We have previously shown that in vitro sensitivity to dexamethasone (DEX) stimulation in human endothelial cells is positively regulated by the glucocorticoid receptor (NR3C1, GR). The present study determined the role of differential GR transcriptional regulation in glucocorticoid sensitivity. We studied 25 human umbilical vein endothelial cells (HUVECs) that had been previously characterized as DEX-sensitive (n = 15), or resistant (n = 10). Real-time PCR analysis of GR 5′UTR mRNA isoforms showed that all HUVECs expressed isoforms 1B, 1C, 1D, 1F, and 1H, and isoforms 1B and 1C were predominantly expressed. DEX-resistant cells expressed higher basal levels of the 5′UTR mRNA isoforms 1C and 1D, but lower levels of the 5′UTR mRNA isoform 1F than DEX-sensitive cells. DEX treatment significantly decreased GRα and GR-1C mRNA isoform expression in DEX-resistant cells only. Reporter luciferase assays indicated that differential GR mRNA isoform expression was not due to differential promoter usage between DEX-sensitive and DEX-resistant cells. Analysis of promoter methylation, however, showed that DEX-sensitive cells have higher methylation levels of promoter 1D and lower methylation levels of promoter 1F than DEX-resistant cells. Treatment with 5-aza-2-deoxycytidine abolished the differential 5′UTR mRNA isoform expression between DEX-sensitive and DEX-resistant cells. Finally, both GRα overexpression and 5-aza-2-deoxycytidine treatment eliminated the differences between sensitivity groups to DEX-mediated downregulation of endothelial nitric oxide synthase (NOS3), and upregulation of plasminogen activator inhibitor 1 (SERPINE1). In sum, human endothelial GR 5′UTR mRNA expression is regulated by promoter methylation with DEX-sensitive and DEX-resistant cells having different GR promoter methylation patterns.

Calorie restriction (CR) has been known to produce many beneficial health effects, and lowered cell proliferation from CR has been shown to produce anti-cancer effects in some tissues. In this study the rate of epidermal cell proliferation in aging Fischer 344 rats from ad libitum fed (AL) and CR colonies was assessed in relation to changes in epidermal thickness with age. Proliferating cell nuclear antigen (PCNA) was detected using immunohistochemical method on paraffin sections in the epidermis of dorsal skin and footpad in these animals obtained from the National Institute on Aging. The proliferating cell index was compared with morphometric measurement of epidermis in young, young adult and old animals (six per group). Data were analyzed by Excel and SPSS 14.0 softwares for statistical evaluation. Two-way analysis of variance (ANOVA) was applied to data to test the effects of age, diet, and age-diet interaction. The following significant effects were noted: (I) age and age-diet effects in dorsal skin epidermal width, and PCNA; (II) age, and diet effect on footpad epidermal thickness, and PCNA index. There was a trend of increasing epidermal thickness in the dorsal skin in normally feeding aging rats which was depressed with CR in the two younger groups. PCNA index showed a trend of attrition from young to old. The thickness of epidermis in foot pad showed a curvilinear trend in both AL and CR groups with lowest mean values in the old group, and more predominant effect in CR-exposed animals. The proliferation index in the foot pad demonstrated a trend of reduction in old specimens with lower mean values in each corresponding CR age group. This report agrees with CR-inhibited cell proliferation reported in many organs by other investigators, and the observed results might have been caused by physiological or endocrine mechanisms affecting the epithelium in these calorie-restricted animals.

Problem To determine if epidermal cell proliferation in colony-raised Fischer 344 rats changes with age and diet. Methods Fischer 344 rats fed ad libitum and calorie-restricted (CR) diets were obtained from the NIA colonies, and young, young adult, and old animals from both groups were used for this study (six in each group). Tissue sections from the dorsal skin (DS) and foot plate (FP) were used for immunohistochemical staining of proliferating cell nuclear antigen (PCNA). The proliferation index (PCNA-I) was computed from counts of stained and total number of keratinocytes. Simultaneous measurements of epidermal thickness were obtained from same sections. Data were analyzed with Excel and SPSS 14.0 software for statistics. Results Two-way analysis of variance (ANOVA) was applied to the data to probe the effect of age, diet, and age-diet interaction. A significant effect of age was noticed in the two parameters i.e., DS PCNA (F 3.96, P .011), FP epidermal width (F 3.37, P .021) and FP PCNA-I (F 9.0, P .000). A significant correlation between DS width and PCNA values was also noted (r 0.5, P .01). Conclusion There is a trend of reduction of PCNA positive cells with increasing age irrespective of thickness of epidermis, and this trend is more apparent in CR rats. Significance This cell proliferation study has implications in relation to CR effect on age-related disease conditions, and biogerontology. Support The study was partially funded by the 2007 Leslie Bernstein grant from AAFPRS foundation.