Hiromitsu Yoshimura

Ursolic acid, an antagonist for transforming growth factor (TGF)-β1

Transforming growth factor‐β (TGF‐β), a multifunctional cytokine which is involved in extracellular matrix modulation, has a major role in the pathogenesis and progression of fibrotic diseases. We now report the effects of ursolic acid on TGF‐β1 receptor binding and TGF‐β1‐induced cellular functions in vitro. Ursolic acid inhibited [125I]‐TGF‐β1 receptor binding to Balb/c 3T3 mouse fibroblasts with an IC50 value of 6.9 ± 0.8 μM. Ursolic acid dose‐dependently recovered reduced proliferation of Minc Mv1Lu cells in the presence of 5 nM of TGF‐β1 and attenuated TGF‐β1‐induced collagen synthesis and production in human fibroblasts. Molecular dynamics simulations suggest that ursolic acid may interact with the hydrophobic region of the dimeric interface and thereby inhibit the binding of TGF‐β1 to its receptor. All these findings taken together show that ursolic acid functions as an antagonist for TGF‐β1. This is the first report to show that a small molecule can inhibit TGF‐β1 receptor binding and influence functions of TGF‐β1.

Molecular cloning and functional expression analysis of a cDNA for human hepassocin, a liver-specific protein with hepatocyte mitogenic activity.

By means of differential cDNA expression cloning, we earlier isolated a novel rat cDNA and its protein, named hepassocin, which is upregulated during liver regeneration. Using the rat cDNA as a probe, we have now isolated human hepassocin cDNA encoding a protein of 312 amino acids, which has 81.4% and 83.8% identity, respectively, to rat hepassocin before and after elimination of its signal peptide. Dot blot analysis revealed that hepassocin mRNA was strongly expressed in adult liver, fairly strongly in fetal liver, and weakly in pancreas, but not in other tissues. Recombinant human hepassocin produced in Chinese hamster ovary (CHO) cells by the dihydrofolate reductase-methotrexate (DHFR--MTX) gene amplification method is a homodimer (68 kDa) and has mitogenic activities in hepatocytes of various animal species including rat, mouse, rabbit and dog, and the activity was lost with 2-mercaptoethanol treatment. These results suggest that hepassocin is a potent regulator in liver cell growth not only in rats but also in humans. Computer searches revealed that human hepassocin as well as rat hepassocin has a characteristic disulfide structure close to that of fibrinogen-gamma. We assume that this newly identified growth factor exerts functions in association with an extracellular matrix such as fibrinogen.

High levels of human recombinant cyclooxygenase-1 expression in mammalian cells using a novel gene amplification method.

We report the expression of a high level of human cyclooxygenase-1 (hCOX-1) in mammalian cells using a novel gene amplification method known as the IR/MAR gene amplification system. IR/MAR-plasmids contain a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) and amplify autonomously without a specific induction process. In this study, the IR/MAR-plasmid pΔBN.AR1 was cotransfected with pCAG-COX1, which expresses hCOX-1, into human HEK293T cells, and G418 and blasticidin S double-resistant cells were obtained in about 1month. Real-time PCR and Western blotting revealed that the expressions of hCOX-1 mRNA and protein in both polyclonal and monoclonal cells were remarkably higher than those in only pCAG-COX1-transfected control cells. Southern blotting demonstrated the amplification of the hCOX-1 gene, and the copy number of clone #43 obtained by the cotransfection of pΔBN.AR1 and pCAG-COX1 was more than 20 copies per cell, though that of clone #14 obtained without using the IR/MAR plasmid pΔBN.AR1 was only two copies. These results indicate that a high level of hCOX-1 expression was achieved as a result of hCOX-1 gene amplification. Furthermore, the crude extract from clone #43 showed a strong COX-1 activity, and the activity was inhibited by the representative COX-1 inhibitor indomethacin, with an IC(50) value of 36nM. These results demonstrate that the IR/MAR gene amplification system is a simple but useful method for generating highly productive mammalian cells.

Effects of kynurenine metabolites on mesangial cell proliferation and gene expression.

In the present study, we examined the effects of kynurenine metabolites on cultured mesangial cells (MCs) and demonstrated for the first time that they affect MC proliferation and gene expression. Anthranilic acid and 3-hydroxy-DL-kynurenine suppressed MC proliferation by 32% and 43%, respectively at 10(-6) M compared to the control. In contrast, quinolinic acid and l-kynurenine promoted MC proliferation by 49% and 35% at 10(-8) M respectively, although promoting activities declined at higher concentrations. In addition, quinolinic acid upregulated the gene expression of platelet-derived growth factor-B, collagen type-Ialpha1, and collagen type-IValpha1. However, the gene expression of hepatocyte growth factor (HGF) was downregulated. We further examined the gene expressions in the glomeruli of high serum IgA (HIGA) mice with IgA nephropathy using microarray technology and found that the gene expression of insulin-like growth factor-1 was higher, but that of HGF was lower at 40 weeks of age compared to 8 weeks of age. In Balb/c mice, the gene expression of three kynurenine pathway enzymes (kynurenine aminotransferase I, kynurenine aminotransferase II, and quinolinate phospho-ribosyltransferase) increased 2- to 3.5-fold, whereas those in HIGA mice did not change significantly. These results suggest that abnormalities in the kynurenine pathway are associated with the dysfunction of MCs and progression of chronic kidney diseases.

Downregulated expression in high IgA (HIGA) mice and the renal protective role of meprinβ

This study discusses the critical role of the metalloproteinase meprinbeta in the progression of glomerulonephritis. Using a microarray technique, the gene expression profiles in glomeruli isolated from high serum IgA (HIGA) mice with a purity of 97% or greater were examined. HIGA mice are a valid model of human IgA nephropathy (IgAN), with the typical pathological features of this condition, including a consistently high serum IgA level as well as dominant mesangial IgA deposition and mesangial enlargement. Among the many upregulated/downregulated genes after the development of IgAN, the downregulation of meprinbeta was intriguing. The expression level of the meprinbeta gene at 40 weeks of age was 52% of that observed at 8 weeks of age (prior to the development of IgAN), although in the control BALB/c mice, a 2.19-fold elevation was seen. These results were also confirmed by semi-quantitative RT-PCR and immunostaining analyses. As meprinbeta is a subunit of metalloproteinase meprins (meprin A, meprin B) and meprins are capable of proteolytically degrading extracellular matrix (ECM) components and proteolytically processing bioactive peptides, the downregulation of meprinbeta may contribute to the progression of glomerulonephritis and the eventual glomerular scarring. This working hypothesis was examined using an in vivo meprinbeta inhibition study. The inhibition of meprins by actinonin exacerbated some parameters of renal injury in mice afflicted with anti-glomerular basement membrane (anti-GBM) antibody-associated nephritis. These in vitro and in vivo results suggest that meprinbeta may play a protective role against the progression of renal injury through the degradation of ECM and bioactive peptides.