Functional importance of estrogen receptors in the periodontium.

The main functions of estrogen are associated with reproduction. However, estrogen has been shown to be of functional importance also in non-classic target organs. Previous studies, especially epidemiologic and clinical ones, have addressed estrogen's influence on periodontitis, suggesting that estrogen has a beneficial effect, but the biological mechanisms have not been identified. Estrogen exerts genomic effects in the target cells by binding to the nuclear receptors, estrogen receptor (ERs), ERalpha and ERbeta. The expression of the two subtypes of ERs varies depending on the tissue. The overall objectives of this thesis were to study the functional importance of estrogen receptors in the periodontium with special focus on inflammation, and stimulators of inflammation and their signaling pathways. The thesis is based on the following five papers. In Paper I, effects of estrogen on E. coli LPS-induced PDL cell production of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and C-reactive protein (CRP) are assessed, by using ELISA. Furthermore, effects of LPS and estrogen on the normal characteristics of the PDL cell such as collagen synthesis and cell proliferation is determined by using L-[3H]proline incorporation and measurement of DNA synthesis, respectively. Key findings: E. coli LPS stimulates PDL cell IL-6 and MCP-1 production but has no effect on the normal physiological properties of PDL cells. LPS-induced IL-6 and MCP-1 is not reversed by estrogen suggesting that estrogen has no anti-inflammatory effect in these experiments. In Paper II, we investigate the effects of ovariectomy and aging on tooth attachment in female mice by using morphometric analysis. Key findings: Withdrawal of female sex hormone production by ovariectomy has no effect on alveolar bone height and apical termination of the junctional epithelium. In a second series of experiments these parameters are similar in mice sacrificed at 8-26 weeks of age, suggesting that tooth attachment is preserved with age in mice within a period of six months. In Paper III, the objective is to investigate the regulation of CCL2/MCP-1, CCL3/MIP-1alpha, and CCL5/RANTES chemokines by estrogen in human PDL cells by determining mRNA transcript levels (using quantitative real-time PCR) and protein levels (using ELISA). Key findings: A physiological concentration of estrogen reduces the expression of CCL3 mRNA by about 40% compared to PDL cells treated with LPS alone. In contrast, inter-individual differences in the effects of estrogen on CCL5 mRNA expression are observed. These findings indicate that estrogen affects chemokine expression in PDL cells showing a complex pattern involving down-regulation as well as up-regulation of chemokines. Estrogen exerts both anti-inflammatory and pro-inflammatory effects through these mechanisms. In Paper IV, ER expression in human gingival biopsies, and effects of estrogen on cultured gingival epithelial cell (HGEP) proliferation, are investigated. Expression of ERalpha and ERbeta is determined by immunohistochemistry and effects of estrogen on HGEP proliferation monitored by measuring DNA synthesis. Key findings: HGEP cells show strong ERbeta immunoreactivity but low ERalpha immunoreactivity both in vivo and in culture, suggesting that ERbeta is the predominant ER subtype in HGEP. High, but not low, concentrations of estrogen attenuates proliferation of gingival epithelial cells, indicating a concentration-dependent mechanism. In Paper V, the objective is to investigate the effects of LPS from Escherichia coli and Porphyromonas gingivalis on IL-6 production in human PDL cells and endothelial cells, and the signaling mechanisms involved. Quantitative real-time PCR is used to determine IL-6 mRNA levels and ELISA to determine IL-6 protein. Key findings: E. coli LPS (but not P. gingivalis LPS) stimulates IL-6 production in PDL cells. Treatment with the non-selective nitric oxide synthase inhibitor L-NAME reduces IL-6 by 30%, while aminoguanidine, an inhibitor of inducible nitric oxide synthase, does not affect IL-6 levels, showing a mechanism probably involving nitric oxide formation via endothelial nitric oxide synthase. Treatment with the glucocorticoid steroid dexamethasone totally prevents-E. coli LPS-induced IL-6 in PDL cells