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Claudin-like protein 24 interacts with the VEGFR-2 and VEGFR-3 pathways and regulates lymphatic vessel development.

The Claudin-like protein of 24 kDa (CLP24) is a hypoxia-regulated transmembrane protein of unknown function. We show here that clp24 knockdown in Danio rerio and Xenopus laevis results in defective lymphatic development. Targeted disruption of Clp24 in mice led to enlarged lymphatic vessels having an abnormal smooth muscle cell coating. We also show that the Clp24(-/-) phenotype was further aggravated in the Vegfr2(+/LacZ) or Vegfr3(+/LacZ) backgrounds and that CLP24 interacts with vascular endothelial growth factor receptor-2 (VEGFR-2) and VEGFR-3 and attenuates the transcription factor CREB phosphorylation via these receptors. Our results indicate that CLP24 is a novel regulator of VEGFR-2 and VEGFR-3 signaling pathways and of normal lymphatic vessel structure.

The Claudin-like protein of 24 kDa (CLP24) is a hypoxiaregulated transmembrane protein of unknown function. We show here that clp24 knockdown in Danio rerio and Xenopus laevis results in defective lymphatic development. Targeted disruption of Clp24 in mice led to enlarged lymphatic vessels having an abnormal smooth muscle cell coating. We also show that the Clp24 À/À phenotype was further aggravated in the Vegfr2 +/LacZ or Vegfr3 +/LacZ backgrounds and that CLP24 interacts with vascular endothelial growth factor receptor-2 (VEGFR-2) and VEGFR-3 and attenuates the transcription factor CREB phosphorylation via these receptors. Our results indicate that CLP24 is a novel regulator of VEGFR-2 and VEGFR-3 signaling pathways and of normal lymphatic vessel structure. The lymphatic vasculature is required for tissue fluid balance and immune system function (Tammela and Alitalo 2010). Dysfunction of the lymphatic system can lead to lymphedema, characterized by swelling of extremities due to fluid accumulation in tissues. Lymphatic vessels also represent the primary route of metastatic spread for many types of human cancers, and they are intimately involved in various inflammatory disorders (Tammela and Alitalo 2010).
The development of the lymphatic vasculature in mice starts at around embryonic day 10.5 (E10.5), after the formation of a functional cardiovascular system (Adams and Alitalo 2007;Tammela and Alitalo 2010). Vascular endothelial growth factor-C (VEGF-C) induces lymphatic sprouting by activating the VEGF receptor-3 (VEGFR-3) on the surface of the first differentiated lymphatic endothelial cells (Karkkainen et al. 2004). VEGFR-3 is expressed initially in all endothelial cells of mouse embryos, but becomes restricted to the lymphatic endothelium during development (Tammela and Alitalo 2010). Some lymphangiogenic signals are also mediated by VEGFR-2, which is expressed in blood vessels, collecting lymphatic vessels, and also lymphatic capillaries undergoing lymphangiogenesis; for example, in tumors (Nagy et al. 2002;Hirakawa et al. 2005;Wirzenius et al. 2007). The further remodeling and maturation of the lymphatic vasculature requires several other molecules (e.g., Nrp2, podoplanin, Foxc2, Syk, SLP76, Angpt2, and Angptl4) (Adams and Alitalo 2007;Tammela and Alitalo 2010).
In order to better understand the development of the lymphatic vasculature, we searched for genes involved in lymphangiogenesis by carrying out a genome-wide gene expression analysis of primary human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs). Here we report on one of the genes enriched in LECs, the hypoxia-inducible Claudin-like protein of 24 kDa (CLP24; TMEM204). We show that CLP24 is essential for lymphatic development in zebrafish, frogs, and mice. Interestingly, CLP24 interacted with VEGFR-2 and VEGFR-3 and attenuated VEGF-and VEGF-Cinduced phosphorylation of the transcription factor CREB, suggesting that CLP24 is a modulator of VEGFR-2 and VEGFR-3 signals required for lymphatic vascular development.

Endothelial expression of CLP24
To search for genes enriched in LECs, we compared the gene expression profiles of cultured LECs and BECs from human skin microvasculature using oligonucleotide arrays. We identified the CLP24 mRNA, which was expressed more abundantly in LECs than BECs cultured in vitro (Supplemental Fig. S1A) and from freshly ex vivo isolated microvascular endothelium (LEC vs. BEC fold: 5.83, P = 0.004) (Wick et al. 2007). All tested human tissues except the bone marrow and peripheral blood contained CLP24 mRNA of 1.9 kb, with enhanced levels in highly vascularized tissues such as the heart, lung, kidney, adrenal gland, and placenta (Supplemental Fig.  S1B). We confirmed that CLP24 is a hypoxia-regulated gene (Supplemental Fig. S1C; Kearsey et al. 2004). CLP24 was conserved in all species, including humans, mice, zebrafish, and frogs (Supplemental Fig. S1D,E).
We found that most of the Clp24 expression occurred in blood vessels at E10.5, E15.5, and E16.5 ( ). In a screen of novel cardiac genes, the Clp24 transcript was detected previously in the developing vascular system before E9.5 (Christoforou et al. 2008). Notably, the expression pattern of Vegfr2 was very similar to that of Clp24. At E10.5, Clp24 and Vegfr2 were both observed in, e.g., intersomitic vessels ( Fig. 1A,B, arrowheads), while only Vegfr2 was detected in larger vessels, such as the cardinal vein ( Fig.  1A,B, arrows). At E10.5, Clp24 was also detected in the developing limb buds and branchial arches. At E15.5 and E16.5, Clp24 and Vegfr2 were prominent in the blood vessels, e.g., in the brain and developing limb bud ( CLP24 has been suggested to be a distant member of the claudin family of transmembrane proteins, which are engaged in homotypic interactions across the cell-cell junctions (Kearsey et al. 2004). We confirmed that overexpressed CLP24 is localized at cell-cell junctions in transfected Madin-Darby canine kidney (MDCK) epithelial cells, but not human dermal microvascular endothelial cells (HDMECs), where CPL24 was distributed uniformly at the plasma membrane in LECs and BECs ( In order to elucidate the biological function of CLP24, we studied it in zebrafish and frog embryos. In situ hybridization (ISH) on early embryos showed that the D. rerio clp24 homolog (tmem204) was expressed initially in the dorsal aorta, and later also in the posterior cardinal vein (PCV) and intersegmental vessels (ISVs) (Fig. 1K,L). The knockdown of clp24 using 6-8 ng of morpholino (MO) directed against the 59 untranslated region (UTR) of clp24 mRNA caused a subtle blood vascular defect, characterized by abnormal extra branching of the ISVs, but only from 4 d post-fertilization (dpf) onward, thus after the initiation of lymphatic development ( Fig. 2A-D). The most striking defect was the impaired formation of the lymphatic thoracic duct (TD) (6 dpf) and its immediate precursor structure, the parachordal lymphangioblasts (48 h post-fertilization [hpf]) in clp24 morphants. The penetrance and severity of these defects were dosedependent ( Fig. 2E,F), and the results were confirmed using a second clp24 MO targeted against the translation start site (Supplemental Fig. S4A,B). Thus, the striking lymphatic defect occurred prior to the appearance of the subtle blood vascular defects.
Twenty nanograms to 60 ng of the clp24 MO resulted in lymphatic and blood vascular defects of X. laevis embryos in a dose-dependent manner (Supplemental Fig. S4A). Live screening at stage 45 ( Fig. 2G,H) showed edema in the heart, gut, and cloaca region in 58% of the clp24 morphants versus 7% of control MO-injected tadpoles (P < 0.0001), and blood flow arrest in 32% of clp24 morphants versus 3% of controls (P < 0.0001) despite normal beating of the heart and lymph hearts. In addition, 21% of the clp24 morphants had blood spots in their tissues (versus 0% of controls; P < 0.0001).
To further characterize the phenotypes, knockdown of clp24 was performed in transgenic Tg(Flk1:eGFP) frogs that express eGFP in their blood and lymphatic vessels. Neither the ventral caudal lymphatic vessel (VCLV) nor the dorsal caudal lymphatic vessel (DCLV) assembled into a compact lymph vessel, and the LECs appeared dispersed and disorganized (Fig. 2I,J) in the clp24 morphants. Lymphangiography showed that only 12.5% of the morphants (n = 8) were able to take up and drain injected dye via the VCLV, as compared with 100% (n = 11) of the control embryos (Fig. 2K,L).
Clp24 gene targeted mice have enlarged lymphatic vessels with abnormal smooth muscle cell (SMC) coverage In order to reveal the in vivo function of Clp24 in mammals, we produced gene targeted Clp24 mice in which the first exon of the gene was flanked by loxP sites (Supplemental Fig. S5). We crossed the Clp24 lox/lox mice with the PGK-Cre mice to delete Clp24 in all tissues (Lallemand et al. 1998). Clp24 À/À mice were born in a normal Mendelian ratio, and survived until adulthood with no obvious health problems. However, immunohistochemical analysis of the vasculature of the Clp24 À/À mice showed enlarged lymphatic vessels in various organs ( Fig. 3A-E; Supplemental Figs. S6, S8I, S9C,D), while the blood capillary network appeared similar to that in wild-type littermates (Supplemental Fig. S9A,B,E-H).
Closer analysis of the ear vasculature showed that the collecting lymphatic vessels located in the deeper layers of the ears of the Clp24 À/À mice were dilated when compared with wild-type littermates, but were equally covered by a continuous layer of SMC actin (SMA)positive perivascular support cells (Fig. 3D,E; Supplemental Fig. S6C,D, arrowheads). In the more superficial layers of the ear, the lymphatic vessels gradually lose the SMC coating, and express LYVE-1 (Supplemental Fig. S6B,D). However, in the Clp24 À/À mice, the enlarged initial lymphatic vessels had increased numbers of associated SMCs when compared with wild type (Fig. 3F; Supplemental Fig S6A-D, arrows). Notably, LYVE-1 expression was down-regulated in the Clp24 À/À lymphatic vessels in the sites that were in contact with SMCs, suggesting that SMCs modulated the lymphatic vessel identity (Supplemental Fig S6A, arrow).
We also mated the Clp24 lox/lox mice with the Tie1-Cre mice, which results in deletion of Clp24 in endothelial cells. Importantly, the lymphatic phenotype of the endothelial cell-specific Clp24 À/À knockout mice (Clp24EC À/À ) was similar to that of the Clp24 À/À mice (Supplemental Fig. S7), strongly suggesting that endothelial cell-expressed Clp24 specifically contributes to the formation of the lymphatic vasculature.
Similarly, down-regulation of clp24 in Xenopus embryos in combination with vegfr3 down-regulation led to an aggravation of the lymphatic phenotype (Supplemental Fig. S10). These results indicated that Clp24 interacts with the Vegfr2 and Vegfr3 pathways in lymphatic vessel patterning.
We next analyzed any possible interactions between CLP24 and VEGFR-2 or VEGFR-3. CLP24 was found to coimmunoprecipitate with both VEGFR-2 and VEGFR-3 from double-transfected human umbilical vein endothelial cells (HUVECs) and 293T cells, in both the presence and absence of ligand stimulation (Fig. 4A,B; data not shown). As a negative control, CLP24 did not coimmunoprecipitate with Tie-2 from double-transfected cells (Fig. 4C).
To further explore the cellular mechanism behind the lymphatic phenotype of the Clp24 À/À mice, we analyzed lung microvascular endothelial cells isolated from the Clp24 À/À mice. Clp24 À/À endothelial cells showed normal morphology and expression of endothelial cell-specific proteins (Supplemental Fig. S11). Ligand stimulation of VEGFRs leads to activation of several downstream signaling molecules-in particular, strong phosphorylation of the transcription factor CREB (Heckman et al. 2008). The VEGF-induced phosphorylation of CREB was increased significantly in Clp24 À/À endothelial cells when compared with wild-type cells, while other signaling proteins, such as p38MAPK, were unaltered ( Fig. 4D-I; data not shown). The VEGFR-2-and VEGFR-3-specific ligands VEGF-E and VEGF-C156S, respectively, induced CREB phosphorylation in the LECs (Fig. 4J-O), which was reduced significantly in CLP24-transfected cells (Fig.  4N,O). These experiments indicated that CLP24 modulates signaling via both VEGFR-2 and VEGFR-3.
Our results show that CLP24 is essential for lymphatic development in frogs, zebrafish, and mice. The lymphatic vessels of the Clp24 À/À mice were dilated and showed abnormal recruitment of SMCs, while in the frog and zebrafish, the lymphatic development appeared to fail altogether. In zebrafish, the parachordal lymphangioblasts were absent from the horizontal myoceptum at 48 hpf, and in stage 35/36 tadpoles, the prox1-positive cells did not bud dorsally from the PCV to form the DCLV. Thus, the early stages of lymphatic development were abrogated in both fish and frogs, while in mice the initial phases of lymphatic development occurred, and vessel abnormalities were detected later. This suggests that, in mice, CLP24 may act during the later stages of lymphatic differentiation, when the SMC-covered collecting lymph vessels are demarcated from lymph capillaries (Tammela and Alitalo 2010). More SMC-covered enlarged lymphatic vessels have also been described in mice deficient of the Foxc2 transcription factor or angiopoietin-2, or having a mutant ephrinB2 Makinen et al. 2005;Dellinger et al. 2008). Although clp24 knockdown caused mild blood vascular defects in zebrafish and frog embryos, such were not found in the Clp24 À/À mice.
It remains to be studied why the fish and frog vasculatures are more susceptible to the loss of clp24 when compared with mice. Although there are no close homologs of Clp24 in mice, it is possible that a compensating mechanism allows Clp24 À/À mice to pass early lymphatic development, thus explaining the differences between the mouse versus the frog and fish phenotypes. In addition, collecting lymphatic vessels have not been reported in fish, and part of the phenotype we describe (difference in SMC recruitment) is associated with their differentiation. It should also be noted that VEGF-C has a more widespread role in fish compared with mice. In mice, VEGF-C is required for all lymphatic sprouting during development, and it also contributes to retinal and tumor angiogenesis (Karkkainen et al. 2004;Tammela et al. 2008), while the zebrafish VEGF-C is also required for venous-derived angiogenesis in the trunk (Hogan et al. 2009). Clp24 knockdown in the fish and frog prevented lymphangiogenic budding from venous endothelium, a process critically dependent on VEGF-C (Hogan et al. 2009). It is possible that CLP24 participates also in VEGF-C-mediated blood vascular development in fish and frog embryos, thus explaining the observed vascular defects in the morphants. Whole-mount staining of lymphatic and blood vessels for LYVE-1 and PECAM-1, respectively, in the ears of Clp24 À/À and Clp24 +/À mice. (B,C) Paraffin sections of the tongue (B) and the small intestine (C) of wild-type, Clp +/À , and Clp24 À/À mice were stained for LYVE-1. Note enlarged lymphatic vessels in Clp24 À/À mice (arrowheads in A-C). (D,E) Whole-mount staining of the ears of Clp24 À/À (D) and wild-type (Clp24 +/+ ) (E) mice for VEGFR-2 and SMA to detect perivascular SMCs. Note enlarged SMC-covered collecting lymphatic vessels (arrows) in Clp24 À/À mice. (F) Quantification of SMC-covered initial lymphatic vessels in the ear periphery in Clp24 À/À and wild-type mice. Shown is the mean of lymphatic vessel-associated SMC clusters per 2.25-mm 2 field. Representative images used for quantification are shown in Supplemental Figure S6, C and D. Bar, 100 mm.
The lymphatic phenotype of Clp24 À/À mice was more pronounced when one allele of Vegfr2 or Vegfr3 was also deleted. Interestingly, CLP24 modulated signaling through VEGFR-2 and VEGFR-3, as phosphorylation of CREB was enhanced when Clp24 was deleted, but was reduced when CLP24 was overexpressed. CREB phosphorylation has been linked to increased cell survival (Lee et al. 2009), which could partly explain the observed enlargement of lymphatic vessels in the Clp24 À/À mice. We found that CLP24 coimmunoprecipitated with VEGFR-2 and VEGFR-3 from endothelial cells, suggesting a possibility that CLP24 directly regulates VEGFR-2/ VEGFR-3 function. However, further studies are required to unravel the mechanism of CLP24-mediated modulation of VEGFR-2/VEGFR-3 signaling.
Overexpression of VEGFs is associated with pathological activation of the VEGFR-2 and VEGFR-3 signaling pathways. Blocking of the VEGF-VEGFR-2 pathway is the first anti-angiogenic therapy that is being increasingly used to treat human diseases (Crawford and Ferrara 2009). The present study discovers CLP24 as a novel modulator of VEGFR-2 and VEGFR-3 signaling, and indicates that CLP24 has an essential function in the lymphatic vasculature, while the possible role of CLP24 in pathological angiogenesis remains to be studied. In conclusion, our results suggest that different effectors are recruited to VEGFR-2 and VEGFR-3 to control the diverse downstream functions of VEGF/VEGF-C signaling in lymphatic versus blood vessels.

Materials and methods
Cell culture, reagents, immunoblotting, immunofluorescence Cell culture, retrovirus production, immunoblotting, and immunofluorescence are described previously (Saharinen et al. 2008), and antibodies and growth factor stimulations are described in the Supplemental Material. TIE2, VEGFR-2, VEGFR-3, CLP24-EGFP, and CLP24-V5 cDNAs were cloned into pMXs vector (a generous gift from Dr. Toshio Kitamura, University of Tokyo). Mouse lung endothelial cells were isolated as explained previously (Reynolds and Hodivala-Dilke 2006) and in the Supplemental Material.

ISH and scoring of TD and parachordal lymphatic precursor formation in zebrafish embryos
Clp24 ISH was performed as described (Chittenden et al. 2006), and live screening and quantification of TD formation was performed as detailed in the Supplemental Material.
ISH, MO injections, and morphometric and general analysis of the development in X. laevis Whole-mount ISH using prox1 probe was performed as described previously (Ny et al. 2005) and in the Supplemental Material. The generation and characterization of the Tg(Flk1:eGFP) line will be reported elsewhere. The cloning of clp24, the X. laevis clp24 ortholog, and the MO oligos are detailed in the Supplemental Material.

ISH of mouse tissues
ISH was performed using digoxigenin-labeled probes on 16-to 30-mm frozen sections of mouse embryos as detailed in the Supplemental Material.