The effectiveness of S-LEC™ solar control film L in laminated glass for reducing the burning sensation caused by near-infrared radiation.

OBJECTIVES: The aim of this study was to examine the potential for S-LEC™ Solar Control Film L, a heat insulating interlayer film for laminated glass developed by Sekisui Chemical Co., Ltd., to reduce the burning sensation caused by near infrared (NIR) radiation. METHODS: A crossover study was performed in 49 male and female subjects, using three different combinations of laminated glass with interlayer films. Subjects placed their right hand under each glass in an NIR irradiation device. The dorsal side of the right hand was irradiated with NIR for 30 s and the onset and degree of a burning sensation was measured. RESULTS: Laminated glass with S-LEC™ Solar Control Film L reduced the burning sensation caused by NIR and delayed the onset of the sensation, compared with the control glasses. CONCLUSION: The use of S-LEC™ Solar Control Film L in the laminated glass of vehicles may improve passenger comfort and prevent skin disorders such as photoaging caused by prolonged exposure to NIR.

Impaired lymphocyte trafficking in mice deficient in the kinase activity of PKN1.

Knock-in mice lacking PKN1 kinase activity were generated by introducing a T778A point mutation in the catalytic domain. PKN1[T778A] mutant mice developed to adulthood without apparent external abnormalities, but exhibited lower T and B lymphocyte counts in the peripheral blood than those of wild-type (WT) mice. T and B cell development proceeded in an apparently normal fashion in bone marrow and thymus of PKN1[T778A] mice, however, the number of T and B cell counts were significantly higher in the lymph nodes and spleen of mutant mice in those of WT mice. After transfusion into WT recipients, EGFP-labelled PKN1[T778A] donor lymphocytes were significantly less abundant in the peripheral circulation and more abundant in the spleen and lymph nodes of recipient mice compared with EGFP-labelled WT donor lymphocytes, likely reflecting lymphocyte sequestration in the spleen and lymph nodes in a cell-autonomous fashion. PKN1[T778A] lymphocytes showed significantly lower chemotaxis towards chemokines and sphingosine 1-phosphate (S1P) than WT cells in vitro. The biggest migration defect was observed in response to S1P, which is essential for lymphocyte egress from secondary lymphoid organs. These results reveal a novel role of PKN1 in lymphocyte migration and localization.

Moesin controls clathrin-mediated S1PR1 internalization in T cells.

The lipid mediator sphingosine 1-phosphate (S1P) regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5), S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM) family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs) and early endosomes, and S1PR1 internalization was blocked when clathrin was pharmacologically inhibited. Stimulating moesin-deficient T cells with S1P failed to induce S1PR1 internalization and CCV formation. Furthermore, treating moesin-deficient mice with FTY720, an S1P receptor agonist known to internalize S1PR1, caused delayed lymphopenia, and lymphocytes isolated from FTY720-treated moesin-deficient mice still responded to S1P ex vivo in chemotaxis assays. These results reveal a novel role for moesin in regulating clathrin-dependent S1PR1 internalization through CCV formation.

Moesin-deficient mice reveal a non-redundant role for moesin in lymphocyte homeostasis.

Moesin is a member of the ezrin-radixin-moesin (ERM) family of cytoskeletal proteins. These proteins organize membrane domains by interacting with plasma membrane proteins and the actin cytoskeleton. Because of their high sequence similarity, ERM proteins are usually thought to be functionally redundant. Lymphocytes express two ERM proteins, ezrin and moesin. Whether each ERM plays a specialized role in lymphocytes, particularly in vivo, remains unknown. Here, we show that moesin has a crucial, non-redundant role in lymphocyte homeostasis. Moesin-deficient mice exhibited decreases in both T and B cells in the peripheral blood and lymph nodes, but not in the spleen. This phenotype was recapitulated in bone marrow (BM) chimeras with a hematopoietic moesin deficiency. Although the T and B cells apparently developed without major defects in the moesin-deficient mice, T cell egress from the thymus and immature B cell egress from the BM were impaired. In the periphery, both T and B cells showed delayed egress from lymphoid organs. We showed that moesin is the primary phosphorylated ERM subject to dynamic regulation during cell shape changes and migration. Our findings identify a previously unknown, non-redundant function of moesin in lymphocyte homeostasis in regulating lymphocyte egress from lymphoid organs.

Ror2/Frizzled complex mediates Wnt5a-induced AP-1 activation by regulating Dishevelled polymerization.

The receptor tyrosine kinase Ror2 acts as a receptor or coreceptor for Wnt5a to mediate Wnt5a-induced activation of the Wnt/JNK pathway and inhibition of the beta-catenin-dependent canonical Wnt pathway. However, little is known about how Ror2 cooperates with another receptor component(s) to mediate Wnt5a signaling. We show here that Ror2 regulates Wnt5a-induced polymerization of Dishevelled (Dvl) and that this Ror2-mediated regulation of Dvl is independent of the cytoplasmic region of Ror2. Ror2 can associate with Frizzled7 (Fz7) via its extracellular cysteine-rich domain to form a receptor complex that is required for the regulation of Dvl and activation of the AP-1 promoter after Wnt5a stimulation. Suppressed expression of Fz7 indeed results in the inhibition of Wnt5a-induced polymerization of Dvl and AP-1 activation. Interestingly, both the DIX and the DEP domains of Dvl are indispensable for Dvl polymerization and subsequent AP-1 activation after Wnt5a stimulation. We further show that polymerized Dvl is colocalized with Rac1 and that suppressed expression of Rac1 inhibits Wnt5a-induced AP-1 activation. Collectively, our results indicate that Ror2/Fz receptor complex plays an important role in the Wnt5a/Rac1/AP-1 pathway by regulating the polymerization of Dvl.

Receptor tyrosine kinase Ror2 mediates Wnt5a-induced polarized cell migration by activating c-Jun N-terminal kinase via actin-binding protein filamin A.

The receptor tyrosine kinase Ror2 has recently been shown to act as an alternative receptor or coreceptor for Wnt5a and to mediate Wnt5a-induced migration of cultured cells. However, little is known about the molecular mechanism underlying this migratory process. Here we show by wound-healing assays that Ror2 plays critical roles in Wnt5a-induced cell migration by regulating formation of lamellipodia and reorientation of microtubule-organizing center (MTOC). Wnt5a stimulation induces activation of the c-Jun N-terminal kinase JNK at the wound edge in a Ror2-dependent manner, and inhibiting JNK activity abrogates Wnt5a-induced lamellipodia formation and MTOC reorientation. Additionally, the association of Ror2 with the actin-binding protein filamin A is required for Wnt5a-induced JNK activation and polarized cell migration. We further show that Wnt5a-induced JNK activation and MTOC reorientation can be suppressed by inhibiting PKCzeta. Taken together, our findings indicate that Wnt5a/Ror2 activates JNK, through a process involving filamin A and PKCzeta, to regulate polarized cell migration.

Filopodia formation mediated by receptor tyrosine kinase Ror2 is required for Wnt5a-induced cell migration.

The receptor tyrosine kinase Ror2 plays important roles in developmental morphogenesis. It has recently been shown that Ror2 mediates Wnt5a-induced noncanonical Wnt signaling by activating the Wnt-JNK pathway and inhibiting the beta-catenin-TCF pathway. However, the function of Ror2 in noncanonical Wnt signaling leading to cell migration is largely unknown. We show, using genetically different or manipulated cultured cells, that Ror2 is critical for Wnt5a-induced, but not Wnt3a-induced, cell migration. Ror2-mediated cell migration requires the extracellular cysteine-rich domain (CRD), which is the binding site for Wnt5a, and the cytoplasmic proline-rich domain (PRD) of Ror2. Furthermore, Ror2 can mediate filopodia formation via actin reorganization, irrespective of Wnt5a, and this Ror2-mediated filopodia formation requires the actin-binding protein filamin A, which associates with the PRD of Ror2. Intriguingly, disruption of filopodia formation by suppressing the expression of either Ror2 or filamin A inhibits Wnt5a-induced cell migration, indicating that Ror2-mediated filopodia formation is essential for Wnt5a-induced cell migration.

The receptor tyrosine kinase Ror2 associates with and is activated by casein kinase Iepsilon.

Ror2, a member of the mammalian Ror family of receptor tyrosine kinases, plays important roles in developmental morphogenesis, although the mechanism underlying activation of Ror2 remains largely elusive. We show that when expressed in mammalian cells, Ror2 associates with casein kinase Iepsilon (CKIepsilon), a crucial regulator of Wnt signaling. This association occurs primarily via the cytoplasmic C-terminal proline-rich domain of Ror2. We also show that Ror2 is phosphorylated by CKIepsilon on serine/threonine residues, in its C-terminal serine/threonine-rich 2 domain, resulting in autophosphorylation of Ror2 on tyrosine residues. Furthermore, it was found that association of Ror2 with CKIepsilon is required for its serine/threonine phosphorylation by CKIepsilon. Site-directed mutagenesis of tyrosine residues in Ror2 reveals that the sites of phosphorylation are contained among the five tyrosine residues in the proline-rich domain but not among the four tyrosine residues in the tyrosine kinase domain. Moreover, we show that in mammalian cells, CKIepsilon-mediated phosphorylation of Ror2 on serine/threonine and tyrosine residues is followed by the tyrosine phosphorylation of G protein-coupled receptor kinase 2, a kinase with a developmental expression pattern that is remarkably similar to that of Ror2. Intriguingly, a mutant of Ror2 lacking five tyrosine residues, including the autophosphorylation sites, fails to tyrosine phosphorylate G protein-coupled receptor kinase 2. This indicates that autophosphorylation of Ror2 is required for full activation of its tyrosine kinase activity. These findings demonstrate a novel role for CKIepsilon in the regulation of Ror2 tyrosine kinase.