Toll-like receptor 3 mediates expression of clusterin/apolipoprotein J in vascular smooth muscle cells stimulated with RNA released from necrotic cells.

Clusterin/Apolipoprotein J is a protein that is upregulated in a broad spectrum of diverse pathological processes. The predominant form is a secreted glycoprotein (sCLU) with cytoprotective and anti-inflammatory properties which shows enhanced expression in vascular smooth muscle cells (VSMC) following aortic injury and in atherosclerotic disease. Recent evidence indicates that during atherosclerosis, Toll-like receptors (TLRs) are activated in vascular cells by endogenous ligands. Here, we analyzed whether CLU expression in VSMC is controlled by TLRs, and stimulated by factors associated with or released by necrotic cells. Activation of TLR3 by the synthetic RNA analogue polyinosinic-polycytidylic acid (poly(I:C)) in CRL2018 VSMC and in mice led to induction of CLU mRNA and protein synthesis, respectively. In TLR3-deficient 10A yolk sac cells, induction of CLU by poly(I:C) challenge depended on the ectopic expression of human TLR3. In mice lacking the TLR3-signaling adaptor protein TRIF (TIR-domain-containing adaptor protein inducing IFN-ß) CLU induction by poly(I:C) was abrogated. In addition to poly(I:C) CLU gene expression in CRL2018 cells was induced by purified cellular RNA and RNA present in necrotic cell lysate. Our data indicate that cellular RNA following its release from necrotic cells in atherosclerotic lesions can act as an endogenous TLR3 ligand to induce CLU expression in VSMC and in vivo. Thus, they expand the view on TLR2 and TLR4 as known pro-atherosclerotic effectors toward TLR3. Conclusively, TLR3 activation induces expression of cytoprotective and anti-inflammatory CLU by VSMC and mice, to potentially counteract atherosclerotic pathology.

Apoptotic cell debris and phosphatidylserine-containing lipid vesicles induce apolipoprotein J (clusterin) gene expression in vital fibroblasts.

The molecular events in cells undergoing programmed cell death (apoptosis) are well studied; however, the response of the surviving neighbor cells to local cell death is largely uncharacterized. Apolipoprotein J (clusterin) is an 80-kDa glycoprotein that has been implied in cytoprotection of the vital cells, presumably by assisting in the clearance of apoptotic vesicles and membrane remnants. Its mRNA is specifically up-regulated in the vital cells of apoptotic tissues. The molecular mechanisms, however, leading to this response are not known. We here show that exposure of vital fibroblasts to apoptotic vesicles, disrupted vital cells, and trypsin-treated membrane remnants induces apoJ mRNA. Moreover, lipid vesicles consisting of phosphatidylserine (PtSer) and dimyristoylphosphatidylcholine (PC), but not liposomes with PC alone nor with dimyristoylphosphatidylethanolamine or phosphatidic acid, did elevate apoJ mRNA level. These results suggest that, apart from mediating the endocytic uptake of the apoptotic vesicles, PtSer also serves as a trigger to stimulate the expression of genes that might be involved in the cellular clearance process.