Akiko Kitamura

A mutation in the immunoproteasome subunit PSMB8 causes autoinflammation and lipodystrophy in humans

Proteasomes are multisubunit proteases that play a critical role in maintaining cellular function through the selective degradation of ubiquitinated proteins. When 3 additional β subunits, expression of which is induced by IFN-γ, are substituted for their constitutively expressed counterparts, the structure is converted to an immunoproteasome. However, the underlying roles of immunoproteasomes in human diseases are poorly understood. Using exome analysis, we found a homozygous missense mutation (G197V) in immunoproteasome subunit, β type 8 (PSMB8), which encodes one of the β subunits induced by IFN-γ in patients from 2 consanguineous families. Patients bearing this mutation suffered from autoinflammatory responses that included recurrent fever and nodular erythema together with lipodystrophy. This mutation increased assembly intermediates of immunoproteasomes, resulting in decreased proteasome function and ubiquitin-coupled protein accumulation in the patients tissues. In the patients skin and B cells, IL-6 was highly expressed, and there was reduced expression of PSMB8. Downregulation of PSMB8 inhibited the differentiation of murine and human adipocytes in vitro, and injection of siRNA against Psmb8 in mouse skin reduced adipocyte tissue volume. These findings identify PSMB8 as an essential component and regulator not only of inflammation, but also of adipocyte differentiation, and indicate that immunoproteasomes have pleiotropic functions in maintaining the homeostasis of a variety of cell types.

Notch2 integrates signaling by the transcription factors RBP-J and CREB1 to promote T cell cytotoxicity

The acquisition of cytotoxic effector function by CD8+ T cells is crucial for the control of intracellular infection and tumor invasion. However, it remains unclear which signaling pathways are required for the differentiation of CD8+ cytotoxic T lymphocytes. We show here that Notch2-deficient T cells had impaired differentiation into cytotoxic T lymphocytes. In addition, dendritic cells with lower expression of the Notch ligand Delta-like 1 induced the differentiation of cytotoxic T lymphocytes less efficiently. We found that the intracellular domain of Notch2 interacted with a phosphorylated form of the transcription factor CREB1, and together these proteins bound the transcriptional coactivator p300 to form a complex on the promoter of the gene encoding granzyme B. Our results suggest that the highly regulated, dynamic control of T cell cytotoxicity depends on the integration of Notch2 and CREB1 signals.

An inherited mutation in NLRC4 causes autoinflammation in human and mice

Kitamura et al. identify NLRC4 as causing familial cold autoinflammatory syndrome using whole exome sequencing on a family with multiple affected family members. They identify a mutation in the NOD domain and show that the mutant protein increases Nlrc4 oligomerization and is associated with increased IL-1β. Transgenic mice with the same NLRC4 mutation are shown to develop a similar FCAS-like syndrome.

Notch signaling drives IL-22 secretion in CD4+ T cells by stimulating the aryl hydrocarbon receptor

CD4+ helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4+ T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4+ T cells increased the production of IL-22 even in the absence of STAT3. CD4+ T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4+ T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch–AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses.

Gene expression of metalloproteinase and its inhibitor in mesangial cells exposed to high glucose

To clarify the roles of metalloproteinases and their inhibitor (TIMP) in diabetic glomerulopathy, we studied the effect of a high glucose concentration on the gene expression of metalloproteinase transin and TIMP as well as collagen type IV and laminin in cultured rat mesangial cells (MCs). In the high glucose group, collagen type IV, laminin, and TIMP mRNA levels were all elevated in a concentration-dependent manner, whereas transin expression was suppressed. Osmotic control of high glucose with mannitol selectively stimulated TIMP expression. We hypothesize that high glucose decreases matrix-degrading activity as well as increases matrix productivity in MCs.

Notch2 Signaling Is Required for Potent Antitumor Immunity In Vivo

CD8+ T cells play a central role in cancer immunosurveillance, and the efficient induction of CTLs against tumor Ags is required for successful immunotherapy for cancer patients. Notch signaling directly regulates the transcription of effector molecules in CTLs. However, it remains unclear whether Notch signaling in CD8+ T cells is required for antitumor CTL responses and whether modulation of Notch signaling can augment antitumor CTL responses. In this study, we demonstrate that signaling by Notch2 but not Notch1 in CD8+ T cells is required for antitumor CTL responses. Notch2flox/flox mice crossed with E8I-cre transgenic (N2F/F-E8I) mice, in which the Notch2 gene is absent only in CD8+ T cells, die earlier than control mice after inoculation with OVA-expressing EG7 thymoma cells. In contrast, Notch1flox/flox mice crossed with E8I-cre transgenic mice inoculated with EG7 cells die comparable to control mice, indicating that Notch2 is crucial for exerting antitumor CTL responses. Injection of anti-Notch2 agonistic Ab or delta-like 1-overexpressing dendritic cells augmented the antitumor response in C57BL/6 mice inoculated with EG7 cells. These findings indicate that Notch2 signaling in CD8+ T cells is required for generating potent antitumor CTLs, thus providing a crucial target for augmenting tumor immune responses.

ERK5 activation enhances mesangial cell viability and collagen matrix accumulation in rat progressive glomerulonephritis

The mitogen-activated protein kinase (MAPK) cascade plays an important role in the regulation of various cellular functions in glomerulonephritis (GN). Here, we investigated whether extracellular signal-regulated kinase 5 (ERK5), a member of the MAPK family, is involved in the pathogenesis of chronic mesangioproliferative GN, using a rat model induced by uninephrectomy and anti-Thy-1 antibody injection. Immunostaining of kidneys obtained at different time points revealed that phospho-ERK5 was weakly expressed in control glomeruli but dramatically increased in a typical mesangial pattern after 28 and 56 days of GN. A semiquantitative assessment indicated that glomerular phospho-ERK5 expression closely paralleled the accumulation of extracellular matrix (ECM), collagen type I, as well as glomerular expression of reactive oxygen species (ROS) and ANG II. On the other hand, phospho-ERK1/2 expression increased on day 7 during the phase of enhanced mesangial cell (MC) proliferation and decreased thereafter. H(2)O(2) and ANG II each induced ERK5 phosphorylation by cultured rat MCs. Costimulation with both H(2)O(2) and ANG II synergistically increased ERK5 phosphorylation in MCs. Cultured MCs transfected with ERK5-specific small interference RNA showed a significant decrease in H(2)O(2) or ANG II-induced cell viability and soluble collagen secretion compared with control cells. Treatment of GN rats with an ANG II type 1 receptor blocker resulted in significant decreases in phospho-ERK5 expression and collagen accumulation accompanied by remarkable histological improvement. Taken together, these results suggest that MC ERK5 phosphorylation by ANG II or H(2)O(2) enhances cell viability and ECM accumulation in an experimental model of chronic GN.

Jagged1 Suppresses Collagen-Induced Arthritis by Indirectly Providing a Negative Signal in CD8+ T Cells

Distinct Notch ligands possess a characteristic ability in terms of functional T cell differentiation. However, the precise role or the therapeutic potential of each Notch ligand in autoimmune diseases is largely unknown. In this study, we examined whether Jagged1 modulates a collagen-induced rheumatoid arthritis (CIA) model by altering T cell responses. The injection of a soluble Jagged1-encoding plasmid, sJag1-P, before or even after initial type II collagen (CII) immunization suppressed the disease severity of CIA. However, this treatment did not suppress CII-specific CD4+ T cell proliferation and CII-specific Ab production. Depletion of either CD4+ or CD8+ T cells ameliorated CIA severity and sJag1-P further improved CIA in CD4+ but not CD8+ T cell-depleted mice. Injection of OVA and Jagged1-encoding plasmids inhibited proliferation of OVA-specific granzyme B-producing CD8+ T cells, although Jagged1 could not directly inhibit CD8+ T cell proliferation in vitro. The blockade of Jagged1 by an anti-Jagged1 Ab exacerbated CIA, whereas this effect was not observed in the absence of CD8+ T cells. These data indicate that Jagged1 is able to deliver an indirect negative signal into CD8+ T cells in vivo, which suggests its therapeutic potential in the treatment of CD8+ T cell-mediated diseases, including rheumatoid arthritis.

Steroid-resistant nephrotic syndrome

Akiko Kitamura, Hiroyasu Tsukaguchi, Kenichi Maruyama, Akemi Shono, Kazumoto Iijima, Shoji Kagami and Toshio Doi Department of Pediatrics, University of Tokushima Graduate School, Tokushima, Japan; Department of Clinical Biology and Medicine, University of Tokushima Graduate School, Tokushima, Japan; Department of Pediatrics, Gunma Children’s Medical Center, Gunma, Japan and Department of Nephrology, National Center for Child Health and Development, Tokyo, Japan

PDGF‐BB enhances α1β1 integrin‐mediated activation of the ERK/AP‐1 pathway involved in collagen matrix remodeling by rat mesangial cells

Platelet‐derived growth factor‐BB (PDGF‐BB) has been implicated in the pathogenesis of progressive glomerulonephritis (GN). Previous studies have reported that PDGF‐BB stimulates mesangial cells (MCs)‐induced collagen matrix remodeling through enhancement of α1β1 integrin‐dependent migratory activity. To determine the cell signaling pathway responsible for abnormal MC‐related mesangial matrix remodeling in progressive GN, we studied the involvement of the extracellular signal‐regulated kinase (ERK)/activator protein‐1 (AP‐1) pathway in PDGF‐BB‐enhanced collagen gel contraction. Western blotting and gel shift assay revealed that MC‐induced gel contraction resulted in ERK activation in parallel with that of AP‐1 binding, peaking at 4 h and lasting at least for 24 h. Application of the MEK inhibitor, U0126, and the c‐jun/AP‐1 inhibitor, curcumin, inhibited gel contraction and AP‐1 activity, respectively, dose dependently. PDGF‐BB enhanced not only gel contraction but ERK phosphorylation and AP‐1 activity by MCs. Marked inhibitory effects on PDGF‐BB‐induced gel contraction and ERK/AP‐1 activity were observed in the presence of either function blocking anti‐α1‐ or anti‐β1‐integrin antibody or U0126. Consistently, AP‐1‐inactive MCs expressing a dominant‐negative mutant of c‐jun showed a significant decrease of PDGF‐BB‐induced gel contraction as compared with mock‐transfected MCs. Finally, migration assay showed that ERK/AP‐1 activity is required for PDGF‐BB‐stimulated α1β1 integrin‐dependent MC migration to collagen I. These results indicated that PDGF‐BB enhances α1β1 integrin‐mediated collagen matrix reorganization through the activation of the ERK/AP‐1 pathway that is crucial for MC migration. We conclude that the ERK/AP‐1 pathway plays an important role in PDGF‐BB‐induced α1β1 integrin‐dependent collagen matrix remodeling; therefore, the inhibition of its pathway may provide a novel approach to regulate abnormal collagen matrix remodeling in progressive GN. J. Cell. Physiol. 198: 470–478, 2004© 2003 Wiley‐Liss, Inc.