Yoshikazu Koyama

Macrophage migration inhibitory factor up-regulates expression of matrix metalloproteinases in synovial fibroblasts of rheumatoid arthritis.

Neutral matrix metalloproteinases (MMPs) are responsible for the pathological features of rheumatoid arthritis (RA) such as degradation of cartilage. We herein show the up-regulation of MMP-1 (interstitial collagenase) and MMP-3 (stromelysin) mRNAs of cultured synovial fibroblasts retrieved from rheumatoid arthritis (RA) patients in response to macrophage migration inhibitory factor (MIF). The elevation of MMP-1 and MMP-3 mRNA was dose-dependent and started at 6 h post-stimulation by MIF, reached the maximum level at 24 h, and was sustained at least up to 36 h. Interleukin (IL)-1β mRNA was also up-regulated by MIF. These events were preceded by up-regulation of c-jun and c-fos mRNA. Tissue inhibitor of metalloproteinase (TIMP)-1, a common inhibitor of these proteases, was slightly up-regulated by MIF. Similarly, mRNA up-regulation of MMP-1 and MMP-3 was observed in the synovial fibroblasts of patients with osteoarthritis. However, their expression levels were much lower than those of RA synovial fibroblasts. The mRNA up-regulation by MIF was inhibited by the tyrosine kinase inhibitors genestein and herbimycin A, as well as the protein kinase C inhibitors staurosporine and H-7. On the other hand, the inhibition was not seen after the addition of the cyclic AMP-dependent kinase inhibitor, H-8. The mRNA up-regulation of MMPs was also inhibited by curcumin, an inhibitor of transcription factor AP-1, whereas interleukin-1 receptor antagonist, an IL-1 receptor antagonist, failed to inhibit the mRNA up-regulation. Considering these results, it is suggested that 1) MIF plays an important role in the tissue destruction of rheumatoid joints via induction of the proteinases, and 2) MIF up-regulates MMP-1 and MMP-3 via tyrosine kinase-, protein kinase C-, and AP-1- dependent pathways, bypassing IL-1β signal transduction.

Effects of fucoxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo

The aim of the present study was to investigate the efficacy of fucoxanthin on endotoxin-induced uveitis (EIU) in rats. The effects of fucoxanthin on endotoxin-induced leucocyte and protein infiltration, nitric oxide (NO), prostaglandin (PG)-E2 and tumour necrosis factor (TNF)-alpha concentrations in rat aqueous humour, as well as on the cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) protein expression in a mouse macrophage cell line (RAW 264.7 cells) were studied. EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). Immediately after the LPS injection, either 0.1, 1 or 10mgkg(-1) of fucoxanthin was injected intravenously. The aqueous humour was collected 24hr later from both eyes, and both the number of cells infiltrating into the aqueous humour and the aqueous humour protein concentration were measured. The levels of PGE2, NO and TNF-alpha were determined by enzyme-linked immunosorbent assay. The RAW 264.7 cells were pretreated with various concentrations of fucoxanthin for 24hr and subsequently incubated with LPS for 24hr. COX-2 and iNOS protein expression was analysed by the Western blotting method. Levels of PGE2, NO and TNF-alpha production were determined. Fucoxanthin suppressed the development of EIU in a dose-dependent fashion. Treatment with fucoxanthin resulted in a reduction in PGE2, NO and TNF-alpha concentrations in the aqueous humour. The expression of COX and iNOS protein in the fucoxanthin treated RAW264.7 cells decreased significantly compared to that the LPS group. It also significantly reduced the concentration of PGE2, NO and TNF-alpha production in the medium of cells. The present result indicate fucoxanthin suppresses the inflammation of EIU by blocking the iNOS and COX-2 protein expression and its anti-inflammatory effect on eye is comparable with the effect of predinisolone used in similar doses.

Macrophage migration inhibitory factor‐deficient mice are resistant to ovariectomy‐induced bone loss

A link between macrophage migration inhibitory factor (MIF) and estrogen has recently emerged. We examined the involvement of MIF in osteoporotic changes in bone after ovariectomy (OVX), and revealed that MIF‐deficient mice (MIF‐KO) were completely protected from this phenomenon. The increase in osteoclast number per bone surface and serum IL‐1β levels, which were observed in wild‐type mice after OVX, did not occur in MIF KO. Our data suggest that MIF plays an important role in the pathogenesis of postmenopausal osteoporosis, and could be a novel target for the treatment of this disease.

Diphosphorylation of the myosin regulatory light chain enhances the tension acting on stress fibers in fibroblasts

Regulation of the contractile force is crucial for cell migration, cell proliferation, and maintenance of cell morphology. Phosphorylation of the myosin II regulatory light chain (MRLC) is involved in these processes. To show whether the diphosphorylation of MRLC increases the tension acting on stress fibers, changes in the stiffness of fibroblasts expressing wild‐type MRLC and a mutant type, which cannot be diphosphorylated, on treatment with lysophosphatidic acid (LPA) were examined by a mechanical‐scanning probe microscope (M‐SPM). The LPA treatment increased cellular stiffness in the wild‐type MRLC expressing cells, while it had no effect on the mutated cells. Immunostaining showed that LPA stimulation induced the diphosphorylation of MRLC. These results suggest that the diphosphorylation of MRLC enhances the tension acting on stress fibers. J. Cell. Physiol. 209: 726–731, 2006.

Transgenic Mice Overexpressing Macrophage Migration Inhibitory Factor (MIF) Exhibit High-Turnover Osteoporosis†

The bone phenotype of mice overexpressing MIF was studied. These mice showed decreased trabecular bone, increased bone formation rate, and increased MMP‐3, −9, and −13 mRNA expression in the femora and tibias. This model provides evidence of the role played by MIF in bone remodeling and balance in vivo.

Vav3 is regulated during the cell cycle and effects cell division

Vav3 is a member of the family of guanine nucleotide exchange factors implicated in the regulation of Rho GTPases. Although the exact in vivo function of Vav3 is unknown, evidence from several studies indicates a role distinct from Vav2 or Vav1. Here we report that the expression of Vav3 is regulated during the cell cycle. Strikingly, Vav3 was transiently up-regulated in HeLa cells during mitosis, whereas enforced expression of Vav3 perturbed cytokinesis and led to the appearance of multinucleated cells. These effects of Vav3 were RhoA-dependent, required phosphorylation of the regulatory tyrosine 173, but were not enhanced by N-terminal truncations. Thus, this report establishes that expression of Vav3 is strictly regulated in a cell cycle-dependent manner and implicates Vav3 in the control of cytokinesis.

Tissue Regeneration Using Macrophage Migration Inhibitory Factor-Impregnated Gelatin Microbeads in Cutaneous Wounds

Migration inhibitory factor (MIF) responds to tissue damage and regulates inflammatory and immunological processes. To elucidate the function of MIF in cutaneous wound healing, we analyzed MIF knockout (KO) mice. After the excision of wounds from the dorsal skin of MIF KO and wild-type (WT) mice, healing was significantly delayed in MIF KO mice compared to WT mice. Lipopolysaccharide treatment significantly increased [(3)H]thymidine uptake in WT mouse fibroblasts compared to MIF KO mouse fibroblasts. Furthermore, there was a significant reduction in fibroblast and keratinocyte migration observed in MIF KO mice after 1-oleoyl-2-lysophosphatidic acid treatment. We subsequently examined whether MIF-impregnated gelatin slow-release microbeads could accelerate skin wound healing. Injection of more than 1.5 microg/500 microl of MIF-impregnated gelatin microbeads around a wound edge accelerated wound healing compared to a single MIF injection without the use of microbeads. MIF-impregnated gelatin microbeads also accelerated skin wound healing in C57BL/6 mice and diabetic db/db mice. Furthermore, incorporating MIF-impregnated gelatin microbeads into an artificial dermis implanted into MIF KO mice accelerated procollagen production and capillary formation. These findings suggest that MIF is crucial in accelerating cutaneous wound healing and that MIF-impregnated gelatin microbeads represent a promising treatment to facilitate skin wound healing.

Thioredoxin suppresses airway inflammation independently of systemic Th1/Th2 immune modulation

Oxidative stress plays an important role in the pathogenesis of asthma via the upregulation of local inflammatory mediators and/or promoting Th2‐skewing during Ag sensitization. Thioredoxin (TRX), a 12 kDa redox‐active protein with antioxidative property, has been recently shown to play a protective role in various inflammatory diseases. Using a mouse model of asthma, we show here that IL‐13 and eotaxin production are decreased in TRX‐Tg mice leading to reduced eosinophils recruitment and mucus metaplasia. The reduction in airway inflammation occurs without the attenuation of systemic Th2 immunity in that comparable levels of Th2‐type cytokines and Ig were detected in LN and serum, respectively, from TRX‐Tg and WT mice. Likewise, CD4+ T cells from both strains of mice developed similar Th1 and Th2 responses in vitro. Asthmatic lungs of TRX‐Tg and WT mice contained similar amounts of GATA‐3+ and Foxp3+ T cells. Finally, production of MIF, an upstream modulator of airway inflammation, was significantly reduced in the lungs of TRX‐Tg mice. Our data suggest that TRX suppresses airway inflammation by inhibiting MIF production thereby limiting the downstream recruitment of eosinophils to the lung independently of modulating systemic Th1/Th2 immunity.

Regulation of cellular contractile force in response to mechanical stretch by diphosphorylation of myosin regulatory light chain via RhoA signaling cascade.

Fibroblasts regulate their contractile force in response to external stretch; however, the detailed mechanism by which the force is regulated is unclear. Here, we show that diphosphorylation and dephosphorylation of myosin regulatory light chain (MRLC) are involved in the stretch-induced force response. Cellular stiffness, which reflects the cellular contractile force, under external stretch was measured by mechanical-scanning probe microscopy. Fibroblasts (NIH-3T3) expressing green fluorescent protein (GFP)-tagged mutant-type MRLC (MRLC(T18A)-GFP), which cannot be diphosphorylated, did not show any stretch-induced stiffness response, whereas the stiffness in cells expressing GFP-tagged wild-type MRLC (MRLC(WT)-GFP) increased immediately after the stretch and subsequently decreased after 1-2 h. Urea-PAGE western blot analysis showed that the proportion of diphosphorylated MRLC (PP-MRLC) transiently increased after the stretch and decreased after 1-2 h. Dominant-negative RhoA (RhoA(N19))-expressing cells did not show the stiffness response to the stretch, whereas wild-type RhoA-expressing cells did. It was concluded that the cellular force response originates in the stretch-induced diphosphorylation and dephosphorylation of MRLC and is regulated via the RhoA signaling cascade.

Identification of macrophage migration inhibitory factor in human leukemia HL‐60 cells and its induction by lipopolysaccharide

Cytokines play an important role in inflammation and immunity. In this study, we examined the expression and presence of human macrophage migration inhibitory factor (MIF) in human myelomonocytic leukemia cell line, HL‐60 by reverse transcription‐polymerase chain reaction (RT‐PCR) and immunological methods (Western blot analysis and immunohistochemistry), respectively. The RT‐PCR showed that MIF mRNA was constitutively expressed, and the expression was further induced by lipopolysaccharide (LPS) stimulation. The expression was up‐regulated by LPS at the range of 10 pg/ml to 10 ng/ml; however, it decreased at doses higher than 100 ng/ml. The expression reached the maximum 12 hr after the stimulation, but substantially decreased by 24 hr. Western blot analysis and immunohistochemistry using an anti‐human MIF antibody revealed the presence of MIF protein in cytoplasm of the cells. The pathophysiological role of MIF in HL‐60 cells has not been fully understood; however, the regulation of MIF mRNA expression by LPS suggests the possibility that the cytokine plays an important role in inflammatory events of leukemia.