Expression of androgen receptor target genes in skeletal muscle.

We aimed to determine the mechanisms of the anabolic actions of androgens in skeletal muscle by investigating potential androgen receptor (AR)-regulated genes in in vitro and in vivo models. The expression of the myogenic regulatory factor myogenin was significantly decreased in skeletal muscle from testosterone-treated orchidectomized male mice compared to control orchidectomized males, and was increased in muscle from male AR knockout mice that lacked DNA binding activity (AR(ΔZF2)) versus wildtype mice, demonstrating that myogenin is repressed by the androgen/AR pathway. The ubiquitin ligase Fbxo32 was repressed by 12 h dihydrotestosterone treatment in human skeletal muscle cell myoblasts, and c-Myc expression was decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle, and increased in AR(∆ZF2) muscle. The expression of a group of genes that regulate the transition from myoblast proliferation to differentiation, Tceal7 , p57(Kip2), Igf2 and calcineurin Aa, was increased in AR(∆ZF2) muscle, and the expression of all but p57(Kip2) was also decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle. We conclude that in males, androgens act via the AR in part to promote peak muscle mass by maintaining myoblasts in the proliferative state and delaying the transition to differentiation during muscle growth and development, and by suppressing ubiquitin ligase-mediated atrophy pathways to preserve muscle mass in adult muscle.

p57Kip2 is a downstream effector of BCR-ABL kinase inhibitors in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is characterized by the expression of BCR-ABL tyrosine kinase, which results in increased cell proliferation and inhibition of apoptosis. In this study, we show that BCR-ABL-positive CML cell lines treated with imatinib (STI571) undergo G1 cell cycle arrest associated with the accumulation of p57(Kip)², a cyclin-dependent kinase inhibitor (CKI). Interestingly, p57(Kip)² increase precedes the reported STI571-dependent upregulation of p27(Kip)¹. A number of complementary approaches allow the demonstration that p57(Kip)² buildup is due to the transcriptional activation of CDKN1C, the p57(Kip)²-encoding gene, while neither p57(Kip)² half-life elongation nor its cell relocalization were observed. We also identified a heretofore undescribed pattern of p57(Kip)² phosphorylated isoforms which, however, did not change in response to STI571 cell treatment. The imatinib-dependent p57(Kip)² upregulation occurs only in STI571-responsive cells, while the CKI accumulation was not evidenced in an imatinib-resistant clone. Nilotinib and dasatinib (second-generation BCR-ABL inhibitors), at concentrations comparable to those used in therapy, increase the CKI but do not affect p27(Kip)¹ level. Finally, CD34(+) cells from CML patients display a clear imatinib-dependent p57(Kip)² upregulation, which was not observed in CD34(+) cells from control subjects. In conclusion, our study points to p57(Kip)² as a novel and precocious effector of BCR-ABL targeting drugs.

Complex regulation of cell-cycle inhibitors by Fbxw7 in mouse embryonic fibroblasts.

The F-box protein Fbxw7 (also known as Fbw7, SEL-10, hCdc4 or hAgo) mediates the ubiquitylation and thereby contributes to the degradation of proteins that positively regulate cell cycle. Conditional ablation of Fbxw7 in mouse embryonic fibroblasts (MEFs) induces cell-cycle arrest accompanied by abnormal accumulation of the intracellular domain of Notch1 (NICD1) and c-Myc. However, the molecular mechanisms by which the accumulation of NICD1 and c-Myc induces cell-cycle arrest have remained unclear. We have now examined the expression of cell-cycle inhibitors in Fbxw7-deficient MEFs and found that the abundance of p27(Kip1) and p57(Kip2) is paradoxically decreased. This phenomenon appears to be attributable to the accumulation of NICD1, given that it was recapitulated by overexpression of NICD1 and blocked by ablation of RBP-J. Conversely, the expression of p16(Ink4a) and p19(ARF) was increased in an NICD1-independent manner in Fbxw7-null MEFs. The increased expression of p19(ARF) was recapitulated by overexpression of c-Myc and abolished by ablation of c-Myc, suggesting that the accumulation of c-Myc is primarily responsible for that of p19(ARF). In contrast, the upregulation of p16(Ink4a) appeared to be independent of c-Myc. These results indicate that cell-cycle inhibitors undergo complex regulation by the Fbxw7-mediated proteolytic system.

Transcriptional upregulation of p57 (Kip2) by the cyclin-dependent kinase inhibitor BMS-387032 is E2F dependent and serves as a negative feedback loop limiting cytotoxicity.

In spite of the fact that cyclin-dependent kinase (cdk) inhibiting drugs are potent transcriptional repressors, we discover that p57 (Kip2, CDKN1C) transcription is significantly upregulated by three small molecule cdk inhibitors, including BMS-387032. Treatment of MDA-MB-231 breast cancer cells with BMS-387032 led to a stabilization of the E2F1 protein that was accompanied by significant increases in the p57 mRNA and protein. This increase did not occur in an E2F1-deficient cell line. An E2F1-estrogen receptor fusion protein activated the endogenous p57 promoter in response to hydroxytamoxifen treatment in the presence of cycloheximide. Luciferase constructs driven by the p57 promoter verified that upregulation of p57 mRNA by BMS-387032 is transcriptional and dependent on E2F-binding sites in the promoter. Expression of exogenous p57 significantly decreased the fraction of cells in S phase. Furthermore, p57-deficient MDA-MB-231 cell lines were significantly more sensitive to BMS-387032-induced apoptosis than controls. The results presented in this manuscript demonstrate that small molecule cdk inhibitors transcriptionally activate p57 dependent upon E2F1 and that this activation in turn serves to limit E2F1's death-inducing activity.