Proliferative status regulates HDAC11 mRNA abundance in nontransformed fibroblasts.

Histone deacetylases (HDACs) are important determinants of gene transcription and other biological processes. HDAC11 is one of the least characterized HDACs and is the only member of the class IV HDAC family. Our studies examined the events that control the expression of the HDAC11 transcript. We show that platelet-derived growth factor (PDGF) rapidly reduces the abundance of HDAC11 mRNA when added to density-arrested Balb/c-3T3 cells, which are nontransformed fibroblasts. Reduction required mRNA and protein synthesis, but not AKT or ERK activity, and resulted from accelerated turnover of the HDAC11 transcript. Reduction was transient in cells receiving PDGF alone but sustained in cells receiving both PDGF and platelet-poor plasma, which together promote G0/G1 traverse and S phase entry. Plasma alone did not appreciably reduce HDAC11 mRNA abundance, nor did epidermal growth factor, insulin-like growth factor, or insulin. HDAC11 mRNA accumulated in Balb/c-3T3 cells exiting the cell cycle due to density-dependent growth inhibition or serum deprivation. Of note, HDAC11 mRNA did not accumulate in a spontaneously transformed Balb/c-3T3 clonal variant (clone 2) that does not density arrest. The HDAC11 promoter was active in Balb/c-3T3 but not clone 2 cells; inactivity in clone 2 cells did not result from methylation of CpG islands. Overexpression of HDAC11 inhibited the cell cycle progression of both transformed and nontransformed fibroblasts. Our studies identify the HDAC11 transcript as a PDGF target and show that HDAC11 mRNA abundance correlates inversely with proliferative status.

Inhibition of p27Kip1 gene transcription by mitogens.

How mitogens reduce the abundance of the cell cycle inhibitor p27(Kip1) is an important question, and regulation of p27(Kip1) translation and turnover has been described. Here we show that platelet-derived growth factor (PDGF) reduces the activity of the p27(Kip1) promoter and the abundance of the p27(Kip1) transcript in density-arrested mouse fibroblasts. Inhibition of p27(Kip1) gene expression by PDGF required protein synthesis and histone deacetylase activity but not Akt or ERK activity. PDGF increased the expression of c-Myc in the absence but not presence of a histone deacetylase inhibitor, and c-Myc inhibited p27(Kip1) promoter activity when ectopically expressed in fibroblasts. c-Myc targeted the same region of the p27(Kip1) promoter as did PDGF (deletion analysis) and interacted with this region in vivo (chromatin immunoprecipitation assay). Collectively, these findings suggest that c-Myc mediates the inhibitory effects of PDGF on the p27(Kip1) promoter. We also demonstrate reductions in p27(Kip1) mRNA abundance in primary splenocytes exposed to concanavalin A and in T cells exposed to interleukin-2 (IL-2). In contrast to PDGF in fibroblasts, IL-2 required Akt activity for maximal reductions in p27(Kip1) promoter activity and mRNA abundance in T cells. Thus, mitogens repress p27(Kip1) gene transcription in multiple systems and by multiple mechanisms.

Histone deacetylase 5 represses the transcription of cyclin D3.

Histone deacetylases (HDACs) modulate the transcription of a subset of genes by various means. HDAC5 is a class II HDAC whose subcellular location is signal-dependent. At present, its known gene targets are few in number. Here we identify a new HDAC5 target: the gene encoding the cell cycle-regulatory protein cyclin D3. When overexpressed in Balb/c-3T3 cells or mouse embryo fibroblasts, HDAC5 substantially reduced the activity of the cyclin D3 promoter and the abundance of endogenous cyclin D3 protein. Conversely, conditions that blocked HDAC5 function increased cyclin D3 expression: treatment of cells with the class I/II HDAC inhibitor trichostatin A (TSA), depletion of HDAC5 from cells by RNA interference, and cytoplasmic sequestration of HDAC5 by co-expression of catalytically active calcium/calmodulin-dependent protein kinase. HDAC5 interacted with the cyclin D3 promoter in vivo, and the HDAC5-responsive element was within 118 base pairs upstream of the transcription start site. Mutation of the Sp1 site and the cyclic AMP response element within this region did not affect the responsiveness of the cyclin D3 promoter to HDAC5 or TSA.

Altered expression of cell cycle regulatory proteins in benign and malignant bone and soft tissue neoplasms.

BACKGROUND: The binding of cyclins to cyclin-dependent kinases regulates cell proliferation. Overexpression of cyclins is believed to deregulate the cell cycle in human tumors. Here the expression of G1 cyclins D1 and D3, and of Ki-67 in a variety of bone and soft tissue sarcomas was assessed as compared to adjacent normal tissue and to a subset of leiomyomas. MATERIALS AND METHODS: Twenty-nine human bone and soft tissue sarcomas were evaluated. Tissue sections from each case were subjected to immunostaining for cyclin D1, cyclin D3 and Ki-67 using the avidin-biotin complex method. RESULTS: Cyclin D1 nuclear positivity was detected in 28% of sarcomas and in none of the leiomyomas. Cyclin D3 nuclear positivity was present in 62% of sarcomas and in none of the leiomyomas. Ki-67 nuclear staining was positive in 86% of sarcomas but in only 16% of leiomyomas. In addition, upregulation of cyclin D1 was observed in leiomyosarcomas, pleomorphic sarcomas and gastrointestinal stromal tumors, but not in liposarcomas or osteosarcomas. Cyclin D3, however, was expressed in all of the sarcoma types including 2 out of 5 liposarcomas and 1 out of 4 osteosarcomas. The normal soft tissue adjacent to the tumors when present (10 cases) was negative for cyclin D1 and D3, and expressed Ki-67 in 5% of the cell nuclei. The expression of cyclin D3 was also noted in human sarcoma cell lines (SKLMS, MG63, SaOS-2 and HT1080) by Western blot. CONCLUSION: The higher expression of cyclin D1 and D3 and of Ki-67 in bone and soft tissue sarcomas, as compared to leiomyomas and peritumoral normal soft tissue, suggests that high cyclin expression may contribute to deregulation of the cell cycle in bone and soft tissue tumors. These data suggest a role of cyclins in the process of human sarcomagenesis.

Cell cycle control: a complex issue.

Do p27Kip1 and p21Cip1 function as activators or inhibitors of D cyclin-cdk4 activity? Attempts to answer this question, and thus to understand how cdk4--a key cell cycle regulator--becomes active, have produced conflicting data. In this perspective, we summarize the results of studies addressing the effects of p27Kip1 and p21Cip1 on the assembly and activation of D cyclin-cdk4 complexes. Emphasis is placed on our experimental findings that support a model of cell cycle control in which p27Kip1 and p21Cip1 stabilize D cyclin-cdk4 complexes but inhibit D cyclin-cdk4 activity.

P27Kip1 and p21Cip1 are not required for the formation of active D cyclin-cdk4 complexes.

Our studies address questions pertaining to the regulation of D cyclin-cdk4 activity, and the following results were obtained. Conditions that increased the abundance of the D cyclins also increased the abundance of enzymatically active D cyclin-cdk4 complexes in mouse embryo fibroblasts (MEFs) lacking both p27(Kip1) and p21(Cip1) (p27/p21(-/-)). Such conditions included ectopic expression of cyclin D1 and inhibition of D cyclin degradation by the proteasome inhibitor MG132. However, as determined by treatment of wild-type MEFs with MG132, maximal accumulation of D cyclin-cdk4 complexes required p27(Kip1) and p21(Cip1) and coincided with the formation of inactive D cyclin-cdk4-p27(Kip1) or -p21(Cip1) complexes. p27(Kip1) or p21(Cip1) also increased the abundance of D cyclin-cdk4 complexes and reduced amounts of cdk4 activity when ectopically expressed in p27/p21(-/-) MEFs. Lastly, increases in the stability of the D cyclins accounted for their greater abundance in wild-type MEFs than in p27/p21(-/-) MEFs. We conclude that (i) D cyclin-cdk4 complexes are formed and become active in the absence of p27(Kip1) and p21(Cip1) and (ii) p27(Kip1) and p21(Cip1) maximize the accumulation but inhibit the activity of D cyclin-cdk4 complexes. We suggest that D cyclin-cdk4 complexes are more stable when bound to p27(Kip1) or p21(Cip1) and that formation of ternary complexes also stabilizes the D cyclins.