Wnt5a regulates multiple intracellular signalling cascades, but how Wnt5a determines the specificity of these pathways is not well understood. This study examined whether the internalization of Wnt receptors affects the ability of Wnt5a to regulate its signalling pathways. Wnt5a activated Rac in the b-catenin-independent pathway, and Frizzled2 (Fz2) and Ror1 or Ror2 were required for this action. Fz2 was internalized through a clathrin-mediated route in response to Wnt5a, and inhibition of clathrindependent internalization suppressed the ability of Wnt5a to activate Rac. As another action of Wnt5a, it inhibited Wnt3a-dependent lipoprotein receptor-related protein 6 (LRP6) phosphorylation and b-catenin accumulation. Wnt3a-dependent phosphorylation of LRP6 was enhanced in Wnt5a knockout embryonic fibroblasts. Fz2 was also required for the Wnt3a-dependent accumulation of b-catenin, and Wnt5a competed with Wnt3a for binding to Fz2 in vitro and in intact cells, thereby inhibiting the b-catenin pathway. This inhibitory action of Wnt5a was not affected by the impairment of clathrin-dependent internalization. These results suggest that Wnt5a regulates distinct pathways through receptor internalizationdependent and -independent mechanisms.

Wnt5a is a representative ligand that activates the b-catenin-independent pathways. Because the b-catenin-independent pathway includes multiple signalling cascades in addition to the planar cell polarity and Ca 2+ pathway, Wnt5a regulates a variety of cellular functions, such as proliferation, differentiation, migration, adhesion and polarity. Consistent with the multiple functions of Wnt5a signalling, Wnt5a knockout mice show various phenotypes, including an inability to extend the embryonic anterior-posterior and proximal-distal axes in outgrowth tissues. Thus, many important roles of Wnt5a in developmental processes have been demonstrated. Moreover, recent reports suggest that the postnatal abnormalities in the Wnt5a signalling are involved in various diseases, such as cancer, inflammatory diseases and metabolic disorders. Therefore, Wnt5a and its signalling pathways could be important targets for the diagnosis and therapy for human diseases.

Wnt5a is a representative ligand that activates the b-catenin-independent pathway in Wnt signaling. Although it has been reported that abnormal activation of the Wnt/ b-catenin-dependent pathway is often observed in human prostate cancer, the involvement of the b-catenin-independent pathway in this cancer is unclear. Abnormal expression of Wnt5a and b-catenin was observed in 27 (28%) and 49 (50%) of 98 prostate cancer cases, respectively, by immunohistochemical analyses. Simultaneous expression of Wnt5a and b-catenin was observed in only five cases, suggesting their exclusive expression. The positive detection of Wnt5a was correlated with high Gleason scores and biochemical relapse of prostate cancer, but that of b-catenin was not. Knockdown and overexpression of Wnt5a in human prostate cancer cell lines reduced and stimulated, respectively, their invasion activities, and the invasion activity required Frizzled2 and Ror2 as Wnt receptors. Wnt5a activated Jun-N-terminal kinase through protein kinase D (PKD) and the inhibition of PKD suppressed Wnt5a-dependent cell migration and invasion. In addition, Wnt5a induced the expression of metalloproteinase-1 through the recruitment of JunD to its promoter region. These results suggest that Wnt5a promotes the aggressiveness of prostate cancer and that its expression is involved in relapse after prostatectomy.

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The Formin proteins are central players in mediating cytoskeletal reorganization and are epistatically positioned in a pathway downstream of Rho activation. These proteins exist in the cytoplasm in an autoinhibited state, which is mediated by intramolecular interactions between the amino-terminal GTPase binding domain (GBD) that encompasses the diaphanous inhibitory domain (DID) and the carboxyl-terminal diaphanous autoregulatory domain (DAD). It Dishevelled ͉ Wnt ͉ Rho D irectional cell migration is required for the development of an organism with proper polarity including dorsoventral, anterior-posterior, and left-right symmetry. Examples of these cell movements include those of gastrulation and neural fold closure. These cell movements are tightly regulated by secreted ligands (1, 2). One of these signaling pathways required for cell movements is the noncanonical Wnt pathway (3-5).Noncanonical Wnt signaling, also termed the planar cell polarity pathway, regulates cell movements through modification of the actin cytoskeleton (1,3,4,6). A number of molecular components for this pathway have been identified including Wnt11, Fz, Dvl, Daam1, Rho, Rac, JNK, Strabismus, and Prickle (reviewed in ref. 5). For noncanonical Wnt signaling, the binding of Wnt to the Frizzled (Fz) receptor stimulates a signal that is transduced to the cytoplasmic phosphoprotein Dishevelled (Dvl). At the level of Dvl, two independent and parallel pathways lead to the activation of the small GTPases Rho and Rac. The first pathway signaling to the small GTPase Rho occurs through the molecule Daam1 (7). This Rho pathway leads to the activation of the Rho-associated kinase Rock and mediates cytoskeletal reorganization (5, 8). The second activates another small GTPase of the Rho-family, Rac, which in turns stimulates JNK activity (9-11). Daam1 is a Formin protein family and has been shown to regulate gastrulation; however, how Daam1 is activated for its function remains unknown.The Formin proteins are central players in regulating cytoskeletal reorganization in mammalian cells (12). The Formin proteins contain three major domains termed the GTPase binding domain (GBD), Formin homology 1 (FH1) domain, and Formin homology 2 (FH2) domain (13). These proteins are proposed to exist in the cytoplasm in an autoinhibited state, which is mediated by a domain termed the diaphanous autoinhibitory domain (DAD) (12). This DAD found in the carboxyl terminus mediates interaction with the amino terminus of the protein and serves to ''lock'' the protein in a folded or closed conformation (12). It is proposed that Rho activation allows for Rho-GTP to bind to the GBD and release this molecule from autoinhibition. The FH1 and FH2 can then bind to effectors to mediate effects on the cytoskeleton. Intriguingly, the FH2 domain has recently been shown to be capable of nucleating actin filaments by itself in vitro, suggesting a complex interplay between the FH1 and FH2 domains along with their effectors for actin polymerization (12). However, it remains unclear how th...

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Abstract-Adiponectin is an antiatherogenic adipokine that inhibits inflammation by mechanisms that are not completely understood. We explored the effect of adiponectin on endothelial synthesis of interleukin-8 (IL-8), a pro-inflammatory chemokine that plays a role in atherogenesis. Adiponectin decreased the secretion of IL-8 from human aortic endothelial cells (HAEC) stimulated with tumor necrosis factor-␣ (TNF-␣). Adiponectin also inhibited IL-8 mRNA expression induced by TNF-␣. Phosphorylation of IB-␣ was decreased by adiponectin, but phosphorylation of ERK, SAPK/JNK, and p38MAPK were unaffected. Adiponectin increased intra-cellular cAMP levels in HAEC in a dose-dependent manner; PKA activity was also increased. The inhibitory effect of adiponectin on TNF-␣-induced IL-8 synthesis was inhibited by pretreatment with Rp-cAMP, a PKA inhibitor. These observations suggest that adiponectin inhibits IL-8 synthesis through inhibition of a PKA dependent NF-B signaling pathway. We also showed that adiponectin enhances Akt phosphorylation.

Growth factor-dependent epithelial morphological changes and proliferation are essential for the formation of tubular structures, but the underlying molecular mechanisms are poorly understood. Co-stimulation with Wnt3a and epidermal growth factor (Wnt3a/ EGF) induced development of tubes consisting of intestinal epithelial cells by inducing expression of Arl4c, an Arf-like small GTPbinding protein, in three-dimensional culture, while stimulation with Wnt3a or EGF alone did not. Arl4c expression resulted in rearrangement of the cytoskeleton through activation of Rac and inactivation of Rho properly, which promoted cell growth by inducing nuclear translocation of Yes-associated protein and transcriptional co-activator with PDZ-binding motif (YAP/TAZ) in leading cells. Arl4c was expressed in ureteric bud tips and pretubular structures in the embryonic kidney. In an organoid culture assay, Wnt and fibroblast growth factor signaling simultaneously induced elongation and budding of kidney ureteric buds through Arl4c expression. YAP/TAZ was observed in the nucleus of extending ureteric bud tips. Thus, Arl4c expression induced by a combination of growth factor signaling mechanisms is involved in tube formation.