Induction of the unfolded protein response by calcineurin inhibitors: a double-edged sword in renal transplantation

Abstract

The endoplasmic reticulum (ER) provides a unique environment for appropriate protein folding and assembly to produce functional, mature proteins. A number of pathophysiological insults cause accumulation of unfolded proteins in the ER, namely, ER stress. In response to ER stress, cells adapt themselves to the stress conditions via the unfolded protein response (UPR), leading to attenuation of translation, induction of ER chaperones and activation of ER-associated degradation (ERAD) to eliminate immature proteins. The UPR is involved in a diverse range of pathophysiological events [1,2], including renal diseases [3]. In response to ER stress, three major branches of the UPR are activated, as summarized in Figure 1A. Those include the RNA-dependent protein kinase-like ER kinase (PERK) pathway, the activating transcription factor 6 (ATF6) pathway and the inositol-requiring ER-to-nucleus signal kinase 1 (IRE1) pathway. Activation of PERK causes phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), which leads to general inhibition of protein synthesis. In response to ER stress, p90ATF6 transits to the Golgi where it is cleaved by the proteases S1P and S2P, yielding an active transcription factor p50ATF6. Similarly, activated IRE1 catalyses removal of a small intron from the mRNA of X-box binding protein 1 (XBP1). This splicing event creates translational frameshift in XBP1 mRNA to produce an active transcription factor. Active p50ATF6 and XBP1 subsequently bind to the ER stress response element (ERSE) and the UPR element (UPRE), leading to expression of target genes including ER chaperones and ERAD factors that degrade unfolded proteins. Cyclosporine A (CsA) and tacrolimus (FK506) are pivotal immunosuppressive agents to prevent allograft rejection in renal transplantation. Use of these immunosuppressants leads to significant reduction in the incidence of acute graft rejection and improvement in the survival of kidney transplants [4]. CsA binds to the cyclophilin family of molecules that have high affinity for calcineurin, a key protein phosphatase in the activation of T cells. FK506 is another calcineurin inhibitor, the mechanism of which is similar to that of CsA. It forms complexes with its cytosolic partner FK506-binding protein 12, and the complexes bind to calcineurin. By blocking calcineurin, CsA and FK506 inhibit phosphatase-controlled translocation of nuclear factor of activated T-cells (NF-AT) into the nucleus and prevent induction of cytokines and their receptors required for activation and proliferation of lymphocytes and other immune cells [5]. These agents are, therefore, regarded as inhibitors of immune cell function. However, several reports have also demonstrated that CsA and FK506 suppress activation of non-immune cells including keratinocytes, endothelial cells, mesangial cells, synovial fibroblasts and hepatocytes. For example, CsA inhibits mitogenesis of endothelial cells and growth factor-driven proliferation of keratinocytes [6,7]. CsA also attenuates induction of cytokines and their receptors in keratinocytes and expression of nitric oxide synthase in mesangial cells [8,9]. Similarly, other investigators reported that FK506 inhibits secretion of TNF-α in keratinocytes, production of matrix metalloproteinase 13 by rheumatoid synovial fibroblasts and generation of reactive oxygen species and nitric oxide in hepatocytes [10–12]. Currently, molecular mechanisms underlying the suppressive effects of CsA and FK506 on the non-lymphoid lineages are largely unknown. Recently, we found that in renal tubular cells, induction of monocyte chemoattractant protein 1 (MCP-1) by inflammatory cytokines is blunted by CsA and FK506 [13]. We identified that this suppressive effect is ascribed, at least in part, to induction of ER stress and consequent UPR. This phenomenon is observed not only in tubular cells but also in other non-immune cells including smooth muscle cells, preadipocytes, mesangial cells and endothelial cells. Indeed, administration with CsA in mice causes rapid induction of the UPR in several organs [13]. In tumour necrosis factor-α (TNF-α)-exposed cells, suppression of MCP-1 by CsA and FK506 is associated with the blunted activation of nuclear factor-κB (NF-κB), and the suppression of NF-κB is reproduced by other UPR inducers. CsA and FK506, as well as other UPR inducers, cause up-regulation