Correlations between different protein species of oral rinse MMP-8 and patient-related factors.

OBJECTIVES: The aim of this study is to examine correlations between different oral rinse matrix metalloproteinase (MMP)-8 protein species in western blot (WB) analysis, quantitative MMP-8 measurements, and patient-related factors. Elevated activated MMP-8 (aMMP-8) associate with periodontitis and a diagnostic point-of-care technology has been developed based on aMMP-8. In WB, different MMP-8 protein species can be analyzed. Relative abundancy of fragmented 20-25 kDa forms in WB has been associated with and reflects MMP-8 activation and related fragmentation and elevated quantitative aMMP-8 measurements. MATERIAL AND METHODS: A random sample of 192 participants from a periodontal disease screening study was used for this study. Oral rinse samples for biomarker analyses were collected before clinical periodontal examinations. aMMP-8 immunofluorometric (IFMA) and WB analysis (utilizing the same monoclonal antibody, 8708), polymorphonuclear leukocyte (PMN) elastase activity test and tissue inhibitor of metalloproteinases (TIMP)-1 ELISA levels were performed from the oral rinse samples. Distinct MMP-8 protein species were differentiated in the WB analysis. Principal component (PC) analysis was conducted to explore correlation patterns between the different species. Adjusted correlation analysis between the extracted PCs of WB and aMMP-8 IFMA levels and multilevel regression analysis were conducted to explore if the other periodontal disease-related biomarkers and clinical surrogate measures and patient-related factors are co-variating with the extracted components. RESULTS: Distinct correlation patterns between the MMP-8 protein species were observed. The first four PCs explained 89% of the whole variance in PC analysis. Statistically significant correlation (p < 0.05) were observed as follows: PC1 positively with 21 kDa (r = .69) and 25 kDa fragments (r = .55) and negatively with 150 kDa complexes (r = -.46). PC2 correlated with 45 (r = .70) and 55 kDa (r = .65) activated forms, PC3 with 70-80 kDa latent proforms (r = .63) and 90-100 kDa complexes (r = .67), and PC4 with 35 kDa fragments (r = .81). There were significant correlations between quantitative (IFMA) aMMP-8 measurements and PC1 (p < 0.001), PC2 (<0.05) and PC3 (<0.05) but not with PC4. In multilevel regression models age, PMN elastase activity, TIMP-1 levels, and a number of 4-5 mm periodontal pockets were associated with PC1, nonsmoking with PC2, age and PMN elastase activity with PC3, and age and smoking with PC4. CONCLUSIONS: Relative abundancy of fragmented 21-25 kDa protein species was correlated with the quantitative aMMP-8 (IFMA) measurements, which is in line with previous results. Different patient-related factors (smoking, age, proteolytic activity) may modify the formation of different MMP-8 protein species in oral rinse samples and may cause variability in quantitative aMMP-8 measurement.

The DNA binding domain of estrogen receptor alpha is required for high-affinity nuclear interaction induced by estradiol.

Estrogen receptor alpha (ER) is a member of the nuclear hormone receptor family, which upon binding estrogen shows increased apparent affinity for nuclear components (tight nuclear binding). The nuclear components that mediate this tight nuclear binding have been proposed to include both ER-DNA interactions and ER-protein interactions. In this paper, we demonstrate that tight nuclear binding of ER upon estrogen occupation requires ER-DNA interactions. Hormone-bound ER can be extracted from the nucleus in low-salt buffer using various polyanions, which mimic the phosphate backbone of DNA. The importance of specific ER-DNA interactions in mediating tight nuclear binding is also supported by the 380-fold lower concentration of the ERE oligonucleotide necessary to extract estrogen-occupied ER from the nucleus compared to the polyanions. We also demonstrate that estrogen-induced tight nuclear binding requires both the nuclear localization domain and the DNA binding domain of ER. Finally, enzymatic degradation of nuclear DNA allows us to recover 45% of tight nuclear-bound ER. We further demonstrate that ER-AIB1 interaction is not required for estrogen-induced tight nuclear binding. Taken together, we propose a model in which tight nuclear binding of the estrogen-occupied ER is predominantly mediated by ER-DNA interactions. The effects of estrogen binding on altering DNA binding in whole cells are proposed to occur through estrogen-induced changes in ER-chaperone protein interactions, which alter the DNA accessibility of ER but do not directly change the affinity of the ER for DNA, which is similar for both unoccupied and occupied ER.