Cyclosporine Inhibits Growth through the Activating Transcription Factor/cAMP-responsive Element-binding Protein Binding Site in the Cyclin D1 Promoter

Abstract

The immunosuppressive agent cyclosporine affects proliferation depending on the cellular system used. In an attempt to study the inhibitory effect of cyclosporine on proliferation of pancreatic acinar cells, we used AR42J cells as a model system. Here we demonstrate that cyclosporine inhibits growth of these cells by inducing G 1 cell cycle arrest. This effect is mediated by the 5 regulatory region of the cyclin D1 gene and leads to a reduction of cyclin D1 mRNA expression and protein abundance. We show that in AR42J cells the proximal cyclin D1 promoter contains a cis-regulated element, which is important for the maintenance of basal transcriptional activity. This element overlaps the described cAMP-responsive element (CRE) and confers cyclosporine sensitivity to the cyclin D1 promoter. Furthermore, the DNA binding activity of the CRE-binding protein (CREB) decreases through cyclosporine treatment and this is mediated by cyclosporine-induced reduction of CREB steady-state levels. These results demonstrate that cyclosporine can inhibit proliferation of acinar cells by targeting the cyclin D1 promoter at the proximal CRE via a reduction of CREB protein abundance.Mammalian cell proliferation is regulated in mid-to late G 1 phase of the cell cycle. The D-type cyclins and the associated cyclin-dependent kinases (CDKs) 1 are thought to be the key regulatory components for progression through G 1 phase and for the commitment of cells to enter S phase. Cyclin D1 and its catalytic partners CDK4 and CDK6 phosphorylate the retinoblastoma tumor suppressor gene product (RB), resulting in the release of E2F transcription factors, which are required for the activation of S phase-specific genes (1, 2). Inhibition of cyclin D1 expression prevents transition of cells from G 1 to S phase, whereas ectopic expression of cyclin D1 shortens the G 1 interval in many cell types, suggesting that cyclin D1 may be ratelimiting for G 1 progression (2, 3). The abundance of cyclin D1 is regulated transcriptionally and through the ubiquitin-proteasome pathway via protein degradation (4). The promoter region of the cyclin D1 gene contains multiple cis-acting elements, including binding sites for activator protein-1 (AP-1), for nuclear factor B (NF-B), for signal transducers and activators of transcription, for SP1, and for activating transcription factor (ATF)/cAMP-responsive element-binding protein (CREB). The ATF/CREB-binding site is implicated in the activation of the cyclin D1 gene by pp60 v-src (5). Furthermore, the estrogeninduced activation of the cyclin D1 promoter depends on the ATF/CREB site in which ATF-2 and c-Jun form heterodimers (6). Moreover, the integrin-linked kinase-dependent induction of the cyclin D1 promoter requires an intact ATF/CREB site, which is important for the maintenance of the basal transcriptional activity, but not for the induction of the cyclin D1 promoter activity in vascular endothelial cells (7,8).The basic leucine zipper transcription factor CREB is ubiquitously expressed and activated by phosphorylatio...

Protein Expression Profiling Identifies Cyclophilin A as a Molecular Target in Fhit-Mediated Tumor Suppression

“…Cyclophilin A is also known to promote calcineurindependent G 1 -S transition by up-regulating cyclin D1 expression and Cdk4 activity in late G 1 phase (21)(22)(23). We confirmed up-regulation of both cyclin D1 and Cdk4 expression by cyclophilin A (10 ng/mL) treatment 60 minutes after exposure in synchronized H1299 cells; however, in the cyclophilin Amediated G 1 -S transition, there was no significant change in p21 WAF1 expression levels (Fig.…”

The Effect of Cyclosporin A on Airway Cell Proinflammatory Signaling and Pneumonia

“…In contrast, CREB activity was substantially reduced by CsA treatment of the airway cells. This is consistent with reports of CsA inhibition of other ATF/CREB promoter elements in the cyclinD1 promoter (30) and effects on CREreporter constructs in pancreatic islet cells and Jurkat cell lines (31,32). CREB is phosphorylated at various sites by a number of kinases, including MAPKs and Ca 2ϩ /calmodulin-dependent protein kinase (CaMKs), which are activated by increases in Ca 2ϩ and may be indirectly affected by CsA/cyclophilin/calcineurin interactions (33).…”

Ca2+ signaling, genes and the cell cycle

“…During the metaphase to anaphase transition at fertilization in Xenopus eggs calcineurin has been shown to play a role although the details remain to be elucidated [43;44]. In pancreatic acinar cells cyclosporine (CsA) treatment inhibits CREB leading to a reduction in cyclin D expression and arrest in the G1-phase of the cell cycle [77]. However, CsA has also been reported to exhibit cell autonomous tumor promoting effects [78], arguing that inhibition of calcineurin activity depending on the cellular context can either lead to cell cycle arrest or promotion of cellular proliferation.…”