Reproducibility of proteomic profiles over 3 years in postmenopausal women not taking postmenopausal hormones.

Most proteomics studies examine one blood specimen per participant; however, it is unknown how well measures at one time point reflect an individual's long-term proteome pattern. Therefore, we examined the stability of the proteome over 3 years in postmenopausal women not taking hormones for at least 3 months using surface-enhanced laser desorption and ionization time-of-flight mass spectrometry. Using the Nurses' Health Study blood cohort, we randomly selected 60 women from a subset providing 2 to 3 blood samples over 3 years. Four different protein chip surfaces/plasma fractions were examined: unfractionated plasma on a CM10 and H50 chip, pH >/= 9, plasma fraction on a CM10 chip, and the organic fraction on the H50 chip, all with a low- and high-energy transfer protocol. Participant and quality control samples were aligned to a reference sample and then peak intensity was assessed for all peaks identified in the reference sample. The average coefficient of variation (CV) of the peak intensity within conditions ranged from 16% (H50, organic, low protocol) to 63% (CM10, pH > or = 9, high protocol). Generally, the CV and mean peak intensity of the quality control samples were inversely correlated (median -0.48). The mean intraclass correlation (ICC) within conditions ranged from 0.37 (H50, unfractionated, low protocol) to 0.68 (CM10, unfractionated, high protocol). For a signal-to-noise cutoff of 2.0, we observed 334 peaks, of which 241 (72%) had an ICC of > or =0.40. Although we observed a large range of CVs and ICCs, sufficient numbers of peaks had reasonable ICCs to suggest that protein peak reproducibility over 3 years was reasonable among postmenopausal women not taking hormones.

ESE-1 is a potent repressor of type II collagen gene (COL2A1) transcription in human chondrocytes.

The epithelium-specific ETS (ESE)-1 transcription factor is induced in chondrocytes by interleukin-1beta (IL-1beta). We reported previously that early activation of EGR-1 by IL-1beta results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE-1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF-kappaB-dependent inhibition by IL-1beta. Of the ETS factors tested, this response was specific to ESE-1, since ESE-3, which was also induced by IL-1beta, suppressed COL2A1 promoter activity only weakly. In contrast, overexpression of ETS-1 increased COL2A1 promoter activity and blocked the inhibition by IL-1beta. These responses to ESE-1 and ETS-1 were confirmed using siRNA-ESE1 and siRNA-ETS1. In transient cotransfections, the inhibitory responses to ESE-1 and IL-1beta colocalized in the -577/-132 bp promoter region, ESE-1 bound specifically to tandem ETS sites at -403/-381 bp, and IL-1-induced binding of ESE-1 to the COL2A1 promoter was confirmed in vivo by ChIP. Our results indicate that ESE-1 serves a potent repressor function by interacting with at least two sites in the COL2A1 promoter. However, the endogenous response may depend upon the balance of other ETS factors such as ETS-1, and other IL-1-induced factors, including EGR-1 at any given time. Intracellular ESE-1 staining in chondrocytes in cartilage from patients with osteoarthritis (OA), but not in normal cartilage, further suggests a fundamental role for ESE-1 in cartilage degeneration and suppression of repair.

The Ets transcription factor ESE-1 mediates induction of the COX-2 gene by LPS in monocytes.

Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins that are major inflammatory agents. COX-2 production is triggered by exposure to various cytokines and to bacterial endotoxins. We present here a novel role for the Ets transcription factor ESE-1 in regulating the COX-2 gene in response to endotoxin and other pro-inflammatory stimuli. We report that the induction of COX-2 expression by lipopolysaccharide (LPS) and pro-inflammatory cytokines correlates with ESE-1 induction in monocyte/macrophages. ESE-1, in turn, binds to several E26 transformation specific (Ets) sites on the COX-2 promoter. In vitro analysis demonstrates that ESE-1 binds to and activates the COX-2 promoter to levels comparable to LPS-mediated induction. Moreover, we provide results showing that the induction of COX-2 by LPS may require ESE-1, as the mutation of the Ets sites in the COX-2 promoter or overexpression of a dominant-negative form of ESE-1 inhibits LPS-mediated COX-2 induction. The effect of ESE-1 on the COX-2 promoter is further enhanced by cooperation with other transcription factors such as nuclear factor-kappa B and nuclear factor of activated T cells. Neutralization of COX-2 is the goal of many anti-inflammatory drugs. As an activator of COX-2 induction, ESE-1 may become a target for such therapeutics as well. Together with our previous reports of the role of ESE-1 as an inducer of nitric oxide synthase in endothelial cells and as a mediator of pro-inflammatory cytokines in vascular and connective tissue cells, these results establish ESE-1 as an important player in the regulation of inflammation.