATc3/c4, however, NFATc1 has not been shown to regulate VEGF transcription in the ECC. 7 By E14.5 in mouse, valve remodeling has begun and expression of VEGF and VEGR2 are lost in valve endothelial cells, supporting a model whereby loss of VEGF signaling in the ECC endothelial cells is associated with the transition from growth to remodeling during valvulogenesis. 14,15
The RANKL/NFATc1 pathway is conserved among vertebrates and is active in endothelial cells of remodeling valves. This study is the first to report that RANKL induces CtsK expression via co-stimulation of Cn/NFATc1 and JNK1/2 pathways in ECC cells. In the skeletal system, NFATc1 is a key regulator of osteoclast differentiation and function in response to RANKL signaling. 17 Upon RANKL binding in osteoclasts, the RANK receptor recruits adaptor molecules that co-stimulate NFAT and JNK1/2 pathways ultimately leading to NFATc1/AP1-mediated activation of ECM remodeling enzymes such as Ctsk and Matrix Metalloproteinase 9 (MMP9). 17 Expression of RANKL/NFATc1 pathway components, RANKL, CtsK and MMP9 are associated with increased pathogenic ECM remodeling and calcification of human valves suggesting this pathway may play a role in valve maturation and disease. 19,26 In contrast, VEGF stimulation of NFATc1 in HPVECs induces endothelial cell proliferation, which implicates NFATc1 in normal homeostasis of the valve endothelium. 16
The work presented here and elsewhere demonstrates that NFATc1 participates in complex regulatory interactions during valve development. In developing osteoclasts and endothelial cells, NFATc1 forms complexes with other NFATs as well as unrelated transcription factors such as Elks, GATAs and AP1, to bind DNA. 8,29,30 Ligand-specific responses to Cn/NFATc1 activation occur through selective co-stimulation of NFATc1 partners in T-cells, where genes associated with increased immune response are targeted by NFATc1/AP1 complexes, while genes associated with dampened immune response are activated by NFATc1 in the absence of AP1. 31 JNK1/2 signaling is important for outflow tract development, but its role in valve development has not been previously reported. 32 MEK1-ERK1/2-activated transcription factors are necessary for EMT and ECC cell proliferation. 33,34 Together these data suggest NFATc1 plays a role in regulating the transition from ECC growth to valve remodeling via partnership with ligand-specific cofactors to elicit gene expression. Further interrogation of NFATc1 and NFATc1 costimulatory pathway functions in valve maturation and homeostasis may reveal new therapeutic targets for prevention and treatment of congenital valve defects and disease.
We are indebted to Alexander Lange for his continued insight and technical support, to Kristen Lipscomb-Sund and Timothy Mead for editorial comments, and to Christina Alfieri, Joy Lincoln, Elaine Shelton and Heather Evans-Anderson for their technical assistance.
Sources of funding
American Heart Association Great Rivers Affiliate Predoctoral Fellowship Award #715107B
NIH/NHLBI SCCOR in Pediatric Cardiology #P50HL074728
Subject Codes: 6, 139, 142, 143, 138