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Cell polarity is essential for various asymmetric cellular events, and the partitioning defective (PAR) protein PAR3 (encoded by PARD3 in mammals) plays a unique role as a cellular landmark to establish polarity. In epithelial cells, PAR3 localizes at the subapical border, such as the tight junction in vertebrates, and functions as an apical determinant. Although we know a great deal about the regulators of PAR3 localization, how PAR3 is concentrated and localized to a specific membrane domain remains an important question to be clarified. In this study, we demonstrate that ASPP2 (also known as TP53BP2), which controls PAR3 localization, links PAR3 and protein phosphatase 1 (PP1). The ASPP2-PP1 complex dephosphorylates a novel phosphorylation site, Ser852, of PAR3. Furthermore, Ser852- or Ser889-unphosphorylatable PAR3 mutants form protein clusters, and ectopically localize to the lateral membrane. Concomitance of clustering and ectopic localization suggests that PAR3 localization is a consequence of local clustering. We also demonstrate that unphosphorylatable forms of PAR3 exhibited a low molecular turnover and failed to coordinate rapid reconstruction of the tight junction, supporting that both the phosphorylated and dephosphorylated states are essential for the functional integrity of PAR3.

In patients with severe hemorrhage, complications such as shock or death may occur if the patient is not treated appropriately and expeditiously. To create a hemostat kit for severe hemorrhage, ultraviolet light irradiation was applied to photocrosslinkable chitosan hydrogel and calcium alginate. As a hemorrhage model, the femoral arteries and veins of anesthetized rats were cut. Hemodynamics and hematological parameters including red blood cell (RBC) count, hemoglobin concentration, hematocrit, white blood cell (WBC) count, and platelet count, and serum parameters including aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured as a marker of hemostasis. In rats for which no procedure was used, death occurred within 30 min. By using the hydrogel hemostat, the survival rate rose to 75% or more. RBC count, hemoglobin, hematocrit, and platelet levels were not significantly changed for 3 days. WBC count increased 1 day after hemostasis. AST and ALT increased 1 day after hemostasis, but it decreased 3 days later. The photocrosslinkable chitosan hydrogel and calcium alginate were biodegraded at 3 and 28 days, respectively, by neutrophils and keratinocyte chemoattractant.

Utrophin is a widely expressed paralogue of dystrophin, the protein responsible for Duchenne muscular dystrophy. Utrophin is a large spectrin-like protein whose C-terminal domain mediates anchorage to a laminin receptor, dystroglycan (DG). The rod domain, composed of 22 spectrin-like repeats, connects the N-terminal actin-binding domain and the C-terminal DG binding domain, and thus mediates molecular linkage between intracellular F-actin and extracellular basement membrane. Previously, we demonstrated that a cell polarity-regulating kinase, PAR-1b, interacts with the utrophin-DG complex, and positively regulates the interaction between utrophin and DG. In this study, we demonstrate that the 8th and 9th spectrin-like repeats (R8 and R9) of utrophin cooperatively form a PAR-1b-interacting domain, and that Ser1258 within R9 is specifically phosphorylated by PAR-1b. Substitution of Ser1258 to alanine reduces the interaction between utrophin and DG, suggesting that the Ser1258 phosphorylation contributes to the stabilization of the utrophin-DG complex. Interestingly, PAR-1b also binds and phosphorylates R8-9 of dystrophin, and colocalizes with dystrophin at the skeletal muscle membrane. These results reveal a novel function of the rod domain of utrophin beyond that of a passive structural linker connecting the N- and C-terminal domain.

Cell polarity depends on extrinsic spatial cues and intrinsic polarity proteins including PAR-aPKC proteins. In mammalian epithelial cells, cell-cell contacts provide spatial cues that activate the aPKC-PAR-3-PAR-6 complex to establish the landmark of the initial cellular asymmetry. PAR-1, a downstream target of the aPKC-PAR-3-PAR-6 complex, mediates further development of the apical and basolateral membrane domains. However, the relationships between the PAR-aPKC proteins and other extrinsic spatial cues provided by the extracellular matrix (ECM) remain unclear. Here, we show that PAR-1 colocalizes with laminin receptors and is required for the assembly of extracellular laminin on the basal surface of epithelial cells. Furthermore, PAR-1 regulates the basolateral localization of the dystroglycan (DG) complex, one of the laminin receptors essential for basement membrane formation. We also show that PAR-1 interacts with the DG complex and is required for the formation of a functional DG complex. These results reveal the presence of a novel inside-out pathway in which an intracellular polarity protein regulates the ECM organization required for epithelial cell polarity and tissue morphogenesis.

Adipose tissue contains a population of cells that have extensive self-renewal capacity and the ability to differentiate along multiple lineages. In addition, adipose tissue-derived stromal cells (ATSCs) are able to differentiate into various cell types that may be useful for autologous cell transplantation for defects of bone, cartilage, adipose, and tendon, etc. Most protocols for in vitro cultures of ATSCs include fetal bovine serum (FBS) as a nutritional supplement. However, in some cell cultures, it involves multiple doses of FBS, which raises a concern over possible infections as well as immunological reactions that are caused by medium-derived FBS proteins, sialic acid, etc. In this study, we were able to expand mouse ATSCs using low mouse serum media containing collagen type I, heparin-carrying polystyrene, and fibroblast growth factor (FGF)-2. These expanded mouse ATSCs maintained their multilineage potential for differentiation into adipocytes, osteoblasts, and chondrocytes. Therefore, this method, which uses autologous cells and low serum media, may be able to be utilized for clinical cell therapies.

The aim of this study was to identify novel angiogenic mechanisms underlying the regenerative process. To that end, interactions between adipose tissue-derived stromal cells (ASCs) and bone marrow cells (BMCs) were initially investigated using real-time fluorescence optical imaging. To monitor cell behavior in mice, we injected green fluorescent protein-positive (GFP+) BMCs into the tail vein and injected PKH26-labeled ASCs behind the ears. Angiogenesis and inflammation were observed at these sites via an optical imaging probe. Injected GFP+ BMCs migrated from the blood vessels into the tissues surrounding the ASC injection sites. Many of the migrating GFP+ BMCs discovered at the ASC injection sites were inflammatory cells, including Gr-1+, CD11b+, and F4/80+ cells. ASCs cocultured with inflammatory cells secreted increased levels of chemokines such as macrophage inflammatory protein (MIP)-1α, MIP-1β, keratinocyte-derived chemokines, and monocyte chemotactic protein 1. Similarly, these ASCs secreted increased levels of angiogenic growth factors such as hepatocyte growth factor and vascular endothelial growth factor. However, when anti-CXC chemokine receptor type 4 antibody was injected at regular intervals, the migration of GFP+ BMCs (especially Gr-1+ and CD11b+ cells) to ASC injection sites was inhibited, as was angiogenesis. The collective influence of the injected ASCs and BMC-derived inflammatory cells promoted acute inflammation and angiogenesis. Together, the results suggest that the outcome of cell-based angiogenic therapy is influenced not only by the injected cells but also by the effect of intrinsic inflammatory cells.

Photocrosslinkable chitosan (Az-CH-LA) contains lactose moieties and photoreactive azide groups, and its viscous solution forms an insoluble hydrogel on exposure to UV irradiation. We previously developed an emergency hemostatic kit using the Az-CH-LA solution, calcium alginate, and a UV irradiation apparatus. However, a suitable UV irradiation apparatus is required to effectively convert the Az-CH-LA solution into a hydrogel, and power supply to use the UV irradiation apparatus may not always be available in a disaster area or battlefield. To address this problem, we produced a portable, battery-powered UV irradiation apparatus constituting a novel hemostatic kit for severe hemorrhage. When the hemostatic kit using the UV irradiation apparatus was examined using a rat model of severe hemorrhage, the survival rate increased up to 73%. Hematological values as markers of hemorrhage did not change significantly over the first 3 days. In this study, we describe the characteristics of a portable UV irradiation apparatus and its use in an emergency hemostatic kit prepared using Az-CH-LA and calcium alginate for severe hemorrhage.

The establishment of epithelial polarity is tightly linked to the dramatic reorganization of microtubules (MTs) from a radial array to a vertical alignment of non-centrosomal MT bundles along the lateral membrane, and a meshwork under the apical and basal membranes. However, little is known about the underlying molecular mechanism of this polarity-dependent MT remodeling. The evolutionarily conserved cell polarity-regulating kinase PAR-1 (known as MARK in mammals), whose activity is essential for maintaining the dynamic state of MTs, has indispensable roles in promoting this process. Here, we identify a novel PAR-1-binding protein, which we call microtubule crosslinking factor 1 (MTCL1), that crosslinks MTs through its N-terminal MT-binding region and subsequent coiled-coil motifs. MTCL1 colocalized with the apicobasal MT bundles in epithelial cells, and its knockdown impaired the development of these MT bundles and the epithelial-cell-specific columnar shape. Rescue experiments revealed that the N-terminal MT-binding region was indispensable for restoring these defects of the knockdown cells. MT regrowth assays indicated that MTCL1 was not required for the initial radial growth of MTs from the apical centrosome but was essential for the accumulation of non-centrosomal MTs to the sublateral regions. Interestingly, MTCL1 recruited a subpopulation of PAR-1b (known as MARK2 in mammals) to the apicobasal MT bundles, and its interaction with PAR-1b was required for MTCL1-dependent development of the apicobasal MT bundles. These results suggest that MTCL1 mediates the epithelial-cell-specific reorganization of non-centrosomal MTs through its MT-crosslinking activity, and cooperates with PAR-1b to maintain the correct temporal balance between dynamic and stable MTs within the apicobasal MT bundles.

We produced a chitosan/fucoidan micro complex-hydrogel as a carrier for controlled release of heparin binding growth factors such as fibroblast growth factor (FGF)-2. Material consisting of a soluble chitosan (CH-LA) mixed with fucoidan yielded a water-insoluble and injectable hydrogel with filamentous particles. In this study, we examined the ability of the chitosan/fucoidan complex-hydrogel to immobilize FGF-2 and to protect its activity, as well as the controlled release of FGF-2 molecules. The chitosan/fucoidan complex-hydrogel has high affinity for FGF-2 (K(d) = 5.4 x 10(-) (9)M). The interaction of FGF-2 with chitosan/fucoidan complex-hydrogel substantially prolonged the biological half-life time of FGF-2. It also protected FGF-2 from inactivation, for example by heat and proteolysis, and enhance FGF-2 activity. When FGF-2-containing complex-hydrogel was subcutaneously injected into the back of mice, significant neovascularization and fibrous tissue formation were induced near the site of injection at 1 week, and the complex-hydrogel was biodegraded and disappeared by 4 weeks. These findings indicate that controlled release of biologically active FGF-2 molecules is caused by both slow diffusion and biodegradation of the complex-hydrogel, and that subsequent induction of vascularization occurs. FGF-2-containing chitosan/fucoidan micro complex-hydrogel is thus useful and convenient for treatment of ischemic disease.

The aim of this study was to evaluate the potential accelerating effects of an adipose tissue-derived stromal cells (ATSC)-containing atelocollagen matrix with silicone membrane (ACMS) for repairing mitomycin C-treated healing-impaired wounds. Mitomycin C was applied to full-thickness skin incisions in this study to create a healing-impaired wound model in rat. After thoroughly washing out the mitomycin C from the wound, ACMS alone or ATSC-containing ACMS was applied to the wounds. Histological sections of the wounds were then prepared at indicated time periods after the treatments. These results indicated significantly advanced granulation tissue and capillary formations in the healing-impaired wounds treated with ATSC-containing ACMS compared with those treated with ACMS alone. Thus, this study suggested that transplantation of inbred ATSC-containing ACMS is effective for repairing healing-impaired wounds.

PAR-3 is a scaffold-like PDZ-containing protein that forms a complex with PAR-6 and atypical protein kinase C (PAR-3-atypical protein kinase C-PAR-6 complex) and contributes to the establishment of cell polarity in a wide variety of biological contexts. In mammalian epithelial cells, it localizes to tight junctions, the most apical end of epithelial cell-cell junctions, and contributes to the formation of functional tight junctions. However, the mechanism by which PAR-3 localizes to tight junctions and contributes to their formation remains to be clarified. Here we show that the N-terminal conserved region, CR1-(1–86), and the sequence 937–1,024 are required for its recruitment to the most apical side of the cell-cell contact region in epithelial Madin-Darby canine kidney cells. We also show that CR1 self-associates to form an oligomeric complex in vivo and in vitro. Further, overexpression of CR1 in Madin-Darby canine kidney cells disturbs the distribution of atypical protein kinase C and PAR-6 as well as PAR-3 and delays the formation of functional tight junctions. These results support the notion that the CR1-mediated self-association of the PAR-3-containing protein complex plays a role during the formation of functional tight junctions.