Characterization and Functional Consequences of Underexpression of Clusterin in Rheumatoid Arthritis

Abstract

We previously compared by microarray analysis gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) tissues. Among the set of genes identified as a molecular signature of RA, clusterin (clu) was one of the most differentially expressed. In the present study we sought to assess the expression and the role of CLU (mRNA and protein) in the affected joints and in cultured fibroblast-like synoviocytes (FLS) and to determine its functional role. Quantitative RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, and Western blot were used to specify and quantify the expression of CLU in ex vivo synovial tissue. In synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in RA tissues compared with OA and healthy synovium. In synovium and in cultured FLS, the overexpression of CLU concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of CLU in RA FLS promoted apoptosis within 24 h. We observed that CLU knockdown with small interfering RNA promoted IL-6 and IL-8 production. CLU interacted with phosphorylated IκBα. Differential expression of CLU by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. We propose that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-κB activation and survival of the synoviocytes.

Fractalkine mediates T cell–dependent proliferation of synovial fibroblasts in rheumatoid arthritis

“…They demonstrate pseudotumorlike overgrowth and resist apoptosis (42)(43)(44). The present study demonstrated the pivotal roles the CX 3 CR1 pathway plays in senescent CD4ϩ,CD28Ϫ T cell and FLS interaction and in the T cell-induced overgrowth of rheumatoid FLS.…”

The clusterin paradigm in prostate and breast carcinogenesis

“…But the effects of NF-kB signalling on CLU expression are more complex, and interestingly it was later shown that CLU regulates NF-kB activity in a negative manner by stabilising the inhibitor IkBs Endocrine-Related Cancer (2010) 17 R1-R17 www.endocrinology-journals.org (Santilli et al 2003, Devauchelle et al 2006, Savkovic et al 2007, Takase et al 2008a. By western blot analysis, we have observed an increased expression level of IkBa and a reduced expression of NF-kB also in PNT1A human immortalised prostate epithelial cells stably overexpressing CLU compared with control cells transfected with empty vector (Bettuzzi et al 2009).…”

Alteration in the activation state of new inflammation-associated targets by phospholipase A2-activating protein (PLAA)

“…Decreased expression of clusterin is involved in rheumatoid arthritis [50]. Disruption of the clusterin gene promotes NF-κB activation [50,51] and IL-6 production [50] A sandwich ELISA assay confirmed the microarray and real-time RT-PCR results. The plaa high cells expressed significantly less secreted clusterin (sCLU) antigen than plaa low cells, both in the cell culture supernatant and in the cytoplasm (Fig 9A).…”

“…9C). Once in the cytoplasm, clusterin may further reduce inflammation by inhibiting NF-κB activation [50,51], thus limiting the expression of COX-2 [53,54] and proinflammatory cytokines [50]. Indeed, disruption of the clusterin gene in cells promotes NF-κB activation [50,51] and IL-6 production [50].…”