Individuals with rheumatoid arthritis frequently have autoantibodies to citrullinated peptides, suggesting the involvement of the peptidylarginine deiminases citrullinating enzymes (encoded by PADI genes) in rheumatoid arthritis. Previous linkage studies have shown that a susceptibility locus for rheumatoid arthritis includes four PADI genes but did not establish which PADI gene confers susceptibility to rheumatoid arthritis. We used a case-control linkage disequilibrium study to show that PADI type 4 is a susceptibility locus for rheumatoid arthritis (P = 0.000008). PADI4 was expressed in hematological and rheumatoid arthritis synovial tissues. We also identified a haplotype of PADI4 associated with susceptibility to rheumatoid arthritis that affected stability of transcripts and was associated with levels of antibody to citrullinated peptide in sera from individuals with rheumatoid arthritis. Our results imply that the PADI4 haplotype associated with susceptibility to rheumatoid arthritis increases production of citrullinated peptides acting as autoantigens, resulting in heightened risk of developing the disease.
Rheumatoid arthritis is a common inflammatory disease with complex genetic components. We investigated the genetic contribution of the cytokine gene cluster in chromosome 5q31 to susceptibility to rheumatoid arthritis in the Japanese population by case-control linkage disequilibrium (LD) mapping using single nucleotide polymorphisms (SNPs). Here we report that there is significant association between rheumatoid arthritis and the organic cation transporter gene SLC22A4 (P = 0.000034). We show that expression of SLC22A4 is specific to hematological and immunological tissues and that SLC22A4 is also highly expressed in the inflammatory joints of mice with collagen-induced arthritis. A SNP affects the transcriptional efficiency of SLC22A4 in vitro, owing to an allelic difference in affinity to Runt-related transcription factor 1 (RUNX1), a transcriptional regulator in the hematopoietic system. A SNP in RUNX1 is also strongly associated with rheumatoid arthritis (P = 0.00035). Our data indicate that the regulation of SLC22A4 expression by RUNX1 is associated with susceptibility to rheumatoid arthritis, which may represent an example of an epistatic effect of two genes on this disorder.
The KK obese mouse is moderately obese and has abnormally high levels of plasma insulin (hyperinsulinemia), glucose (hyperglycemia) and lipids (hyperlipidemia). In one strain (KK/San), we observed abnormally low plasma lipid levels (hypolipidemia). This mutant phenotype is inherited recessively as a mendelian trait. Here we report the mapping of the hypolipidemia (hypl) locus to the middle of chromosome 4 and positional cloning of the autosomal recessive mutation responsible for the hypolipidemia. The hypl locus encodes a unique angiopoietin-like lipoprotein modulator, which we named Allm1. It is identical to angiopoietin-like protein 3, encoded by Angptl3, and has a highly conserved counterpart in humans. Overexpression of Angptl3 or intravenous injection of the purified protein in KK/San mice elicited an increase in circulating plasma lipid levels. This increase was also observed in C57BL/6J normal mice. Taken together, these data suggest that Angptl3 regulates lipid metabolism in animals.
Studies with KK/San, obese and diabetic model mice having a unique hypotriglyceridemia phenotype, revealed that angiopoietin-like protein 3 (ANGPTL3) regulates lipid metabolism in mice. To determine the lipid-modulating role of other ANGPTLs, we focused on ANGPTL4, which overall shows a significant similarity to ANGPTL3. Surprisingly, an intravenous injection of the ANGPTL4 protein in KK/San mice rapidly increased the circulating plasma lipid levels at a higher rate than ANGPTL3 protein. Furthermore, the ANGPTL4 protein inhibited the lipoprotein lipase activity in vitro. Angiopoietin-like proteins, ANGPTL3 and ANGPTL4, have been well characterized as secretory proteins. ANGPTL4 has been shown to be a downstream target of peroxisome proliferator-activated receptor-gamma (PPAR ␥ ) and possibly has a role in the regulation of systemic lipid metabolism or glucose homeostasis since PPAR target genes identified so far are largely involved in fatty acid metabolism (1). ANGPTL4 could also be a novel endocrine signal involved in the regulation of metabolism, especially under fasting conditions, as demonstrated by the fact that ANGPTL4 mRNA is strongly up-regulated during fasting in white adipose tissue and liver (2). Nevertheless, we still do not know the exact role of the ANGPTL4 protein.Studies with KK/San mice that model obesity and diabetes but with significantly lower plasma lipid levels have shown that a functional loss of ANGPTL3 at the hypolipidemia ( hypl ) locus leads to abnormally low plasma lipid levels (3). Overexpression of the ANGPTL3 protein in KK/San mice reverses the hypolipidemia phenotype (3). These findings incited us to examine whether other ANGPTLs have an up-regulating effect on lipid metabolism.Here, we report a novel lipid modulating function of ANGPTL4, the closest resembling protein of ANGPTL3. The ANGPTL4 protein potently increased plasma lipid levels in mice. This unique functional feature of ANGPTL4 is quite similar to that of ANGPTL3 (3). The ANGPTL4 protein also inhibited lipoprotein lipase (LPL) activity in vitro. We considered that ANGPTL4 and ANGPTL3 are functionally unique members of the angiopoietins. MATERIALS AND METHODS Vector constructionThe cDNA clones used for human ANGPTL4 (GenBank Accession No. BF337379) were obtained from IMAGE consortium. The primers used to amplify full-length human ANGPTL4 (26-406 aa) were 5 Ј -TCCCCCGGGGGACCCGTGCAGTCCAAG-3 Ј as the sense primer and 5 Ј -CCGCTCGAGGGAGGCTGCCTCTGC-TGC-3 Ј as the anti-sense primer. PCR was performed with 30 cycles at 94 Њ C for 30 s, 60 Њ C for 30 s, and 72 Њ C for 90 s. The PCR products were digested with Sal I/ Xho I and cloned into the corresponding site of pGEX-4T-2 (Amersham). All constructs were confirmed by sequencing. Several independent clones were tested for their protein expression level and the most suitable clone was used for a larger preparation of the protein. Preparation of GST fusion proteinsThe protein was expressed in Escherichia coli BL21CodonPlus (DE3)-RIL strain (Stratagene) by induction of log...
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