Hiroki Kawasaki

c-Fos/activator protein-1 transactivates wee1 kinase at G1/S to inhibit premature mitosis in antigen-specific Th1 cells

M‐phase promoting factor is a complex of cdc2 and cyclin B that is regulated positively by cdc25 phosphatase and negatively by wee1 kinase. We isolated the wee1 gene promoter and found that it contains one AP‐1 binding motif and is directly activated by the immediate early gene product c‐Fos at cellular G1/S phase. In antigen‐specific Th1 cells stimulated by antigen, transactivation of the c‐fos and wee1 kinase genes occurred sequentially at G1/S, and the substrate of wee1 kinase, cdc2‐Tyr15, was subsequently phosphorylated at late G1/S. Under prolonged expression of the c‐fos gene, however, the amount of wee1 kinase was increased and its target cdc2 molecule was constitutively phosphorylated on its tyrosine residue, where Th1 cells went into aberrant mitosis. Thus, an immediate early gene product, c‐Fos/AP‐1, directly transactivates the wee1 kinase gene at G1/S. The transient increase in c‐fos and wee1 kinase genes is likely to be responsible for preventing premature mitosis while the cells remain in the G1/S phase of the cell cycle.

Human wee1 kinase is directly transactivated by and increased in association with c-Fos/AP-1: rheumatoid synovial cells overexpressing these genes go into aberrant mitosis.

Wee1 kinase downregulates the M-phase promoting factor, a complex of cdc2 and cyclin B kinase, that controls mitotic cell division. We isolated human wee1 kinase gene promoter and found that it contained one AP-1-binding motif in its promoter region (5′-CGAGTCA-3′; −823/−817), through which wee1 kinase gene was directly transactivated by c-Fos/AP-1. In rheumatoid synovial cells, wee1 kinase was increased in conjunction with the increase of c-Fos/AP-1 and the substrate of wee1, cdc2, was phosphorylated. The amount of wee1 and c-Fos and the phosphorylation of cdc2 were decreased after treatment of the cells with an inhibitor of AP-1, curcumin. A significant proportion of cultured synovial cells of the patients with rheumatoid arthritis, but not those of osteoarthritis, shifted to a tetraploid (4C) state upon long-term culture. Thus, human wee1 kinase gene is directly transactivated by and increased in association with c-Fos/AP-1, and rheumatoid synovial cells overexpressing these genes go into aberrant mitosis.

p21waf1/cip1 is down-regulated in conjunction with up-regulation of c-Fos in the lymphocytes of rheumatoid arthritis patients.

Features characteristic to rheumatoid arthritis (RA) including synovial overgrowth and joint destruction are experimentally produced by augmenting c-fos gene expression. We show that cyclin dependent kinase inhibitor p21waf1/cip1, that inhibits cell proliferation, is down-regulated in conjunction with up-regulation of c-fos in the lymphocytes of patients with RA. As to the mechanism of down-regulation of p21waf1/cip1 gene expression, transfection studies in U937 cells showed that c-fos down-regulated phosphorylation and dimerization of signal transducers and activators of transcription (STAT) 1, thereby inhibiting interferon -induced transactivation of p21waf1/cip1. Phosphorylation of STAT1 was indeed decreased in the lymphocytes of patients with RA. Thus, under overexpression of c-fos gene, c-Fos inactivates STAT1 to down-regulate p21waf1/cip1 gene expression in the lymphocytes of patients with RA, and in this way may enhance proliferation of lymphocytes.

Spontaneous and cytokine regulated c-fos gene expression in rheumatoid synovial cells: resistance to cytokine stimulation when the c-fos gene is overexpressed

OBJECTIVE To study the effect of cytokines on the transactivation of the c-fos gene in relation to the contribution of overexpression of c-fos/AP-1 in rheumatoid joint destruction. METHODS The promoter region (−447 to +109) of the human c-fosgene was integrated upstream of the chloramphenicol acetyltransferase (CAT) reporter gene, and the effect of cytokines on the expression of the c-fos gene was studied in the rheumatoid synovial cells of early (3–4) or late (14–18) passages, in the presence or absence of cytokines, by the transient transfection assay. RESULTS Expression of c-fos gene was enhanced by tumour necrosis factor α (TNFα) and interleukin 6 (IL6) in the synovial cells of early passage, whereas it was not enhanced in the synovial cells of late passage. The c-fos gene expression was also enhanced by 13-O-tetradecanoyl phorbol-13-acetate (TPA) in early passage but was somewhat suppressed in the late passage. It was found that the c-fos gene and c-Fos protein were both increased in the synovial cells of late passage. Similarly, c-fos gene expression was also not increased by TPA or cytokine stimulation in the stablec-fos transformants (fos-pH8) or H-ras transformed NIH3T3 cells (NIH H-ras cells) that constitutively expressed c-fos genes. CONCLUSIONS Although TNFα and IL6 augmented c-fos gene expression of rheumatoid synovial cells, transactivation of c-fos gene became resistant against cytokine stimulation under prolonged expression of c-fos gene, which may impart a tumour-like characteristic to rheumatoid synovial cells.

A variant of death-receptor 3 associated with rheumatoid arthritis interferes with apoptosis-induction of T cell

Rheumatoid arthritis (RA) is a chronic polyarthritis of unknown etiology. To unravel the molecular mechanisms in RA, we performed targeted DNA sequencing analysis of patients with RA. This analysis identified a variant of the death receptor 3 (DR3) gene, a member of the family of apoptosis-inducing Fas genes, which contains four single-nucleotide polymorphisms (SNPs) and a 14-nucleotide deletion within exon 5 and intron 5. We found that the deletion causes the binding of splicing regulatory proteins to DR3 pre-mRNA intron 5, resulting in a portion of intron 5 becoming part of the coding sequence, thereby generating a premature stop codon. We also found that this truncated DR3 protein product lacks the death domain and forms a heterotrimer complex with wildtype DR3 that dominant-negatively inhibits ligand-induced apoptosis in lymphocytes. Myelocytes from transgenic mice expressing the human DR3 variant produced soluble truncated DR3, forming a complex with TNF-like ligand 1A (TL1A), which inhibited apoptosis induction. In summary, our results reveal that a DR3 splice variant that interferes with ligand-induced T cell responses and apoptosis may contribute to RA pathogenesis.

Notes on the Disease Mechanism and Genetics of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic polyarthritis of unknown etiology that affects about 1% of the population worldwide. The risk of the disease in the siblings of affected individuals (λ s ) is significantly increased in RA, suggesting that both genetic and environment factors are important in the pathogenesis of RA. Previous studies in this laboratory have shown that features characteristic to RA, synovial overgrowth and bone resorption, can be experimentally reproduced by augmenting the expression of the c-fos protooncogene. We have searched the human genome for genes that predispose to RA using fluorescence-based microsatellite marker analysis and affected sib-pair linkage study. A panel of 41 Japanese families, each with at least two affected siblings, was typed for genome-wide 358 polymorphic microsatellite marker loci. Three principal chromosome regions of linkage, D1S253/214, D8S556 and DXS1232, have been identified which we call RAI, RA2 and RA3 for rheumatoid arthritis disease loci.