Efficient and targeted sperm motility is essential for animal reproductive success. Sperm from mammals and echinoderms utilize a highly conserved signaling mechanism in which sperm motility is stimulated by pH-dependent activation of the cAMP-producing enzyme soluble adenylyl cyclase (sAC). However, the presence of this pathway in early-branching metazoans has remained unexplored. Here, we found that elevating cytoplasmic pH induced a rapid burst of cAMP signaling and triggered the onset of motility in sperm from the reef-building coral Montipora capitata in a sAC-dependent manner. Expression of sAC in the mitochondrial-rich midpiece and flagellum of coral sperm support a dual role for this molecular pH sensor in regulating mitochondrial respiration and flagellar beating and thus motility. In addition, we found that additional members of the homologous signaling pathway described in echinoderms, both upstream and downstream of sAC, are expressed in coral sperm. These include the Na+/H+ exchanger SLC9C1, protein kinase A, and the CatSper Ca2+ channel conserved even in mammalian sperm. Indeed, the onset of motility corresponded with increased protein kinase A activity. Our discovery of this pathway in an early-branching metazoan species highlights the ancient origin of the pH-sAC-cAMP signaling node in sperm physiology and suggests that it may be present in many other marine invertebrate taxa for which sperm motility mechanisms remain unexplored. These results emphasize the need to better understand the role of pH-dependent signaling in the reproductive success of marine animals, particularly as climate change stressors continue to alter the physiology of corals and other marine invertebrates.

Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-β (TGF-β) signaling, and mice lacking components of the TGF-β signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-β pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4(NULL) mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4(NULL) mice, resolution of inflammation was delayed, and expression of β4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4(NULL) skin. The induced level of phosphorylated Smad2 in response to TGF-β was reduced in cultured CLIC4(NULL) keratinocytes relative to in wild-type cells, and CLIC4(NULL) keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-β. CLIC4(NULL) keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-β pathway.

SUMMARY Wnts are a family of 19 extracellular ligands that regulate cell fate, proliferation and migration during metazoan embryogenesis and throughout adulthood. Wnts are acylated post-translationally at a conserved serine and bind the extracellular cysteine-rich domain (CRD) of Frizzled (FZD) seven-pass transmembrane receptors. Although crystal structures suggest that acylation is essential for Wnt binding to FZDs, we show here that several Wnts can promote signaling in Xenopus laevis and Danio rerio embryos – as well as in an in vitro cell culture model – without acylation. The non-acylated Wnts are expressed at similar levels to wild-type counterparts and retain CRD binding. By contrast, we find that certain other Wnts do require acylation for biological activity in Xenopus embryos – although not necessarily for FZD binding. Our data argue that acylation-dependence of Wnt activity is context-specific. They further suggest that acylation may underlie aspects of ligand/receptor selectivity and/or control other aspects of Wnt function.

C o m m e n t a r y2 Divergent functions for Dickkopf-1Dickkopf-1 (DKK-1) was first identified in Xenopus as an extracellular antagonist of canonical Wnt signaling (1), a fundamental pathway that controls cell fate, survival, and proliferation. Aberrant activation of Wnt signaling also drives tumorigenesis in colon, breast, hepatocellular, and many other cancers (2-4). As a Wnt signaling inhibitor, DKK-1 suppresses tumorigenesis in some cancer cells, including renal cell carcinoma (5), breast carcinoma (6), and colon cancer cells (7-9). However, DKK-1 is often elevated in cancers, is associated with aggressive disease, and promotes proliferation in multiple cell types (10, 11), suggesting that DKK-1 also functions independently of Wnt inhibition. In this issue, Kimura and colleagues investigate the molecular events that lead to DKK-1-induced cancer cell proliferation (12). Their work has identified a Wnt-independent signaling role for DKK-1 through interaction with the type-II transmembrane protein cytoskeleton-associated protein 4 (CKAP4; also known as p63, ERGIC-63, or CLIMP-63). These findings also indicate that the DKK-1/CKAP4 interaction represents a promising therapeutic target for cancer.CKAP4 is a DKK-1 receptor that activates AKT signaling DKK family proteins contain two cysteinerich domains (CRDs). The C-terminal CRD (CRD-2) of DKK-1 inhibits Wnt signaling by directly binding to the Wnt coreceptors LRP5/6 (LDL-related proteins 5 and 6), thereby preventing interaction with Wnts, and by binding to the transmembrane protein KREMEN to promote LRP5/6 internalization (11, 13-15). Kimura et al. have now demonstrated that CRD-1 of DKK-1 binds to CKAP4 at the apical surface of epithelial cells (Figure 1 and ref. 12). Furthermore, DKK-1 binds to CKAP4 with high affinity that is comparable to but independent of its interaction with LRP5/6. Upon DKK-1 binding, CKAP4 recruits phosphatidylinositol-3 kinase (PI3K) to its cytoplasmic domain through interaction with the SH3 domain of the p85α subunit of PI3K. Through this mechanism, DKK-1 activates AKT and promotes proliferation. These findings substantially expand our understanding of DKK-1 signaling mechanisms and define a previously unrecognized input to the PI3K/AKT pathway.The findings of Kimura et al. also raise questions about the potential functions of other DKK family members (12). The DKK family comprises four conserved proteins, DKK-1 through -4 (and DKK-like 1, which lacks the CRDs). DKK-1 antagonizes Wnt/β-catenin signaling, activates AKT (as shown here), and may also activate JNK signaling (16); however, the functions of other DKK family members are less well characterized. DKK-2 and -4 antagonize Wnt signaling, similar to DKK-1, but DKK-2 can also activate canonical Wnt signaling depending on the cellular context (11,17). In contrast, DKK-3 positively regulates Wnt signaling (18) and binds to integrin α6b to induce myogenesis in zebrafish (19). As Kimura and colleagues point out (12), CRD-1 is highly conserved among the four Dkk genes, raising the possib...

Highlights d WNT-binding CRDs of ROR family receptors differ structurally from those in FZDs d The WIF domain of the RYK ortholog Derailed lacks WIF-1's lipid binding site d Drosophila WNT-5 binds ROR and RYK family receptors despite lacking an acyl chain d WNT-binding receptor tyrosine kinases recognize different WNT features than FZDs