In this study, we propose different biological roles of DKK1 and DKK2 in regulating angiogenesis. Expression of DKK1 and DKK2 was reciprocally and specifically regulated depending on the status of ECs, such as proliferation and morphogenetic differentiation. DKK2 promoted angiogenesis, whereas DKK1 suppressed it. DKK1 and DKK2 have similar primary structures and were originally identified as soluble WNT antagonists (29
). These proteins regulate various biological functions through inhibition of the WNT signaling pathway (22
). Interestingly, DKK2 is also suggested to work as an activator depending on cell context (29
). However, the biological function of DKK2 as an LRP5 or LRP6 agonist in vivo and the underlying signaling mechanism remain to be determined. Our finding that DKK2 promotes angiogenesis provides a new insight into the agonist function of DKK2 in vivo.
Our data suggest that the angiogenic activity of DKK2 is correlated with activation of EC dynamics. The vascular-specific DKK2 Tg mice exhibited significantly increased vessel branching, EC density, and proliferation, with significantly enhanced numbers of tip cells and filopodia at the vascular front of the retina (Figure ). Interestingly, these retinal vascular phenotypes of DKK2 Tg mice were comparable to those of Jag1 Tg mice as described previously (16
). Since our study is based on the gain of function, it is limited to arguing whether DKK2 is involved in tip cell and stalk cell specification as Jag1 is. However, it is most likely that the angiogenic sprouting and vascular patterning governed by DKK2 might be correlated with its stimulatory function in tip cell dynamics, as clearly indicated in aortic ring imaging (Figure , J–M, and Supplemental Video 1). Our data showed that DKK2 is rapidly induced by EC recognition of laminins, major components of the basement membrane. Notably, it has been shown that laminins were produced by stalk cells and tip cells during sprouting angiogenesis (31
) and that laminin-1 induces expression of Jag1 in ECs (32
). We also observed that both Jag1 and DKK2 are induced in a similar way during morphogenesis of HUVECs on Matrigel (data not shown). Thus, the expression pattern and Tg phenotypes point to a potential relationship between Jag1 and DKK2 in sprouting angiogenesis. Recent in silico analysis suggested that DKK2 is a Notch signaling target in intestinal stem cells (33
). However, Notch-response elements were detected in the promoter region of DKK2 of human, chimpanzee, and rat, but not in cow and mouse (33
). Thus, it remains unclear whether angiogenic action of DKK2 is functionally correlated with Notch signaling.
This study further provides insight into the mechanism by which DKK2 promotes sprouting angiogenesis. Filopodia extending from tip cells lead the angiogenic sprout by sensing guidance cues and migrating along a specific path in response (7
). Previous studies have shown that Rho GTPases represent an important class of molecules involved in vascular morphogenesis (34
). DKK2, but not DKK1, consistently induced Cdc42 activation in ECs. Although cultured ECs expressed both LRP5 and LRP6, which are potential signaling receptors for DKK2, depletion of LRP6, but not LRP5, resulted in a significant reduction in DKK2-induced Cdc42 activation and EC morphogenesis. LRP5 and LRP6 share 73% identity in protein sequences but the ligand-binding repeats show only 50% similarity (36
), which suggests that they may not bind ligands with similar affinity. Indeed, it has been shown that LRP6 works differently from LRP5 in the WNT signaling pathway. Overexpression of LRP6, but not LRP5, induces axis duplication in Xenopus (37
), and only LRP6 cooperates with several WNT-Frizzled fusion proteins to activate WNT-responsive promoters (38
). Similarly, our data suggest that the response of LRP5 and LRP6 to DKK2 in ECs is distinct and LRP6 may act as a cognate DKK2 receptor that mediates Cdc42 activation and angiogenic sprouting.
We further asked how the association of LRP6 with DKK2 activates Cdc42. APC is known to be involved in WNT signaling downstream of LRP6 through its binding to β-catenin (39
). Recent studies have uncovered an additional function for APC, in which it activates Cdc42 via direct binding to Asef2 (28
). Interestingly, APC/Asef2 complex formation was enhanced by DKK2, and the reduction of APC or Asef2 expression inhibited DKK2-induced Cdc42 activation and EC morphogenesis, suggesting functional significance of the APC/Asef2 system in linking DKK2/LRP6 to Cdc42 activation in ECs. Some WNT members are known to activate Cdc42 via noncanonical pathways, promoting cell polarity (41
). However, treatment with sFRP, a decoy antagonist of WNT, did not inhibit DKK2-induced Cdc42 activation (Figure E) and DKK2 treatment did not increase β-catenin–mediated transcription levels (Supplemental Figure 23), suggesting that DKK2 may activate Cdc42 independent of the WNT pathway.
Norrin/LRP5/Fzd4 signaling is well known in regulating retinal angiogenesis. EC-specific knockout mice of Norrin or Fzd4 showed reduced retina vessel growth and endothelial-mural cell interaction (42
). However, blocking of Norrin/LRP5/Fzd4 signaling in vitro by Fzd4 CRD IgG did not interrupt Cdc42 activation by DKK2. In addition, Cdc42 activation was not affected by Norrin treatment (Supplemental Figure 24). In summary, CDC42 activation by the DKK2/LRP6 pathway does not seem to be affected by Norrin/LRP5/Fzd4 signaling.
Our data also demonstrate an inhibitory function of DKK1 in angiogenesis with 2 possible mechanisms. First, unlike the WNT-independent mechanism of DKK2 in vascular morphogenesis, DKK1 appears to directly affect WNT signaling in ECs. Previous studies have demonstrated roles for the WNT signaling pathway in angiogenesis and vascular stability (18
). Signals transmitted from the Frizzled family of receptors upon binding of its ligands, WNT and Norrin, influence angiogenic processes such as EC proliferation and migration (17
). Moreover, mice deficient in Lef1 and Ctnnb1, both of which control transcription of target genes of the canonical WNT signaling pathway, displayed significant reduction in vascular density in retinas (44
). Our data show that DKK1 inhibits WNT-responsive promoter activity and proliferation of ECs (Figure D and Supplemental Figure 25), and endothelium-specific overexpression of DKK1 in mice results in delayed peripheral vascularization and reduced vascular density in retinas (Supplemental Figure 26). Alternatively, DKK1 may counteract DKK2 action through competitive binding to LRP6. This mechanism is supported by our data showing that upregulation of DKK1 antagonizes DKK2-induced angiogenic sprouting and Cdc42 activation (Supplemental Figure 26).
Although knockout of DKK1 and DKK2 showed unique phenotypes in head formation and osteoblast differentiation (45
), respectively, no obvious vascular phenotype in mutant mice has been reported so far. This may be due to the compensation for DKK1 and DKK2 deficiency by other members of DKK family (47
) or other regulatory factors during normal vascular development. And also, considering the complexity and diversity of blood vessels in different organs and pathological states, careful reexamination of vascular phenotypes in DKK-deficient mice may be valuable for further understanding the precise roles of DKKs in the vasculature.
The feature of DKK2-induced vessels, which were structurally organized and well covered by pericytes and SMCs, was different from those induced by VEGF as shown by the corneal assay. DKK2 also provided profound therapeutic effects in 2 ischemic disease models mentioned above. Although the discovery of several angiogenic factors has led to the development of therapeutic strategies for ischemic diseases, the success of therapeutic angiogenesis in clinical trials has been limited due to insufficient efficacy and pronounced adverse effects (48
). Given that DKK2 is a relatively small and soluble ligand, our finding that DKK2 alone can induce new and functional vessel formation in vivo may provide a novel strategy for developing therapeutic strategies to treat ischemic vascular diseases.