Foxc2 Transcription Factor: a Novel Regulator of Lymphangiogenesis

Lymphology 44 (2011) 35-41

FOXC2 TRANSCRIPTION FACTOR: A NOVEL REGULATOR OF LYMPHANGIOGENESIS

X. Wu, N.-F. Liu

Lymphology Center of Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, People’s Republic of China

ABSTRACT life. Although embryonic vein endothelial cells sprout and incorporate to form primary Lymphangiogenesis is the critical process lymph sacs and primary lymphatic plexus (1), of forming new lymphatic vessels under subsequent processes of vascular remodeling physiological and pathological conditions and and maturation gives rise to a functional involves both molecular and morphological network of lymphatic vessels, including changes. Despite evidence that lymphangio- lymphatic capillaries responsible for absorp- genic factors, including vascular endothelial tion of interstitial fluid and collecting lymph growth factors (VEGFs) and Prox1, regulate vessels that transport the lymph back to the lymphangiogenesis, the molecular mechanisms blood circulation. While lymphangiogenesis underlying gene regulation in lymphatic vessel occurs normally in almost all tissues under remodeling and maturation are not fully physiological conditions (with the notable understood. Importantly, recent studies exceptions of the central nervous system, demonstrate that Forkhead transcription bone marrow, cartilage, cornea and factor FOXC2 controls later steps of lymphatic epidermis), elucidation of the mechanisms vascular development and is responsible for involved in pathological lymphangiogenesis establishing a collecting lymphatic vessel such as solid tumor metastasis, inflammation identity by regulating expression of down- and lymphedema is clearly of great impor- stream genes involved in lymphangiogenesis, tance. Recent work has discovered several including PDGF-ß, Delta-like 4 (Dll4) and regulators including the transcription factors angiopoietin (Ang)-2. Thus, FOXC2 is now Prox1 (2), VEGFR-3/ VEGF-C (3), Tie/Ang2 recognized as a novel regulator of lymphatic (4), and EphrinB2 (5), which contribute to vascular formation and remodeling. This early lymphangiogenesis. However, the later review summarizes current knowledge about steps of lymphatic vascular development the function of FOXC2 in lymphangiogenesis including the maturation of the primary and discusses prospects for future research in lymphatic plexus into functional collecting FOXC2-mediated pathological lymphangio- vessels and capillaries, remain largely genesis in lymphatic-related disease. unknown and need further investigation. Here we discuss the rapidly accumulating Keywords: FOXC2, remodeling and evidence that transcription factor FOXC2 is maturation, lymphangiogenesis. a key regulator during lymphatic remodeling and maturation. Lymphatic network formation is vital for embryonic development as well as postnatal Physiological Roles of FOXC2 during

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Development and in the Adult form lymphatic valves and increased mural cell investment of lymphatic capillaries in a FOXC2 is a member of the forkhead/ mouse model, indicating Foxc2’s role in winged-helix family of transcription factor remodeling and maturation of lymphatic genes that is located on the long (q) arm of vessels (19). Further investigations using chromosome 16 at position 24.1 (6). The gene, models of animal development showed that with a single exon which is highly GC rich, change in Foxc2 expression is the critical first encodes for a 2.2 Kb protein (7). Forkhead event of the maturation process of lymphatic box proteins are characterized by the vessels (18), configuring Foxc2 as an forkhead box, a DNA binding Motif with a important transcriptional regulator to control sequence of 80 to 100 amino acids (8). In expression of multiple genes in the process of humans, the FOX family of transcription lymphatic maturation. This conclusion is factors is present in various embryonic organs supported by the observation that Foxc2 and tissues during development and plays inhibited SMC coverage of initial lymphatic crucial roles in a variety of processes such as vessels through suppressing the expression of proliferation, differentiation and survival (9). PDGF-ß, a potent chemoattractant for FOXC2 protein, also known as forkhead- vSMCs, in lymphatic vessels (19,22). Notably, related protein FKHL14 (FKHL14), Foxc2 regulated the transcriptional network transcription factor FKH-14, or mesenchyme in this process in cooperation with nuclear forkhead protein (MFH1), belongs to the “C” factor of activated T cells (NFATc)-1 that has subfamily and is required for cardiovascular been found to control the morphogenesis of development (10) as well as the development cardiac valves (18,23). of the lungs (11), eyes (12), kidneys (13) and Interestingly, patients with Lymphedema- urinary tract (14). Recently, FOXC2 has been Distichiasis syndrome (LD) [Online shown to be involved in cancer angiogenesis Mendelian Inheritance in Man (OMIM)], a and metastasis (15) and is particularly disease caused by mutations in FOXC2, have implicated in cancer progression through its a similar phenotype to Foxc2 null (19) and induction of epithelial-to-mesenchymal haploinsufficient mice (20). So far, mutations transition (16). Moreover, suppression of in FOXC2 have been found to be the only FOXC2 expression using shRNA in a highly known cause of LD. Moreover, frameshift metastatic breast cancer model blocked mutations that involve a probable loss-of- metastatic ability (17), indicating FOXC2 function mechanism (24-26) were the might be another therapeutic target for predominant mutations, whereas nonsense cancer therapy. and missense mutations were rare (7,26,27). While genotype/phenotype correlation has Role of Foxc2 in Lymphatic Vascular not been clearly delineated, all mutations Development including frameshift and nonsense mutations that are responsible for LD truncate the Recently, Foxc2 has been shown to be protein, leading to FOXC2 haploinsufficiency implicated in lymphatic vascular develop- (24,26,28,29). Clinically, affected patients ment and disease (18,19). The fact that Foxc2 commonly have bilateral lymphedema of the is highly expressed in the developing lower limbs that usually develops around lymphatic vessels as well as lymphatic valves puberty and abnormal eyelashes (a double in adult mice raises the possibility that it row of eyelashes) (7). In addition to these serves a function in the development and specific signs of LD, other associated compli- maintenance of the lymphatic vasculature cations may include ptosis (31%), varicose (19-21). Moreover, loss of Foxc2 leads to veins (25%), congenital heart diseases (6.8- abnormal lymphatic patterning, failure to 10%), and cleft palate (4-10%). Scoliosis,

Permission granted for single print for individual use. Reproductiom not permitted without permission of Journal LYMPHOLOGY. 37 renal anomalies, hydrocele, strabismus have roles in development of some organs (33-36), also been reported but are less common (26), Foxc1 has been shown to be absent in suggesting the phenotypic spectrum of LD lymphatics (21). These observations suggest could be variable. that Foxc2 regulates the maturation of Further investigation of the lymphedema- lymphatic vessels, whereas early lymphatic distichiasis phenotype through lymphscin- specification depends to some extent on tigraphy demonstrates lymph reflux in the proper Foxc1 activity. large lymphatic vessels of the leg, suggesting Although deficiency of forkhead agenesis or absence of valves in the collecting transcription factor Foxc2 results in defects lymphatic vessels (7). In addition, in contrast in lymphatic remodeling and failure to form to the lymphatic vessel hypoplasia or aplasia lymphatic valves (19), the precise function of seen in other forms of primary lymphedema, Foxc2 in this process has yet to be defined. FOXC2 haploinsufficient humans and mice Recently, it has been reported that integrin-α9 exhibit a hyperplastic lymphatic system mutant mice have a similar phenotype to (7,18,20,22,30). Indeed, failure of downregu- Foxc2-null mice, displaying abnormal lation of VEGFR3 and active VEGFR3 lymphatic valves and retrograde lymph flow signaling have been observed in FOXC2- and impaired fluid transport. Integrin-α9 has deficient lymphatic vessels (18).This finding been shown to be implicated in controlling indicates that reduced FOXC2 expression the formation of lymphatic valve leaflets (37). disrupts the normal balance between Further analysis shows that upregulation of lymphatic vessel growth promoting and Foxc2 transcription factor initiated the inhibiting genes. Interestingly, histological formation of the lymphatic valve followed by study revealed that the major proportion of the development of the valve leaflet, which skin lymphatic vessels was abnormally was initiated by upregulation of integrin-α9 invested by smooth muscle cells in patients expression during embryogenesis (18,37). On with FOXC2 mutations (19), which is in the other hand, Foxc2 like integrin-α9 is agreement with the findings in Foxc2-/- mice present in mature and developing lymphatic and indicates that FOXC2 is essential for the valves (37). In this light, it is tempting to morphogenesis of lymphatic valves and the speculate that Foxc2 acts upstream of establishment of a pericyte-free initial integrin-α9 in the formation of lymphatic lymphatic network (19). valves. But this possibility needs to be further In addition, varicose veins are another investigated. typical sign of LD. Recently FOXC2 mutations have been shown to be implicated in Foxc2 Regulates Notch Signaling in primary valve failure in veins (31), suggesting Lymphangiogenesis? a possible developmental role for FOXC2 in both venous and lymphatic systems. Notch signaling is an evolutionarily Recently, Seo et al have reported that conserved pathway that modulates arterial compound null mouse mutant embryos for cell-fate decisions (38,39). Interestingly, Foxc2 and related Foxc1, have reduced Notch, in addition to its role in blood vessel sprouting of Prox1 positive cells from the morphogenesis and arterial development cardinal vein by reducing mesenchymal (40,41), also regulates lymphatic develop- Vegf-C expression (32), indicating Forkhead ment. Notch1 and Notch4 have been found to transcription factors might act upstream of be present in some murine lymphatics as well Vegf-C in the formation of primary lymph as tumoral lymphatics of human mammary sacs. On the other hand, while Foxc1 and carcinomas (42). Moreover, some in vitro Foxc2 have very similar expression patterns studies have displayed that notch signaling in various embryonic tissues and overlapping participates in and regulates

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Fig. 1. Proposed upstream location of FOXC2 and NFAT1c in lymphatic remodeling and maturation. lymphangiogenesis (43), and a recent in vivo arterial endothelial differentiation in part study using gene silencing methods in through regulation of ephrinB2 expression in zebrafish demonstrated a role for Notch arterial endothelium (46). Equally, Dll4/ signaling in lymphangiogenesis (44). Notch increased expression levels of ephrinB2 Knockdown of Dll4 or its receptors (Notch-1b in the lymphatic endothelium in vitro. or Notch-6) in zebrafish resulted in impaired Moreover, ephrinB2 has recently been shown lymphangiogenesis at multiple steps during to contribute to remodeling of the lymphatic early lymphatic vascular development, plexus into lymphatic capillaries and indicating critical roles for Notch signaling collecting vessels (5). It is thus possible that in the formation and wiring of the lymphatic Foxc2 might mediate later steps of lymphatic network (45). vascular remodeling and maturation via Recently, it has been found that Foxc1 Notch- ephrinB2 pathway (Fig. 1). and Foxc2 upregulate the expression of Notch1, Notch4, and Dll4, as well as the Foxc2-Dependent Autocrine Effects on arterial specific markers, ephrinB2 in mouse Lymphangiogenesis embryonic endothelial cells (MEECs) (32). Furthermore, recent studies showed that Recent studies have demonstrated that Foxc2 directly induces the transcription of Foxc2 indirectly controls angiogenesis as delta-like4 (a novel ligand for notch recep- well as remodeling and maturation of the tors) by activating its promoters, suggesting vasculature by inducing Ang2 expression in that Foxc2 transcription factors act upstream adipocytes in a paracrine manner (47). Ang2 of Notch signal to mediate arterial specifica- is a ligand for the tyrosine kinase receptor tion. Thus, these findings lead to speculation Tie2 on endothelial cells and plays an of a similar relationship between Foxc2 and important role in remodeling and maturation Notch signaling in lymphatic endothelial of blood vessels as well as lymphatic vessels cells. On the other hand, Notch promotes (4,48-50). Ang2 is produced by LECs in vivo

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(18,29). Moreover, the activation of FOXC2 processes involved in the interactions results in release of Ang-2 from Weibel- between FOXC2 and VEGFR3? How does Palade bodies in endothelial cells (51,52). such an interaction affect gene expression These findings suggest that FOXC2 promotes in lymphatic endothelial cells? lymphangiogenesis in an autocrine manner in Taken together, further analysis of the lymphatic endothelial cells. On the other role of FOXC2 in lymphangiogenesis may hand, Ang2 and Foxc2 mutant mice exhibit promote a new treatment approach for LD some similarities in lymphatic phenotype, and perhaps secondary lymphedemas. e.g., improper recruitment of smooth muscle cells into lymphatic capillaries (4,19,53). ACKNOWLEDGMENT Could the lymphatic defects in Foxc2 mutant mice be in part due to decreased Ang2 The study is funded by the National expression as well? Natural Science Foundation (grant no. 30772262) as well as Shanghai Science and CONCLUSION Technology Committee (grant no. 09410706400, 10411964100, 10440711000). Accumulated studies on FOXC2’s role in lymphangiogenesis, using animal models REFERENCES development as well as clinical investigations, have revealed complicated functions of 1. Karina, Y, I Sumio, C Daniel, et al: Live FOXC2 in lymphangiogenesis. It is well imaging of lymphatic development in the zebrafish. Nat Med. 12 (2006), 711-716. recognized that FOXC2 regulates later steps 2. Wigle, JT, G Oliver: Prox1 function is of lymphatic vascular development, the required for the development of the murine maturation of the primary lymphatic plexus lymphatic system. Cell 98 (1999), 769-778. into functional collecting vessels and 3. Kaipainen, A, J Korhonen, T Mustonen, et al: capillaries in cooperation with NFATc1 by Expression of the fms-like tyrosine kinase 4gene becomes restricted to lymphatic controlling expression of multiple genes in endothelium during development. Proc. Natl. critical aspects of lymphangiogenesis. In Acad. Sci USA 92 (1995), 3566-3570. addition to PDGF-ß, FOXC2 might also 4. Gale, NW, G Thurston, GD Yancopoulos, contribute to lymphatic vascular remodeling et al: Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and and maturation via Notch- ephrinB2 pathway only the latter role is rescued by Angiopoietin- and Ang2. Elevated FOXC2 expression in 1. Dev. Cell. 3 (2002), 411-423. the developing lymphatic vessels as well as in 5. Makinen, T, RH Adams, J Bailey, et al: PDZ lymphatic valves in adults have established interaction site in ephrinB2 is required for the FOXC2 as an attractive therapeutic target remodeling of lymphatic vasculature. Genes Dev. 19 (2005), 397-410. for lymphatic-related diseases. 6. Shaw-Smith, C: Genetic factors in esophageal Future studies should focus on identifying atresia, tracheo-esophageal fistula and the the direct FOXC2 target genes and enhancing VACTERL association: Roles for FOXF1 and understanding of the interplay of FOXC2 the 16q24.1 FOX transcription factor gene with other prolymphangiogenic molecules cluster, and review of the literature. Eur. J. Med. Genet. 53 (2010), 6-13. such as VEGFR3, which is linked to a 7. Brice, G, S Mansour, R Bell, et al: Analysis of subset of primary congenital lymphedema the phenotypic abnormalities in lymphoedema- syndromes (Milroy disease; OMIM 153100) distichiasis syndrome in 74 patients with (54,55). Although it has been reported that FOXC2 mutations or linkage to 16q24. J. Foxc2 may act downstream of Vegfr3 in Med. Genet. 39 (2002), 478-483. 8. Arden, KC: Multiple roles of FOXO transcrip- blocking SMC coverage to lymphatic tion factors in mammalian cells point to capillaries (19), many key questions remain. multiple roles in cancer. Exp. Gerontol. 41 What are the cellular and molecular (2006), 709-717.

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