CCM3 signaling through sterile 20-like kinases plays an essential role during zebrafish cardiovascular development and cerebral cavernous malformations

Abstract

Cerebral cavernous malformation is a common human vascular disease that arises due to loss-of-function mutations in genes encoding three intracellular adaptor proteins, cerebral cavernous malformations 1 protein (CCM1), CCM2, and CCM3. CCM1, CCM2, and CCM3 interact biochemically in a pathway required in endothelial cells during cardiovascular development in mice and zebrafish. The downstream effectors by which this signaling pathway regulates endothelial function have not yet been identified. Here we have shown in zebrafish that expression of mutant ccm3 proteins (ccm3Delta) known to cause cerebral cavernous malformation in humans confers cardiovascular phenotypes identical to those associated with loss of ccm1 and ccm2. CCM3Delta proteins interacted with CCM1 and CCM2, but not with other proteins known to bind wild-type CCM3, serine/threonine protein kinase MST4 (MST4), sterile 20-like serine/threonine kinase 24 (STK24), and STK25, all of which have poorly defined biological functions. Cardiovascular phenotypes characteristic of CCM deficiency arose due to stk deficiency and combined low-level deficiency of stks and ccm3 in zebrafish embryos. In cultured human endothelial cells, CCM3 and STK25 regulated barrier function in a manner similar to CCM2, and STKs negatively regulated Rho by directly activating moesin. These studies identify STKs as essential downstream effectors of CCM signaling in development and disease that may regulate both endothelial and epithelial cell junctions.

Figure 1. Zebrafish ccm3 proteins are highly conserved with the human ortholog CM3, and ccm3 is necessary during early zebrafish development.

(A) Alignment of the predicted amino acid sequences of zebrafish ccm3a and ccm3b with that of human CCM3. Identical residues are shaded in gray. Vertical lines indicate exon boundaries. The amino acids encoded by CCM3 exon 5 (hExon5) and ccm3a/b exon 3 (zExon3) are indicated. (B) Expression pattern of ccm3a and ccm3b in zebrafish embryos. ccm3a and ccm3b are ubiquitously expressed at the 60% epiboly and 12-second stages. At 24 hpf, ccm3a and ccm3b are expressed most strongly in the head. (C–G) Loss of ccm3a+ccm3b or ccm3b alone results in early embryonic lethality in zebrafish. Shown are 24-hpf zebrafish embryos following injection of ccm3aX2 mismatch control morpholino (C, 5 ng/embryo); a morpholino (ccm3a/bATG) designed to block translation of both ccm3a and ccm3b (D, 0.5 ng/embryo); or morpholinos specifically interfering the splicing of ccm3a or ccm3b: ccm3bX4 morpholino (E, 4 ng/embryo), ccm3aX2 morpholino (F, 3 ng/embryo), and ccm3aX4 morpholino (G, 4 ng/embryo). C, F, and G are composites of 2–3 images taken of the same embryos.

Figure 2. Expression of ccm3 proteins lacking the 18 amino acids encoded by exon 3 confers cardiovascular phenotypes characteristic of heg, ccm1, and ccm2 deficiency.

(A) Light images of the hearts of 48-hpf zebrafish control embryos, ccm2 mutant embryos, and embryos injected with morpholinos that block splicing into exon 3 of ccm3a only (ccm3aX3, 3 ng/embryo), ccm3b only (ccm3bX3, 3 ng/embryo), both ccm3a and ccm3b [ccm3(a+b)X3], or exon 2 of ccm3a (ccm3aX2, 3 ng/embryo) are shown. Arrows indicate the embryo hearts. (B) Fluorescence images of the hearts of transgenic embryos in which myocardial cells express GFP following injection of the indicated morpholinos. ccm2MO indicates a morpholino that blocks splicing of the ccm2 gene. (C) Thinned myocardium in ccm3(a+b)X3 morphants is identical to that seen in ccm2 mutants. Shown are hematoxylin/eosin-stained sagittal sections of the indicated 48-hpf embryos. a, atrium; v, ventricle; hw, heart wall. (D) Angiography of 48-hpf control and ccm3(a+b)X3 morphant embryos reveals blocked circulation at the cardiac outflow tract. (E) Vascular endothelial patterning as revealed in Tg (fli1a:EGFP)^y1 embryos is undisturbed in ccm3(a+b)X3 morphant embryos. The images are composites of 2–3 images taken of the same embryos. (F) The big heart phenotype conferred by morpholinos that block splicing into exon 3 of ccm3a and ccm3b is rescued by coinjection of cRNAs (100 pg/embryo) encoding either ccm3a or ccm3b (right 2 bars). Shown are mean and SEM. The number of embryos examined is indicated above each bar, and the number of injections performed for each group shown in parentheses. ***P < 0.001 by Student’s t test. Scale bars: 20 μm.

Figure 3. ccm3Δ proteins form a complex with ccm1 and ccm2 but fail to bind the GCK-III family of sterile 20–like kinases.

(A) A working model in which HEG receptors bind CCM1, CCM1 binds CCM2, and CCM2 binds CCM3 is shown. (B) Co-immunoprecipitation of a zebrafish ccm1/ccm2/ccm3 protein complex. FLAG-ccm1, HA-ccm2, and myc-ccm3 were coexpressed and ccm3 immunoprecipitations performed (left). Protein expression is shown by immunoblot analysis (right). The white line indicates the boundary of a noncontiguous lane run on the same gel. (C) Formation of the zebrafish ccm1/ccm2/ccm3a complex requires the ccm2 PTB domain but not the 18 amino acids encoded by ccm3 exon 3. ccm proteins were coexpressed and ccm3 immunoprecipitations performed. ccm2L197R contains a point mutation in the PTB domain that blocks interaction with ccm1 (10). ccm3aΔ proteins lack the 18 amino acids encoded by exon 3. (D) Zebrafish ccm3a interaction with STK24 and STK25 requires the 18 amino acids encoded by exon 3. ccm3 immunoprecipitations were performed as described above, and co-immunoprecipitated endogenous human STK24, STK25, and MST4 were detected with specific antibodies. (E) Zebrafish ccm3b interaction with STK24, STK25, and MST4 requires the 18 amino acids encoded by exon 3. (F) Human CCM3 interaction with STK24, STK25, and MST4 requires the 18 amino acids encoded by exon 5. CCM3 and CCM3Δ double-tagged with Flag and HA (FH) were expressed and CCM3-STK co-immunoprecipitation experiments performed as described for D and E. The red asterisk indicates a background band present in all lanes.

Figure 4. ccm3 and GCK-III family STKs interact in the CCM pathway in vivo.

(A) STK deficiency confers the big heart phenotype characteristic of CCM deficiency in zebrafish embryos. Conferral of the big heart phenotype was scored following injection of low-dose morpholinos targeting stk24 (stk24X6, 3 ng/embryo), stk25a (stk25aX6, 3 ng/embryo), stk25b (stk25bX6, 3 ng/embryo) alone or a combination of all 3 morpholinos. Shown are mean and SEM. The number of embryos examined is indicated above each bar, and the number of separate injections performed with the indicated morpholinos is indicated in parentheses. ***P < 0.001 by Student’s t test. (B) ccm3 and STKs functionally interact in the CCM pathway in zebrafish embryos. Low-dose morpholinos against ccm3a (ccm3aX3, 3 ng/embryo) and stk24 (stk24X3+stk24X6, 2 ng/embryo each), stk25a (stk25aX3+stk25aX6, 2 ng/embryo each) or stk25b (stk25bX6, 2 ng/embryo) were injected separately or in combination into wild-type embryos and the presence of the big heart phenotype characteristic of CCM signaling deficiency was scored at 48 hpf. ***P < 0.001 by Student’s t test. (C–F) Representative light microscopic images of the big heart phenotypes conferred by the morpholinos described in B. The atrial margins are outlined by white dashed lines. (G–I) Representative histologic cross sections of 48-hpf morphant (ccm3aX3 and ccm3aX3+stk25bX6) and mutant (ccm2) embryos stained with hematoxylin and eosin. h, heart. (J–L) High-power images of the boxed regions of hearts shown in G–I. Scale bars: 20 μm.

Figure 5. CCM3 and STKs regulate endothelial junctions.

(A) Basal TEER was measured in HMVEC monolayers following treatment with siRNAs targeting the indicated genes. (B) The change in TEER in response to VEGF was measured in HMVECs treated with the indicated siRNAs. n = 12. ***P < 0.001, **P < 0.01, *P < 0.05 by 1-way ANOVA and Bonferroni’s correction. Data shown are representative of 4 independent experiments.

Figure 6. STK deficiency, like CCM deficiency, results in actin stress fiber formation and elevated Rho-A activity in human endothelial cells.

(A) Phalloidin staining of actin stress fibers in HUVECs treated with control siRNA and siRNAs targeting CCM2, CCM3, STK24, STK25, or STK24+STK25 is shown. (B) The percentage of cells with central actin stress fibers following the siRNA treatments described in A is shown. Shown are mean and SEM. n = 6; *P < 0.05, ***P < 0.0001 by Student’s t test. (C) Upregulation of Rho-A activity is associated with deficiency of CCM proteins or STKs. Rho-A–GTP levels were measured in endothelial cells treated with the indicated siRNAs (top bands) and compared with total cellular Rho-A (bottom bands). (D) Fold change in Rho-A activation following treatment of HUVECs with siRNAs targeting the indicated genes or with LPA (10 μM, 30 minutes). Shown are mean and SEM. n = 3. *P < 0.05, **P < 0.01 by Student’s t test. Scale bars: 20 μm.

Figure 7. STK24 and STK25 directly activate moesin, and loss of moesin increases Rho-A activity in endothelial cells.

(A) In vitro kinase assays were performed using purified STK24 or STK25 and HA-moesin or HA-moesin T558A and phosphorylation at the moesin T558 detected using an antibody against phospho-moesin (top panel). The total moesin protein used in the reactions was detected using anti-moesin antibody (bottom panel). (B) Expression of STK25 raises endothelial phospho-T558 moesin levels. BAECs transfected with plasmids to drive expression of FLAG-STK25 were stained with anti-FLAG (Alexa Fluor 488) and anti–phospho-T558 moesin (Alexa Fluor 594) antibodies as well as DAPI to detect cell nuclei. (C) Inhibition of CCM3 and STK25 reduces phospho-T558 moesin levels. siRNA was used to knock down CCM3 and/or STK25 in HMVECs and phospho-T558 moesin detected using immunofluorescence. (D) Loss of moesin increases activated Rho-A levels. siRNA was used to reduce expression of moesin (MSN) and/or ezrin (EZR) in HMVECs and activated Rho-A measured as in Figure 5D. Shown are mean and SEM. n = 2. *P < 0.05, **P < 0.01 by Student’s t test. Scale bars: 20 μm.