p21(Waf1/Cip1/Sdi1) mediates retinoblastoma protein degradation.

Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21(Waf1) (CDKN1A). Inhibition of cyclin-dependent kinase (CDK)2 and CDK4/6 by p21 leads to dephosphorylation and activation of Rb. We now show that ectopic p21 expression in human HT1080 fibrosarcoma cells causes not only dephosphorylation but also depletion of Rb; this effect was p53-independent and susceptible to a proteasome inhibitor. CDK inhibitor p27 (CDKN1B) also caused Rb dephosphorylation and depletion, but another CDK inhibitor p16 (CDKN2A) induced only dephosphorylation but not depletion of Rb. Rb depletion was observed in both HT1080 and HCT116 colon carcinoma cells, where p21 was induced by DNA-damaging agents. Rb depletion after DNA damage did not occur in the absence of p21, and it was reduced when p21 induction was inhibited by p21-targeting short hairpin RNA or by a transdominant inhibitor of p53. These results indicate that p21 both activates Rb through dephosphorylation and inactivates it through degradation, suggesting negative feedback regulation of damage-induced cell-cycle checkpoint arrest.

Age-related alterations in the inflammatory response to dermal injury.

Previous studies have documented that the ability to heal wounds declines with age. Although many factors contribute to this age-associated deficit, one variable that has not been carefully examined is leukocyte recruitment and function in wounds. This investigation compares the inflammatory response in excisional wounds of young (age 8 wk) and aged (age 22 mo) mice. In the early inflammatory response, neutrophil content of wounds was similar for both aged and young mice. In contrast, macrophage levels were 56% higher in aged versus young mice (81 +/- 20 vs 52 +/- 13 cells per mm2). In the later inflammatory response, wounds of aged mice exhibited a delay in T cell infiltration, with maximum T cell levels at day 10 in aged mice versus day 7 in young mice. Despite this delay, the eventual peak concentration of T cells was 23% higher in the wounds of aged mice (152 +/- 11 cells per mm2 vs 124 +/- 21cells per mm2). The observed alterations in inflammatory cell content suggested that chemokine production might be altered with age. An elevation of monocyte chemoattractant protein (MCP-1) levels was observed in wounds of aged mice. RNase protection studies, however, revealed that the production of most chemokines, including MIP-2, MIP-1alpha, MIP-1beta, and eotaxin, tended to decline with age. Because optimal wound healing requires both appropriate macrophage infiltration and phagocytic activity, phagocytosis was examined. Compared to young mice, wound macrophages from aged mice exhibited a 37%-43% reduction in phagocytic capacity. Taken together, the data demonstrate age-related shifts in both macrophage and T cell infiltration into wounds, alterations in chemokine content, and a concurrent decline in wound macrophage phagocytic function. These alterations may contribute to the delayed repair response of aging.

Impaired wound repair and delayed angiogenesis in aged mice.

Wound repair is a multistep process consisting of hemostasis, inflammatory cell infiltration, tissue regrowth, and remodeling. In aged individuals, this progression of events is altered, resulting in wounds that heal more slowly than wounds in the young. These studies were designed to examine the proliferative phase of repair in young and aged mice, with attention to the angiogenic process. Using a standardized excisional injury model, wound re-epithelialization, collagen accumulation, and angiogenesis were examined. Re-epithelialization and collagen synthesis were substantially delayed in aged mice as compared with young mice. Angiogenesis in wounds from aged mice was also delayed, with significantly more capillary growth in wounds from young mice than aged mice. In addition, wounds from aged mice contained significantly less of the angiogenic mediators fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) than wounds from young animals (p < 0.05). Because macrophages are a rich source of angiogenic factors in wounds, macrophage production of VEGF was examined. Macrophages from aged mice produced significantly less VEGF than cells from young mice. To examine the in vivo endothelial cell responsiveness, a defined amount of rFGF-2 was suspended in Matrigel and placed subcutaneously in either young or aged mice. In response to FGF-2, capillary growth into Matrigel was significantly less in aged than young mice. The results suggest that a decline in angiogenic growth factor production, as well as a decline in endothelial responsiveness to specific factors, may account for the delayed wound angiogenesis in aged mice. These results also indicate that age-related alterations in macrophage function might partially account for the overall delay in the wound repair process.