Junko Inagaki

Increased activity and expression of histone deacetylase 1 in relation to tumor necrosis factor-alpha in synovial tissue of rheumatoid arthritis

IntroductionThe purpose of this study was to investigate the profile of histone deacetylase (HDAC) expression in the synovial tissue of rheumatoid arthritis (RA) compared with that of normal control and osteoarthritis (OA), and to examine whether there is a link between HDAC activity and synovial inflammation.MethodsHDAC activity and histone acetyltransferase (HAT) activity were determined in nuclear extracts of total synovial tissue surgically obtained from normal, OA and RA joints. The level of cytoplasmic tumor necrosis factor a (TNFα) fraction was measured by ELISA. Total RNA of synovial tissue was used for RT-PCR of HDAC1-8. In synovial fibroblasts from RA (RASFs), the effects of TNFα on nuclear HDAC activity and class I HDACs (1, 2, 3, 8) mRNA expressions were examined by quantitative real-time PCR. The protein expression and distribution of class I HDACs were examined by Western blotting.ResultsNuclear HDAC activity was significantly higher in RA than in OA and normal controls and correlated with the amount of cytoplasmic TNFα. The mRNA expression of HDAC1 in RA synovial tissue was higher than in OA and normal controls, and showed positive correlation with TNFα mRNA expression. The protein level of nuclear HDAC1 was higher in RA synovial tissue compared with OA synovial tissue. Stimulation with TNFα significantly increased the nuclear HDAC activity and HDAC1 mRNA expression at 24 hours and HDAC1 protein expression at 48 hours in RASFs.ConclusionsOur results showed nuclear HDAC activity and expression of HDAC1 were significantly higher in RA than in OA synovial tissues, and they were upregulated by TNFα stimulation in RASFs. These data might provide important clues for the development of specific small molecule HDAC inhibitors.

IgG anti-laminin-1 autoantibody and recurrent miscarriages.

PROBLEM: The present study assesses the clinical significance of anti‐laminin‐1 auto‐antibodies (auto‐Abs) in recurrent miscarriages.
 METHOD OF STUDY: A total of 207 recurrent aborters with a history of two or more consecutive first‐trimester miscarriages were tested for the presence of anti‐laminin‐1 Abs, β2‐glycoprotein I‐dependent anticardiolipin Abs, lupus anticoagulants, anti‐DNA Abs, and anti‐nuclear Abs, before they had conceived again. Recurrent aborters then were followed up during subsequent pregnancies and their outcomes were evaluated relative to their blood test results prior to pregnancy.
 RESULTS: Fifty‐five (31.1%) women out of 177 recurrent aborters were positive for IgG anti‐laminin‐1 auto‐Abs. The levels of IgG anti‐laminin‐1 auto‐Abs in recurrent aborters were significantly higher than those in healthy pregnant women and in healthy non‐pregnant women (P=0.0043 and 0.0073, respectively). The live birth rate of subsequent pregnancies in IgG anti‐laminin‐1 auto‐Abs‐positive recurrent aborters was significantly lower than the IgG anti‐laminin‐1 auto‐Abs‐negative recurrent aborters (P=0.0320). There were no specifically significant relationships observed between IgG anti‐laminin‐1 auto‐Abs and other tested auto‐Abs.
 CONCLUSION: IgG anti‐laminin‐1 auto‐Abs are associated with recurrent miscarriages and the subsequent pregnancy outcome of recurrent aborters.

Pregnancy Loss and Endometriosis: Pathogenic Role of Anti‐Laminin‐1 Autoantibodies

Abstract: Laminin‐1 is a major multifunctional glycoprotein that forms an integral part of the scaffolding network of basement membranes, and is the earliest synthesized component during embryogenesis. This protein (α1β1γ1) plays an important role in basement membrane assembly and epiblast differentiation during embryonic development. Anti‐laminin‐1 autoantibodies are known to cause infertility and recurrent spontaneous abortion in animals. Recently, we reported that the presence of IgG anti‐laminin‐1 antibodies (Abs) in the blood is significantly associated with recurrent first‐trimester miscarriages and subsequent negative pregnancy outcomes. Interestingly, these antibodies are also strongly associated with infertility, especially infertility caused by endometriosis. Laminin‐α1, laminin‐β1, and laminin‐γ1 mRNAs were also detected in 90% of endometriotic lesions, and all laminin‐α1, laminin‐β1, and laminin‐γ1 chains were localized to the basement membranes of glandular epithelium in endometriotic peritoneal lesions. ELISA showed specific reactivity of the autoantibodies to a particular region of the laminin‐1 molecule, that is, the α1 chain G domain. IgM monoclonal anti‐laminin‐1 Abs, which we recently established, also recognized the G domain and cross‐reacted with human α1 chain located in the basement membrane of the glandular epithelium of human endometrium. We also established an animal model that produced high titers of anti‐laminin‐1 Abs after immunization with mouse laminin‐1. Anti‐laminin‐1 Abs from the immunized mice caused a higher fetal resorption rate with lower embryonic and placental weights. Thus, anti‐laminin‐1 Abs may be important in the development of autoimmune‐mediated reproductive failures, and the assessment of the such antibodies may provide a novel means for noninvasive diagnosis of endometriosis.

Type IV collagen induces expression of thrombospondin‐1 that is mediated by integrin α1β1 in astrocytes

Following brain injury, thrombospondin‐1 (TSP‐1) is involved in angiogenesis and synaptic recovery. In this study, we used a cold injury‐model and found that TSP‐1 mRNA was markedly upregulated after brain injury. Immunohistochemistry showed that TSP‐1 was upregulated in both the core of the lesion and in the perilesional area of injured brain tissue. Numerous astrocytes immunopositive for glial fibrillary acidic protein (GFAP) were found in the perilesional area, and TSP‐1 was also expressed in almost all astrocytes surrounding blood vessels at 4 days after injury. Next, we examined the influence of vascular basement membrane components on TSP‐1 expression. When astrocytes were cultured on type IV collagen, TSP‐1 was significantly upregulated compared with the expression when cells were grown on laminin, fibronectin, or poly‐L‐lysine. This increase occurred exclusively when astrocytes were grown on the native form of type IV collagen but not on the heat‐denatured form or the non‐collagenous 1 domain. Further, integrin α1 and β1 mRNAs were upregulated concomitantly with GFAP mRNA, and integrin α1 protein was localized to the endfeet of astrocytes that surrounded blood vessels in the injured brain. Using function‐blocking antibodies, we found that the effectof type IV collagen was attributed to integrin α1β1 in primary astrocytes. Collectively, our results suggest that vascular basement membrane components substantially impact gene expression in astrocytes during brain tissue repair.

Immunization of naïve mice with mouse laminin-1 affected pregnancy outcome in a mouse model.

PROBLEM: Laminins have important roles during placental and embryonic development. The aim of our study was to determine if active immunization of mice with laminin‐1 could elicit an autoimmune response, and induce features of reproductive failure.

Tumor growth inhibitory effect of ADAMTS1 is accompanied by the inhibition of tumor angiogenesis.

Angiogenesis plays an important role in tumor progression. Several reports have demonstrated that a disintegrin and metalloproteinase with thrombospondin motifs1 (ADAMTS1) inhibited angiogenesis via multiple mechanisms. The aim of this study was to investigate the effect of ADAMTS1 on endothelial cells in vitro and on tumor growth with regard to angiogenesis in vivo. We examined the effects of the transfection of ADAMTS1 using two constructs, full‐length ADAMTS1 (full ADAMTS1) and catalytic domain‐deleted ADAMTS1 (delta ADAMTS1). Transfection of both the full ADAMTS1 and delta ADAMTS1 gene constructs demonstrated the secretion of tagged‐ADAMTS1 protein into the conditioned medium, so we examined the effects of ADAMTS1‐containing conditioned medium on endothelial cells. Both types of conditioned media inhibited endothelial tube formation, and this effect was completely abolished after immunoprecipitation of the secreted protein from the medium. Both types of conditioned media also inhibited endothelial cell migration and proliferation. We then examined the impact of ADAMTS1 on endothelial cell apoptosis. Both conditioned media increased the number of Annexin V‐positive endothelial cells and caspase‐3 activity and this effect was attenuated when z‐vad was added. These results indicated that ADAMTS1 induced endothelial cell apoptosis. We next examined the effects of ADAMTS1 gene transfer into tumor‐bearing mice. Both full ADAMTS1 and delta ADAMTS1 significantly inhibited the subcutaneous tumor growth. Collectively, our results demonstrated that ADAMTS1 gene transfer inhibited angiogenesis in vitro and in vivo, likely as a result of the induction of endothelial cell apoptosis by ADAMTS1 that occurs independent of the protease activity.

Anti-laminin-1 autoantibodies, pregnancy loss and endometriosis

Laminin-1 is a major component and multifunctional glycoprotein of basement membranes that consists of three different subunits, α1, β1 and γ1 chains. It is the earliest synthesized network-forming protein during embryogenesis and plays an important role in embryonic development, embryonic implantation and placentation. We have recently shown that IgG anti-laminin-1 antibodies were significantly associated with recurrent first-trimester miscarriages and with subsequent pregnancy outcome. Interestingly, these antibodies were also observed in patients with endometriosis-associated infertility but not in patients with other causes of infertility, including tubal factors, hormonal and uterine abnormalities. Laminin-α1, -β1 and -γ1 mRNAs have been detected in 90% of endometriotic lesions and all laminin-α1, -β1 and -γ1 chains were localized in the basement membranes of glandular epithelium in endometriotic peritoneal lesions. Western blot analysis showed that anti-laminin-1 antibodies from those patients reacted with all laminin-1s chains. ELISA also confirmed that one of the target epitopes for these antibodies was located in a particular region of the laminin-1 molecule, i.e. the carboxyl-terminal globular G domain of α1 chain. IgM monoclonal anti-laminin-1 autoantibody, that we recently established, also recognized the G domain. Anti-laminin-1 antibodies from mice immunized with –mouse— laminin-1, caused a higher fetal resorption rate with lower embryonic and placental weights. Thus, anti-laminin-1 antibodies may be important in development of autoimmune-mediated reproductive failures and the assessment of the antibodies may provide a novel non-invasive diagnosis of endometriosis.

A Possible Mechanism of Autoimmune‐Mediated infertility in Women with Endometriosis

Citation Inagaki J, Hao L, Nakatsuka M, Yasuda T, Hiramatsu Y, Shoenfeld Y, Matsuura E. A possible mechanism of autoimmune‐mediated infertility in women with endometriosis. Am J Reprod Immunol 2011; 66: 90–99

The presequence of the precursor to the nucleus-encoded 30 kDa protein of photosystem II in Euglena gracilis Z includes two hydrophobic domains

A cDNA clone for the extrinsic 30 kDa protein (OEC30) of photosystem II in Euglena gracilis Z was isolated and characterized. The open reading frame of the cDNA encoded a polypeptide of 338 amino acids, which consisted of a long presequence of 93 amino acids and a mature polypeptide of 245 amino acids. Two hydrophobic domains were identified in the presequence, in contrast to the presence of a single hydrophobic domain in the presequence of the corresponding proteins from higher plants. At the N- and C-terminal regions, respectively, of the presequence, a signal-peptide-like sequence and a thylakoid-transfer domain were identified. The presence of a long and unique presequence in the precursor to OEC30 is probably related to the complexity of the intracellular processes required for the synthesis and/or transport of the protein in Euglena.

ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor

Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy.