Yosuke Kanno

Pathogen recognition by Toll-like receptor 2 activates Weibel-Palade body exocytosis in human aortic endothelial cells

The endothelial cell-specific granule Weibel-Palade body releases vasoactive substances capable of modulating vascular inflammation. Although innate recognition of pathogens by Toll-like receptors (TLRs) is thought to play a crucial role in promotion of inflammatory responses, the molecular basis for early-phase responses of endothelial cells to bacterial pathogens has not fully been understood. We here report that human aortic endothelial cells respond to bacterial lipoteichoic acid (LTA) and synthetic bacterial lipopeptides, but not lipopolysaccharide or peptidoglycan, to induce Weibel-Palade body exocytosis, accompanied by release or externalization of the storage components von Willebrand factor and P-selectin. LTA could activate rapid Weibel-Palade body exocytosis through a TLR2- and MyD88-dependent mechanism without de novo protein synthesis. This process was at least mediated through MyD88-dependent phosphorylation and activation of phospholipase Cγ. Moreover, LTA activated interleukin-1 receptor-associated kinase-1-dependent delayed exocytosis with de novo protein synthesis and phospholipase Cγ-dependent activation of the NF-κB pathway. Increased TLR2 expression by transfection or interferon-γ treatment increased TLR2-mediated Weibel-Palade body exocytosis, whereas reduced TLR2 expression under laminar flow decreased the response. Thus, we propose a novel role for TLR2 in induction of a primary proinflammatory event in aortic endothelial cells through Weibel-Palade body exocytosis, which may be an important step for linking innate recognition of bacterial pathogens to vascular inflammation.

Arginine-specific gingipains from Porphyromonas gingivalis deprive protective functions of secretory leucocyte protease inhibitor in periodontal tissue

Chronic periodontitis is correlated with Porphyromonas gingivalis infection. In this study, we found that the expression of secretory leucocyte protease inhibitor (SLPI), an endogenous inhibitor for neutrophil‐derived proteases, was reduced in gingival tissues with chronic periodontitis associated with P. gingivalis infection. The addition of vesicles of P. gingivalis decreased the amount of SLPI in the media of primary human gingival keratinocytes compared to untreated cultures. We therefore investigated how arginine‐specific gingipains (Rgps) affect the functions of SLPI, because Rgps are the major virulence factors in the vesicles and cleave a wide range of in‐host proteins. We found that Rgps digest SLPI in vitro, suppressing the release of SLPI. Rgps proteolysis of SLPI disrupted SLPI functions, which normally suppresses neutrophil elastase and neutralizes pro‐inflammatory effects of bacterial cell wall compounds in cultured human gingival fibroblasts. The protease inhibitory action of SLPI was not exerted towards Rgps. These results suggest that Rgps reduce the protective effects of SLPI on neutrophil proteases and bacterial proinflammatory compounds, by which disease in gingival tissue may be accelerated at the sites with P. gingivalis infection.

Interleukin (IL)-17 enhances tumor necrosis factor-α-stimulated IL-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

Inflammatory cytokines are well known to play crucial roles in the pathogenesis of rheumatoid arthritis. Among them, interleukin (IL)‐17 is a cytokine that is mainly synthesized by activated T cells and its receptors are present in osteoblasts. The synthesis of IL‐6, known to stimulate osteoclastic bone resorption, is reportedly responded to bone resorptive agents such as tumor necrosis factor‐α (TNF‐α) in osteoblasts. It has been reported that IL‐17 enhances TNF‐α‐stimulated IL‐6 synthesis in osteoblast‐like MC3T3‐E1 cells. We previously showed that sphingosine 1‐phosphate (S1‐P) mediates TNF‐α‐stimulated IL‐6 synthesis in these cells. In the present study, we investigated the mechanism of IL‐17 underlying enhancement of IL‐6 synthesis in MC3T3‐E1 cells. IL‐17 induced phosphorylation of p38 mitogen‐activated protein (MAP) kinase. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, significantly reduced the enhancement by IL‐17 of TNF‐α‐stimulated IL‐6 synthesis. IL‐17 also amplified S1‐P‐stimulated IL‐6 synthesis, and the amplification by IL‐17 was suppressed by SB203580. Anisomycin, an activator of p38 MAP kinase, which alone had no effect on IL‐6 level, enhanced the IL‐6 synthesis stimulated by TNF‐α. SB203580 and PD169316 inhibited the amplification by anisomycin of the TNF‐α‐induced IL‐6 synthesis. Taken together, our results strongly suggest that IL‐17 enhances TNF‐α‐stimulated IL‐6 synthesis via p38 MAP kinase activation in osteoblasts.

The Absence of uPAR Is Associated with the Progression of Dermal Fibrosis

The fibrinolytic system is considered to play an important role in the degradation of extracellular matrices (ECM). However, the detailed mechanism regarding how this system affects fibrosis remains unclear. Urokinase-type plasminogen activator receptor (uPAR) not only functions as a proteinase receptor but also plays a role in cellular adhesion, differentiation, proliferation, and migration through intracellular signaling. To investigate the effect of uPAR on dermal fibrosis, the skin of wild-type mice was compared with uPAR-deficient (uPAR(-/-)) mice. The results showed that the absence of uPAR increases dermal thickness. In addition, collagen synthesis as well as the number of myofibroblasts was greater in the skin of uPAR(-/-) mice than in the skin of uPAR(+/+) mice. Moreover, we showed that the absence of uPAR attenuates the activity of matrix metalloproteinases (MMP)-2, 9 in the skin. In conclusion, this study suggests that the absence of uPAR not only regulates fibrosis-related gene expression and MMP activity but also results in ECM deposition. Therefore, the absence of uPAR induces dermal fibrosis. These findings provide new insights into the role of uPAR on dermal fibrosis.

Lack of alpha2-antiplasmin improves cutaneous wound healing via over-released vascular endothelial growth factor-induced angiogenesis in wound lesions.

Summary.  Background: The fibrinolytic system is supposed to play an important role in the degradation of extracellular matrices for physiological and pathological tissue remodeling; however, the detailed mechanism regarding how this system affects cutaneous wound healing remains to be clarified. Methods and results: We performed experimental cutaneous wounding in mice with a deficiency of α2‐antiplasmin (α2AP), which is a potent and specific plasmin inhibitor. We found that an accelerated wound closure was observed in α2AP‐deficient (α2AP−/−) mice in comparison with wild type (WT) mice. Moreover, we observed that a greater increase of angiogenesis occurred in the process of wound healing in α2AP−/− mice than in the WT mice. Intriguingly, mRNA expression of vascular endothelial growth factor (VEGF), which is the best characterized positive regulator of angiogenesis, in wound lesions was found to show a greater increase in the early phase of the healing process in α2AP−/− mice than in WT mice. In addition, the amount of released‐VEGF from the explanted fibroblasts of α2AP−/− mice increased dramatically more than in the WT mice. Finally, the intra‐jugular administration of anti‐VEGF antibody clearly suppressed the increased angiogenesis and accelerated wound closure in the wound lesion of α2AP−/− mice. Conclusion: The lack of α2AP markedly causes an over‐release of VEGF from the fibroblasts in cutaneous wound lesions, thereby inducing angiogenesis around the area, and thus resulting in an accelerated‐wound closure. Conclusions: This is the first report to describe the crucial role that α2AP plays following angiogenesis in the process of wound healing. Our results provide new insight into the role of α2AP on cutaneous wound healing.

α2-Antiplasmin is involved in the production of transforming growth factor β1 and fibrosis

Background: Fibrotic disease occurs in most tissues. Transforming growth factor (TGF)‐β is the major inducer of fibrosis. The fibrinolytic system is considered to play an important role in the degradation of extracellular matrices. However, the detailed mechanism of how this system affects fibrosis remains unclear. Methods and results: We examined experimental fibrosis in mice with a deficiency of α2‐antiplasmin (α2AP), which is a potent and specific plasmin inhibitor. We found that the lack of α2AP attenuated bleomycin‐induced TGF‐β1 synthesis and fibrosis. In addition, the production of TGF‐β1 from the explanted fibroblasts of α2AP−/− mice decreased dramatically as compared to that in wild‐type mice. Moreover, we found that α2AP specifically induces the production of TGF‐β1 in fibroblasts. Conclusion: The lack of α2AP attenuated TGF‐β1 synthesis, thereby resulting in attenuated fibrosis. This is the first report to describe the crucial role that α2AP plays in TGF‐β1 synthesis during the process of fibrosis. Our results provide new insights into the role of α2AP in fibrosis.

Plasminogen/Plasmin Modulates Bone Metabolism by Regulating the Osteoblast and Osteoclast Function

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg−/− mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg−/− OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg−/− mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg−/− OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg−/− mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.

Bovine milk lactoferrin induces synthesis of the angiogenic factors VEGF and FGF2 in osteoblasts via the p44/p42 MAP kinase pathway

Lactoferrin (LF) belongs to the transferrin family and is present in several physiological fluids, including milk and colostrum. LF has recently been identified as an anabolic factor for bone. Here we investigated whether bovine LF (bLF) induces synthesis of angiogenic factors by osteoblasts. If so, we examined the underlying mechanism. We found that bLF purified from milk increased the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) in murine osteoblast-like MC3T3-E1 cells and primary murine osteoblasts in a time- and dose-dependent manner. Furthermore, bLF increased VEGF and FGF2 protein levels in MC3T3-E1 cells. In addition, treatment of MC3T3-E1 cells with bLF rapidly induced phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase. The bLF-mediated increases in VEGF and FGF2 mRNA and protein were inhibited by U0126, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Taken together, our results strongly suggest that bLF induces VEGF and FGF2 synthesis in a p44/p42 MAP kinase-dependent manner in MC3T3-E1 cells.

Adenylyl cyclase-cAMP system inhibits thyroid hormone-stimulated osteocalcin synthesis in osteoblasts

It is generally recognized that thyroid hormone modulates osteoblast cell function. We have previously shown that triiodothyronine (T(3)) activates p38 mitogen-activated protein (MAP) kinase, resulting in the synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on thyroid hormone-stimulated osteocalcin synthesis in these cells. Dibutyryl-cAMP (DBcAMP) reduced the osteocalcin synthesis stimulated by T(3). Forskolin and cholera toxin suppressed the osteocalcin synthesis while dideoxyforskolin, a forskolin derivative that does not activate adenylyl cyclase, had little effect on the synthesis. KT5720, a selective inhibitor of protein kinase A, reversed the inhibitory effect of forskolin or DBcAMP. DBcAMP and forskolin markedly reduced the phosphorylation of p38 MAP stimulated by T(3). Pituitary adenylate cyclase-activating polypeptide (PACAP) significantly inhibited the T(3)-stimulated osteocalcin synthesis. These results strongly suggest that the adenylyl cyclase-cAMP system has an inhibitory role in thyroid hormone-stimulated osteocalcin synthesis via suppression of p38 MAP kinase activation in osteoblasts.

Alpha2-antiplasmin regulates the development of dermal fibrosis in mice by prostaglandin F2α synthesis through adipose triglyceride lipase/calcium-independent phospholipase A2

OBJECTIVE Systemic sclerosis (SSc) is characterized by fibrosis of the skin and visceral organs. Patients with SSc have enhanced plasma levels of the plasmin-α2-antiplasmin (α2AP) complex, and we recently implicated α2AP in the development of fibrosis through transforming growth factor β (TGFβ) production. This study was undertaken to clarify how α2AP induces TGFβ production and the development of fibrosis. METHODS To clarify the detailed mechanism by which α2AP induces TGFβ production, we focused on adipose triglyceride lipase (ATGL)/calcium-independent phospholipase A(2) (iPLA(2)) and examined whether ATGL/ iPLA(2) is associated with α2AP-induced TGFβ production. The mouse model of bleomycin-induced SSc was used to evaluate the role of α2AP in the development of fibrosis. Dermal thickness and collagen content were determined in mouse skin treated with phosphate buffered saline or bleomycin. Moreover, we cultured SSc-like fibroblasts from the bleomycin-treated mouse skin and examined the production of TGFβ and prostaglandin F(2α) (PGF(2α)). RESULTS We found that α2AP binding to ATGL promoted PGF(2α) synthesis through iPLA(2) in fibroblasts, and the PGF(2α) synthesis that was promoted by α2AP induced TGFβ production in fibroblasts. In addition, the neutralization of α2AP attenuated the production of TGFβ and PGF(2α) in SSc-like fibroblasts from mice. The α2AP deficiency attenuated bleomycin-induced fibrosis and PGF(2α) synthesis, while the administration of PGF(2α) to α2AP-deficient mice facilitated α2AP deficiency-attenuated fibrosis. CONCLUSION These findings suggest that α2AP regulates the development of fibrosis by PGF(2α) synthesis through ATGL/iPLA(2). The inhibition of α2AP-initiated pathways might provide a novel therapeutic approach to fibrotic diseases.