ICER Reverses Tumorigenesis of Rat Prostate Tumor Cells without affecting Cell Growth

BACKGROUND. Inducible cAMP early repressor (ICER) is an important mediator of cAMP antiproliferative activity that acts as a putative tumor suppressor gene product. ICER is a transcriptional repressor that negatively regulates cAMP-mediated gene expression. Here, we report the effect of ectopically increasing the expression of ICER on in vitro and in vivo proliferation of the highly metastatic and androgen-insensitive AT6.3 rat prostate cells. METHODS. The proliferative potential of stable AT6.3 cell clones expressing ICER was studied by cell counts, thymidine incorporation, flow cytometry, colony formation in soft agar, and growth in immunodeficient nude mice. RESULTS. cAMP inhibits the growth of AT6.3 cells. ICER mRNA and protein levels were markedly induced by cAMP in AT6.3 cells. Forced expression of ICER in AT6.3 cells did not affect cell growth, thymidine incorporation, or the cell cycle. However, these ICER-bearing AT6.3 cells were rendered unable to grow in soft agar or to form tumors in nude mice. CONCLUSION. These results show that ICER specifically affects the tumorigenicity of prostate cancer cell without affecting their growth. Therefore, the manipulation of ICER expression could be used for the treatment of androgen-insensitive prostate tumors without causing undesirable toxicity to the cells

Evidence for a Regulatory Role of Inducible Camp Early Repressor in Protein Kinase a-Mediated Enhancement of Vitamin D Receptor Expression and Modulation of Hormone Action

Parathyroid hormone (PTH) or activators of protein kinase A (PKA) up-regulate the vitamin D receptor (VDR) and augment the induction by 1,25-dihydroxyvitamin D3 of the expression of target genes (24-hydroxylase and osteopontin) in osteoblastic cells. To understand regulatory mechanisms involved, we asked whether the inducible cAMP early repressor (ICER), which serves as a dominant negative regulator of cAMP-induced transcription in other endocrine systems, may similarly play a role in modulation of vitamin D hormone action. In this study we demonstrate that PTH or 8-bromo-cAMP rapidly induces ICER mRNA and protein in osteoblastic cells. In UMR 106 osteoblastic cells transfected with an expression vector containing the ICER II-γ coding sequence, cAMP or PTH enhancement of 1,25-dihydroxyvitamin D3-induced osteopontin and 24-hydroxylase mRNA and transcription is inhibited. The vitamin D response element is sufficient for the PKA enhancement of VDR-mediated transcription and is also sufficient to observe the inhibitory effect of ICER. Our data indicate that the mechanism of the inhibitory effect of ICER involves an inhibition of PKA-induced VDR transcription, and this inhibition may be mediated in part by binding of ICER to a cAMP response element-like sequence in the VDR promoter. This study provides evidence for the first time that ICER has a key regulatory role in the PKA enhancement of VDR transcription and therefore in the cross-talk between the PKA signaling pathway and the vitamin D endocrine system

Mitogen-Activated Protein Kinase Phosphorylates and Targets Inducible Camp Early Repressor to Ubiquitin-Mediated Destruction

Inducible cAMP early repressor (ICER) is an important mediator of cAMP antiproliferative activity that acts as a putative tumor suppressor gene product. In this study, we examined the regulation of ICER protein by phosphorylation and ubiquitination in human choriocarcinoma JEG-3 and mouse pituitary AtT20 cells. We found that cAMP stabilized ICER protein by inhibiting the mitogen-activated protein kinase (MAPK) cascade. Activation of the MAPK pathway increased ICER phosphorylation. ICER phosphorylation was abrogated by inhibition of the MAPK pathway either by cAMP or directly by the MAPK inhibitor PD098059. The MAPKs extracellular signal-regulated kinases 1 and 2 physically interact with ICER and mediated the phosphorylation of ICER on a critical serine residue (Ser-41). A mutant form of ICER in which Ser-41 was substituted by alanine had a half-life 4-5 h longer than its wild-type counterpart. This alteration in stability was due to the inability of the Ser-41-mutant ICER to be efficiently ubiquitinated and degraded via the ubiquitin-proteasome pathway. These results present a novel cell signaling cross-talk mechanism at the cell nucleus between the MAPK and cAMP pathways, whereby MAPK targets a repressor of the cAMP-dependent gene expression for ubiquitination and proteasomal degradation

ICER-Ilγ Is a Tumor Suppressor that Mediates the Antiproliferative Activity of Camp

The second messenger cAMP inhibits the proliferation of most cell types. The nuclear response of cAMP is mediated by transcription factors like the cAMP-Responsive Element Modulator (CREM) gene. One of the products of the CREM gene, the transcriptional repressor Inducible cAMP Early Repressor-IIγ (ICER-IIγ), is induced by cAMP. ICER-IIγ blocks cells at the G2/M boundary of the cell cycle. Here we show that ICER-IIγ dramatically inhibits the growth and DNA synthesis of mouse pituitary tumor cells and human choriocarcinoma cells. This alteration in cell growth is coupled with reduced ability of these cells to grow in an anchorage-independent manner and to form tumors in mice. These data demonstrate that ICER-IIγ is a tumor suppressor gene product mediating the antiproliferative activity of cAMP

Inducible Camp Early Repressor (ICER) Is a Negative-Feedback Regulator of Cardiac Hypertrophy and an Important Mediator of Cardiac Myocyte Apoptosis in Response to Β-Adrenergic Receptor Stimulation

Although stimulation of the β-adrenergic receptor increases levels of cAMP and activation of the cAMP response element (CRE) in cardiac myocytes, the role of the signaling mechanism regulated by cAMP in hypertrophy and apoptosis is not well understood. In this study we show that protein expression of inducible cAMP early repressor (ICER), an endogenous inhibitor of CRE-mediated transcription, is induced by stimulation of isoproterenol (ISO), a β-adrenergic agonist with a peak at ≈12 hours and persisting for more than 24 hours in neonatal rat cardiac myocytes. ICER is also upregulated by phenylephrine but not by endothelin-1. Continuous infusion of ISO also increased ICER in the rat heart in vivo. Overexpression of ICER significantly attenuated ISO- and phenylephrine-induced cardiac hypertrophy but did not inhibit endothelin-1-induced cardiac hypertrophy. Overexpression of ICER also stimulated cardiac myocyte apoptosis. Antisense inhibition of ICER significantly enhanced β-adrenergic hypertrophy, whereas it significantly inhibited β-adrenergic cardiac myocyte apoptosis, suggesting that endogenous ICER works as an important regulator of cardiac hypertrophy and apoptosis. Inhibition of CRE-mediated transcription by dominant-negative CRE binding protein inhibited cardiac hypertrophy, whereas it stimulated cardiac myocyte apoptosis, thereby mimicking the effect of ICER. Both ISO and ICER reduced expression of Bcl-2, an antiapoptotic molecule, whereas antisense ICER prevented ISO-induced downregulation of Bcl-2. These results suggest that ICER is upregulated by cardiac hypertrophic stimuli increasing CRE-mediated transcription in cardiac myocytes and acts as a negative regulator of hypertrophy and a positive mediator of apoptosis, in part through both inhibition of CRE-mediated transcription and downregulation of Bcl-2

Constitutive Expression of Inducible Cyclic Adenosine Monophosphate Early Repressor (ICER) in Cycling Quiescent Hematopoietic Cellsimplications for Aging Hematopoietic Stem Cells

Despite extensive insights on the interaction between hematopoietic stem cells (HSCs) and the supporting bone marrow (BM) stroma in hematopoietic homeostasis there remains unanswered questions on HSC regulation. We report on the mechanism by which HSCs attain cycling quiescence by addressing a role for inducible cyclic AMP early repressor (ICER). ICER negatively transcriptional regulators of cAMP activators such as CREM and CREB. These activators can be induced by hematopoietic stimulators such as cytokines. We isolated subsets of hematopoietic cells from ten healthy donors: CD34+CD38−/c-kit+ (primitive progenitor), CD34+CD38+/c-kitlow (mature progenitor) and CD34−CD38+/−/c-kitlow/− (differentiated lineage-). The relative maturity of the progenitors were verified in long-term culture initiating assay. Immunoprecipitation indicated the highest level of ICER in the nuclear extracts of CD34+/CD38− cells. Phospho (p)-CREM was also present suggesting a balance between ICER and p-CREM in HSC. ICER seems to be responsible for decrease in G1 transition, based on reduced Cdk4 protein, decreased proliferation and functional studies with propidium iodide. There were no marked changes in the cycling inhibitors, p15 and p-Rb, suggesting that ICER may act independently of other cycling inhibitors. The major effects of ICER were validated with BM mononuclear cells (BMNCs) in which ICER was ectopically expressed, and with BMNCs resistant to 5-fluorouracil- or cyclophosphamide. In total, this study ascribes a novel role for ICER in G1 checkpoint regulation in HSCs. These findings are relevant to gene therapy that require engineering of HSCs, age-related disorders that are associated with hematopoietic dysfunction and other hematological disorders

Transcriptional Regulation of Cyclin D2 By the PKA Pathway and Inducible Camp Early Repressor in Granulosa Cells

Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis