p21^CIP1 Controls Proliferating Cell Nuclear Antigen Level in Adult Cardiomyocytes

Abstract

Cell cycle withdrawal associated with terminal differentiation is responsible for the incapability of many organs to regenerate after injury. Here, we employed a cell-free system to analyze the molecular mechanisms underlying cell cycle arrest in cardiomyocytes. In this assay, incubation of S phase nuclei mixed with cytoplasmic extract of S phase cells and adult primary cardiomyocytes results in a dramatic reduction of proliferating cell nuclear antigen (PCNA) protein levels. This effect was blocked by the proteasome inhibitors MG132 and lactacystin, whereas actinomycin D and cycloheximide had no effect. Immunodepletion and addback experiments revealed that the effect of cardiomyocyte extract on PCNA protein levels is maintained by p21 but not p27. In serum-stimulated cardiomyocytes PCNA expression was reconstituted, whereas the protein level of p21 but not that of p27 was reduced. Cytoplasmic extract of serum-stimulated cardiomyocytes did not influence the PCNA protein level in S phase nuclei. Moreover, the hypertrophic effect of serum stimulation was blocked by ectopic expression of p21 and the PCNA protein level was found to be upregulated in adult cardiomyocytes derived from p21 knockout mice. Our data provide evidence that p21 regulates the PCNA protein level in adult cardiomyocytes, which has implications for cardiomyocyte growth control.The idea of eliciting tissue repair by regenerative growth has incited concerted effort within the past decade aimed at the identification of mechanisms which maintain cell cycle arrest in terminally differentiated cells. Progression of eukaryotic cells through the cell cycle is controlled by the specific activation of a series of cyclin-dependent kinases (cdk's) (38). cdk's are known to phosphorylate tumor suppressor pocket proteins (Rb, p107, and p130), resulting in a release of E2F transcription factors and thus enabling the transcription of E2F-dependent genes required for S phase entry (15). One mechanism that downregulates the activity of cdk's leading to cell cycle arrest involves the binding of inhibitory proteins. Cyclin/cdk complexes are regulated by two families of cdk inhibitors (cki's), the INK4 family and the CIP/KIP family, including p21 and p27. INK4 proteins and CIP/KIP proteins are structurally distinct and interact with cyclins and cdk's in different ways. Whereas members of the INK4 family specifically inhibit cdk4 and cdk6, members of the CIP/KIP family are general inhibitors of cdk's involved in the G 1 /S transition and S phase (36, 49). Furthermore, p21 and p27 are involved in the differentiation of intestinal epithelial cells, keratinocytes, PC12 cells, glioma cells, and skeletal and cardiac muscle cells (53). The biochemical activities of cki's and their ability to promote differentiation implicate these proteins as mediators of cell cycle exit and differentiation. However, it is unclear why p21 and p27 are highly expressed in most differentiated cells, while cdk's as their main targets are downregulated.In a previous study we established a new my...

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