Yoshiharu Takayama

Transmembrane Phosphoprotein Cbp Positively Regulates the Activity of the Carboxyl-terminal Src Kinase, Csk

Csk (carboxyl-terminalSrc kinase) is a cytoplasmic tyrosine kinase that phosphorylates a critical tyrosine residue in each of the Src family kinases (SFKs) to inhibit their activities. Recently, we identified a transmembrane protein, Cbp (Csk-binding protein), that, when phosphorylated, can recruit Csk to the membrane where the SFKs are located. The Cbp-mediated relocation of Csk to the membrane may play a role in turning off the signaling events initiated by SFKs. To further characterize the Csk-Cbp interaction, we have generated a reconstituted system using soluble, highly purified proteins. Csk and phosphorylated Cbp were co-purified as a large protein complex consisting of at least four Csk·Cbp units. The addition of the phosphorylated, but not nonphosphorylated, Cbp to an in vitro assay stimulated Csk activity toward Src. Csk was also activated by a phosphopeptide containing the tyrosine in Cbp that binds to Csk (Tyr-314). Kinetic analysis revealed that Cbp or the phosphopeptide induced up to a 6-fold reduction in the K m for Src, indicating that the Csk·Cbp complex has a greater affinity for Src than free Csk. These findings suggest that Cbp is involved in the regulation of SFKs not only by relocating Csk to the membrane but also by directly activating Csk.

Structure of the carboxyl-terminal Src kinase, Csk.

The carboxyl-terminal Src kinase (Csk) is an indispensable negative regulator for the Src family tyrosine kinases (SFKs) that play pivotal roles in various cell signalings. To understand the molecular basis of the Csk-mediated regulation of SFKs, we elucidated the crystal structure of full-length Csk. The Csk crystal consists of six molecules classified as active or inactive states according to the coordinations of catalytic residues. Csk assembles the SH2 and SH3 domains differently from inactive SFKs, and their binding pockets are oriented outward enabling the intermolecular interaction. In active molecules, the SH2-kinase and SH2-SH3 linkers are tightly stuck to the N-lobe of the kinase domain to stabilize the active conformation, and there is a direct linkage between the SH2 and the kinase domains. In inactive molecules, the SH2 domains are rotated destroying the linkage to the kinase domain. Cross-correlation matrices for the active molecules reveal that the SH2 domain and the N-lobe of the kinase domain move as a unit. These observations suggest that Csk can be regulated through coupling of the SH2 and kinase domains and that Csk provides a novel built-in activation mechanism for cytoplasmic tyrosine kinases.

Effect of lactoferrin-embedded collagen membrane on osteogenic differentiation of human osteoblast-like cells

Lactoferrin (LF) has the ability to promote the proliferation and differentiation of osteoblasts, suggesting its potential utility as an osteogenic growth factor in bone tissue engineering. However, this type of application requires improved drug delivery system (DDS) technology at the target site. In this study, we report enhanced calcium deposition and alkaline phosphatase (ALP) activity using the type I collagen membrane during osteogenic differentiation of MG63 human osteoblast-like cells, indicating that type I collagen not only acts as a site for calcification but also promotes the expression of differentiated phenotypes. We also used this membrane as a drug delivery carrier for bovine LF. Approximately 27% of LF embedded on the type I collagen membrane was released within the first hour in cell-free condition. This initial burst release of LF was followed by a slower release from the collagen membrane. Bovine LF embedded in the type I collagen membrane promoted its calcification during osteogenic differentiation of MG63 cells without the loss of LF bioactivity. Taken together, ALP activity and osteocalcin production were enhanced in the MG63 cells plated on the LF-embedded collagen membrane, suggesting that LF incorporated in the collagen membrane promoted bone-like tissue formation by MG63 cells. These observations suggest that the type I collagen membrane is useful as a drug delivery carrier for LF in bone tissue engineering.

Effects of lactoferrin on collagen gel contractile activity and myosin light chain phosphorylation in human fibroblasts

When fibroblasts are plated on a type I collagen gel they reduce the size of the gel and the extent of collagen gel contraction reflects the motile activity of the fibroblasts. We found that both bovine and human lactoferrin (Lf) enhanced the collagen gel contractile activity of WI‐38 human fibroblasts. Rho inhibitor (exoenzyme C3), Rho kinase inhibitor (Y‐27632), myosin light chain kinase inhibitor (ML‐7), MEK inhibitor (PD98059) and Src family tyrosine kinase inhibitor inhibited the Lf‐enhanced collagen gel contraction. Treatment of fibroblasts with Lf induced the phosphorylation of myosin light chain (MLC) within 30 min. Lf‐enhanced MLC phosphorylation was inhibited by Y‐27632 and ML‐7. These results suggest that Lf promotes the motility of fibroblasts by regulating MLC phosphorylation.

Effect of Bovine Lactoferrin on Extracellular Matrix Calcification by Human Osteoblast-Like Cells

Osteoblast-mediated calcium deposition to the extracellular matrix (ECM) is a critical step in bone tissue generation. Bovine lactoferrin enhanced the calcium deposition by MG63 human osteoblast-like cells cultured on collagen-coated plates. Lactoferrin also promoted the alkaline phosphatase activity and osteocalcin production during the calcification process, whereas it had little effect on the growth of the cells on the collagen-coated plates.

A Protein-Permeable Scaffold of a Collagen Vitrigel Membrane Useful for Reconstructing Crosstalk Models between Two Different Cell Types

Soft and turbid collagen gel disks were previously converted into strong and transparent gel membranes utilizing a concept for the vitrification of heat-denatured of proteins. This novel stable and transparent gel has been termed ‘vitrigel’. By encompassing the collagen vitrigel membrane in a nylon frame, it can be easily handled with tweezers, and functions as an excellent scaffold for three-dimensional cell culture models, as cells can be cultured on both sides. Here, we investigated the molecular permeability of the collagen vitrigel membrane in a time course-dependent manner using glucose and serum proteins. Glucose penetrated through the collagen vitrigel membrane to the opposite side, and concentrations on each side were found to be equilibrated within 24 h. Serum proteins up to a molecular weight >100 kDa also gradually passed through the collagen vitrigel membrane. In addition, human microvascular endothelial cells (HMVECs) were cultured on one surface of the collagen vitrigel membrane with a nylon frame, and human dermal fibroblasts (HDFs) or HT-29 (a human colon carcinoma cell line) cells were cocultured on the opposite surface. Histomorphological observations revealed the formation of three-dimensional crosstalk models composed of HMVECs and HDFs or HMVECs and HT-29 cells. Resulting data suggest that the protein-permeable scaffold composed of the collagen vitrigel membrane is useful for the reconstruction and/or modeling of ‘crosstalk’ between two different cells types. Hereafter, such crosstalk models in vitro could be applied to research not only of paracrine factors, but also to epithelial- or endothelial-mesenchymal transitions.

The bovine lactoferrin region responsible for promoting the collagen gel contractile activity of human fibroblasts

We have reported that bovine lactoferrin (bLf) promotes the contractile activity of collagen gels by WI-38 human fibroblasts via the phosphorylation of myosin light chain (MLC). To identify the region of bLf that is responsible for this activity, we prepared bLf fragments by limited proteolysis using trypsin and investigated the effects of each fragment on gel contractile activity. Lf consists of a single polypeptide chain containing two lobes that are independent globular structures termed the N- and C-lobes. The fragment corresponding to the C-lobe of bLf (amino acids 341-689) had a more prominent effect on collagen gel contractile activity than did that of either native bLf or its N-lobe (1-284). Further hydrolysis of the C-lobe with either pepsin or trypsin resulted in a loss of this activity. The effect of the C-lobe on collagen gel contraction by fibroblasts was dose-dependent and was associated with the elevation of MLC phosphorylation.

Co-treatment with dexamethasone and octanoate induces adipogenesis in 3T3-L1 cells

We report here that octanoate, a medium chain fatty acid, induces adipocyte differentiation in 3T3‐L1 cells by co‐treatment with dexamethasone, although octanoate has been known not to stimulate 3T3‐L1 adipogenesis. A low concentration of exogenous glucose prevented 3T3‐L1 adipogenesis induced by 1‐methyl 3‐isobutylxanthine, dexamethasone, and insulin (MDI) treatment (a common protocol for adipocyte differentiation). In contrast, co‐treatment with dexamethasone and octanoate (D‐OCT) induced adipogenesis under the same conditions. These findings imply that octanoate, rather than glucose, is the source of accumulated lipids in D‐OCT‐induced adipogenesis. D‐OCT increased expression of the differentiation markers peroxisome proliferator‐activated receptor (PPAR)γ2 and caveolin‐1. A specific inhibitor of p38 mitogen‐activated protein (MAP) kinase inhibited D‐OCT‐induced adipogenesis. These results suggest that the p38 MAP kinase pathway followed by up‐regulation of PPARγ2 may be involved in 3T3‐L1 adipocyte differentiation induced by D‐OCT, as well as by MDI.

Omics Studies of the Murine Intestinal Ecosystem Exposed to Subchronic and Mild Social Defeat Stress

The microbiota-gut-brain axis plays an important role in the development of stress-induced mental disorders. We previously established the subchronic and mild social defeat stress (sCSDS) model, a murine experimental model of depression, and investigated the metabolomic profiles of plasma and liver. Here we used omics approaches to identify stress-induced changes in the gastrointestinal tract. Mice exposed to sCSDS for 10 days showed the following changes: (1) elevation of cholic acid and reduction of 5-aminovaleric acid among cecal metabolites; (2) downregulation of genes involved in the immune response in the terminal ileum; (3) a shift in the diversity of the microbiota in cecal contents and feces; and (4) fluctuations in the concentrations of cecal metabolites produced by gut microbiota reflected in plasma and hepatic metabolites. Operational taxonomic units within the family Lachnospiraceae showed an inverse correlation with certain metabolites. The social interaction score correlated with cecal metabolites, IgA, and cecal and fecal microbiota, suggesting that sCSDS suppressed the ileal immune response, altering the balance of microbiota, which together with host cells and host enzymes resulted in a pattern of accumulated metabolites in the intestinal ecosystem distinct from that of control mice.

Lactoferrin promotes hyaluronan synthesis in human dermal fibroblasts

Bovine lactoferrin (BLF) enhanced production of hyaluronan in normal human dermal fibroblasts. The elevation of hyaluronan was accompanied by elevation of HAS2 (hyaluronan synthase 2) mRNA transcription and HAS2 protein expression. The promoting effect of BLF was not observed for HAS1. In addition, COL1A1 transcription and collagen synthesis were enhanced by BLF. These observations suggest that BLF promotes wound healing by increasing hyaluronan and type-I collagen synthesis.