Yutaka Kanamaru

Constitutive phosphorylation and nuclear localization of Smad3 are correlated with increased collagen gene transcription in activated hepatic stellate cells

Hepatic stellate cells (HSC) are the main producers of type I collagen in fibrotic liver, and transforming growth factor‐β (TGF‐β) plays critical roles in stimulating collagen gene expression in the cells mainly at the level of transcription. We have previously identified an upstream sequence of α2(I) collagen gene (COL1A2) that is essential for its basal and TGF‐β‐stimulated transcription in skin fibroblasts and HSC. We designated this region the TGF‐β‐responsive element (TbRE). Recently Smad3, an intracellular mediator of TGF‐β signal transduction, has been shown to bind to the TbRE and stimulate COL1A2 transcription when overexpressed in skin fibroblasts. In the present study, we demonstrate increased transcription of COL1A2 and plasminogen activator inhibitor‐1 (PAI‐1) genes and low response to TGF‐β in an activated HSC clone derived from a cirrhotic liver. Western blot analyses indicated constitutive phosphorylation of Smad3 in the cells. Immunofluorescence studies revealed that, in contrast to Smad2 that translocated from the cytoplasm to the nucleus upon TGF‐β treatment, Smad3 and Smad4 were present in the nucleus irrespective of ligand stimulation. Increased COL1A2 and PAI‐1 gene transcription in the cells was not affected by overexpression of inhibitory Smad7. Altogether, the results correlate abnormality in TGF‐β/Smad signaling with pathologically accelerated collagen gene transcription in activated HSC.

Blockade of TGF-β Signaling in T Cells Prevents the Development of Experimental Glomerulonephritis

Anti-glomerular basement membrane (GBM) Ab-induced glomerulonephritis (GN) at late stage is thought to be mediated by T cells. However, signaling pathways of T cells that are involved in the development of anti-GBM Ab-induced GN are unclear. We have recently established transgenic mice expressing Smad7, an inhibitor of TGF-β signaling, in mature T cells, where signaling by TGF-β was blocked specifically in T cells. In this study, we showed that anti-GBM Ab-induced GN was suppressed in several measures in the transgenic mice including the severity of glomerular changes, proteinuria, renal function, and CD4 T cell infiltration into the glomeruli without down-regulation of CD62 ligand (CD62L) (L-selectin) expression on CD4 T cells. Furthermore, treatment with the soluble fusion protein of CD62L and IgG enhanced anti-GBM Ab-induced GN. These findings indicated that blockade of TGF-β signaling in T cells prevented the development of anti-GBM Ab-induced GN. Because CD62L on T cells appears to be inhibitory for the development of anti-GBM Ab-induced GN, persistent expression of CD62L on CD4 T cells may explain, at least in part, the suppression of anti-GBM Ab-induced GN in the transgenic mice. Our findings suggest that the development of anti-GBM Ab-induced GN requires TGF-β/Smad signaling in T cells.

Smad3 Deficiency in Mast Cells Provides Efficient Host Protection against Acute Septic Peritonitis

Mast cells play an important role in innate immunity as well as in allergic reaction. However, regulatory mechanisms underlying mast cell-mediated innate immune responses remain largely unknown. Here we determined whether Smad3, a major signal transducer of TGF-β, regulates innate immune response by mast cells against Gram-negative bacteria. Bone marrow-derived mast cells (BMMC) obtained from Smad3 null mutant mice showed augmented capacity to produce proinflammatory cytokines upon stimulation with a Gram-negative bacteria-associated product, LPS. In acute septic peritonitis model induced by cecal ligation and puncture, mast cell-deficient W/Wv mice reconstituted with Smad3 null BMMC had significantly higher survival rate than W/Wv mice reconstituted with wild-type BMMC, which was associated with higher production of proinflammatory cytokines in the peritoneal cavity. These in vitro and in vivo results suggest that Smad3 in mast cells functions as inhibitory for mast cell-mediated innate immune response against Gram-negative bacteria. Suppression of Smad3 expression in mast cells may thus have therapeutic potential for Gram-negative bacterial infection such as acute septic peritonitis by augmenting innate immune responses of mast cells.

Involvement of p300 in TGF-β/Smad-Pathway-Mediated α2(I) Collagen Expression in Mouse Mesangial Cells

Background: Transforming growth factor beta 1 (TGF-β1) induces α2(I) collagen gene (COL1A2) expression in mesangial cells through physical and functional cooperation of Smad proteins and Sp1. A transcriptional coactivator, p300, is also suggested to play an important role in TGF-β1/Smad signal transduction. However, the role of p300 in TGF-β1/Smad-pathway-mediated transcriptional activation of the COL1A2 gene in mesangial cells is still obscure. Methods: Endogenous p300 expression and its modulation by TGF-β1 were evaluated by Western blotting and immunofluorescence. The physical interaction of p300 with Smad2/3 was examined by immunoprecipitation followed by Western blotting. The functional role of p300 in TGF-β1/Smad-pathway-mediated COL1A2 transcription was investigated in cotransfection experiments using a COL1A2 promoter-luciferase reporter gene construct and p300 expression plasmids. Results: TGF-β1 induced COL1A2 gene expression in cultured mouse mesangial cells which was blocked by overexpression of inhibitory Smad7. In addition, TGF-β1-induced nuclear export of endogenous Smad7 was observed in mouse mesangial cells. Endogenous p300 was expressed in the nucleus of the cells. TGF-β1 induced interaction of endogenous p300 with Smad2/3, and a dominant negative construct of p300 inhibited the TGF-β1-induced COL1A2 expression in cultured mouse mesangial cells. Conclusions: p300 may be involved in TGF-β1/Smad-pathway-mediated type I collagen gene transcription in mouse mesangial cells. Our findings would reveal a molecular basis of TGF-β1-induced type I collagen gene transcription in mouse mesangial cells.

Chronic Graft-versus-Host Autoimmune Disease in Fc Receptor γ chain-deficient Mice Results in Lipoprotein Glomerulopathy

Lipoprotein glomerulopathy (LPG) is a unique renal disease characterized by intraglomerular lipoprotein thrombi associated with severe proteinuria and frequent progression to renal failure. The histologic hallmark of LPG is the presence of laminated thrombi, consisting of lipid droplet, within the lumina of dilated glomerular capillaries. The findings of thrombi consisting of lipoproteins raised the possibilities that LPG might be related to a primary abnormality in lipid metabolism. However, the precise pathogenic basis of LPG remains unresolved. It was herein found that chronic graft-versus-host disease (GVHD) induced by the transfer of Ia-incompatible spleen cells from B6.C-H2(bm12) into coisogenic C57BL/6 mice with deficiency of Fc receptor gamma chain (FcRgamma) resulted in glomerulopathy that resembled LPG. The uptake of acetylated LDL was partially decreased in peritoneal macrophages isolated from FcRgamma-deficient mice compared with wild-type mice, suggesting that partial impairment of modified LDL uptake might contribute to the development of LPG associated with chronic GVHD in FcRgamma-deficient mice. LPG has been suggested to be a disorder of primary abnormality in lipid metabolism; these findings would therefore provide novel insight into the disease process.

IgA Fc receptor I is a molecular switch that determines IgA activating or inhibitory functions.

IgA Fc receptor I, FcalphaRI or CD89, an ITAM-bearing receptor, has revealed unique genetic, structure and function features among immunoreceptors. While all genes encoding for the human Fc receptors are located in chromosome 1, the FcalphaRI gene has been found in a cluster in 19q13 that includes the killer inhibitory receptors (KIRs) and the leukocyteimmunoglobulin- like receptors (LIRs). FcalphaRI-IgA complexes display an original crystal structure and a 2:1 stoichiometry. Our results show that intrinsic signals elicited by FcalphaRI have outlined the particularity of this receptor, since it could function in two opposite ways: cell activation and inhibition of a heterologous FcR. Contrary to the dogma that receptor multimerization is necessary to ensure signaling, we demonstrated that monomeric targeting of FcalphaRI is sufficient to trigger a low-intensity signaling cascade that leads to cell desensitization by recruiting the tyrosine phosphatase SHP-1. By contrast, multimerization of FcalphaRI allows a high-intensity signaling pathway that leads to the recruitment of tyrosine kinase Syk and cell activation. Both types of signals require the FcRgamma-ITAM motif. This dual function is unique among ITAM-bearing FcR and led us to postulate that ITAM motifs could deliver an unexpected array of signaling intensity. These findings redefine FcalphaRI as a molecular switch of the immune system that mediates both pro- and anti-inflammatory functions of IgA.

B lymphocytes undergo apoptosis because of FcγRIIB stress response to infection : A novel mechanism of cell death in sepsis

Sepsis is predominantly characterized by proinflammatory signs in its initial phase, but can be also associated with immune suppression that can be a consequence of apoptotic cell death. The role of Fc receptors (FcRs) is poorly understood in this disease, and it was recently shown that, in addition to the promotion of opposite inflammatory responses, they are implicated in apoptosis. Using a model of peritonitis in mice that do not express activating FcRs, we tested the hypothesis that FcγRIIb, the only known immunoglobulin G receptor capable of inducing apoptosis, would participate in the induction of this kind of cell death during serious infection. The blocking of this receptor by a monoclonal antibody significantly decreased the number of apoptotic splenic B cells, demonstrating its involvement in apoptosis. FcγRIIb-mediated apoptosis was neither the result of increased TNFα levels nor was it associated with IL-10 production. Finally, the decreased apoptosis after mice treatment with FcγRIIb-blocking antibody was not sufficient to increase its survival. Thus, we conclude that although apoptosis is a multifactorial phenomenon in sepsis, one of these factors is the inhibitory immunoglobulin G receptor FcγRIIb. FcγRIIb stress response to infection is a novel mechanism that contributes to the comprehension of apoptosis in sepsis.

Targeted IgA Fc receptor I (FcαRI) therapy in the early intervention and treatment of pristane-induced lupus nephritis in mice

The Fc receptor I for IgA (FcαRI) down‐regulates humoral immune responses and modulates the risk of autoimmunity. This study aimed to investigate whether FcαRI targeting can affect progression of pristine‐induced lupus nephritis. In the first experiment (early intervention), four groups of animals were evaluated: untreated FcαRI/FcRγ transgenic (Tg) mice and Tg mice administered control antibody (Ctr Fab), saline and anti‐FcαRI Fab [macrophage inflammatory protein (MIP)‐8a], respectively, three times a week for 29 weeks, after being injected once intraperitoneally with 0·5 ml pristane. In the second experiment, antibody injection started after the onset of nephritis and was carried out for 2 months, with similar groups as described above. MIP‐8a improved proteinuria, decreased the amounts of glomerular injury markers, serum interleukin (IL)‐6, IL‐1 and monocyte chemoattractant protein (MCP)‐1, and F4/80 macrophages in the interstitium and glomeruli, in both experiments. When MIP‐8a was used as early intervention, a decrease in mouse serum anti‐nuclear antibody (ANA) titres and reduced deposition of immunoglobulins in glomeruli were observed. This effect was associated with reduced serum titres of immunoglobulin (Ig)G2a but not IgG1, IgG2b and IgG3. Furthermore, pathological analysis showed lower glomerular activity index and less fibronectin in MIP‐8a treated mice. This study suggests that FcαRI targeting could halt disease progression and lupus activation by selective inhibition of cytokine production, leucocyte recruitment and renal inflammation. Our findings provide a basis for the use of FcαRI as a molecular target for the treatment of lupus.

Role of transforming growth factor-β/Smad signalling pathway in enhanced production of glomerular extracellular matrix in IgA nephropathy of high-serum-IgA ddY mice

SUMMARY: The high‐IgA inbred strain (HIGA) of ddY mice, an animal model of human IgA nephropathy, shows consistently high serum IgA levels, progressive mesangial sclerosis and IgA deposition, and elevated renal expression of transforming growth factor (TGF)‐β. In the present study the role of the TGF‐β/Smad signalling pathway in extracellular matrix (ECM) production was assessed in cultured mesangial cells derived from HIGA mice. The production of type I and type IV collagens in response to TGF‐β1, expression of Smad2, Smad4, Smad7, and Sp1 and p300, and phosphorylation of Smad2 by TGF‐β1 were assessed in cultured mesangial cells derived from HIGA mice at the age of 10 weeks by comparison with age‐matched C57BL/6 mice as controls. In addtion, the expression of p300 and type I and type IV collagens in renal tissues of HIGA mice at the age of 60 weeks was determined. The production of type I and type IV collagens by cultured mesangial cells in HIGA mice was markedly upregulated compared with that in C57BL/6 mice. Although protein expression levels of Smad2, Smad4, Smad7, and Sp1 in the mesangial cells were similar in the two mouse strains, upregulation of p300 was marked in HIGA mice. Expression of p300 in renal tissues of HIGA mice was also enhanced in HIGA mice when compared with C57BL/6 mice. In HIGA mice, p300 expression was upregulated in the mesangial cells both in vitro and in vivo. It appears that the upregulation of p300 may be related to glomerular sclerosis associated with IgA nephropathy in HIGA mice.

Negative regulation of inflammatory responses by immunoglobulin A receptor (FcαRI) inhibits the development of Toll-like receptor-9 signalling-accelerated glomerulonephritis.

Myeloid FcαRI, a receptor for immunoglobulin (Ig)A, mediates cell activation or inhibition depending on the type of ligand interaction, which can be either multivalent or monovalent. Anti‐inflammatory signalling is triggered by monomeric targeting using anti‐FcαRI Fab or IgA ligand binding, which inhibits immune and non‐immune‐mediated renal inflammation. The participation of Toll‐like receptors (TLRs) in kidney pathology in experimental models and various forms of human glomerular nephritis has been discussed. However, little is known about negative regulation of innate‐immune activation. In the present study, we generated new transgenic mice that express FcαRIR209L/FcRγ chimeric protein and showed that the monovalent targeting of FcαRI exhibited inhibitory effects in an in vivo model of TLR‐9 signalling‐accelerated nephritis. Mouse monoclonal anti‐FcαRI MIP8a Fab improved urinary protein levels and reduced the number of macrophages and immunoglobulin deposition in the glomeruli. Monovalent targeting using MIP8a Fab attenuates the TLR‐9 signalling pathway and is associated with phosphorylation of extracellular signal‐related protein kinases [extracellular signal‐regulated kinase (ERK), P38, c‐Jun N‐terminal kinase (JNK)] and the activation of nuclear factor (NF)‐κB. The inhibitory mechanism involves recruitment of tyrosine phosphatase Src homology 2 domain‐containing phosphatase‐1 (SHP‐1) to FcαRI. Furthermore, cell transfer studies with macrophages pretreated with MIP8a Fab showed that blockade of FcαRI signalling in macrophages prevents the development of TLR‐9 signalling‐accelerated nephritis. These results suggest a role of anti‐FcαRI Fab as a negative regulator in controlling the magnitude of the innate immune response and a new type of anti‐inflammatory drug for treatment of kidney disease.