Inhibition of autoantigen expression by (-)-epigallocatechin-3-gallate (the major constituent of green tea) in normal human cells.

Autoimmune disorders, characterized by inflammation and apoptosis of target cells leading to tissue destruction, are mediated in part by autoantibodies against normal cellular components (autoantigens) that may be overexpressed. For example, antibodies against the autoantigens SS-A/Ro and SS-B/La are primary markers for systemic lupus erythematosus and Sjögren's syndrome. Recently, studies in animals demonstrated that green tea consumption may reduce the severity of some autoimmune disorders, but the mechanism is unclear. Herein, we sought to determine whether the most abundant green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), affects autoantigen expression in human cells. Cultures of pooled normal human primary epidermal keratinocytes and of an immortalized human salivary acinar cell line were incubated with 100 microM EGCG (a physiologically achievable level for topical application or oral administration) for various time periods and then analyzed by cDNA microarray analysis, reverse transcription-polymerase chain reaction, and Western blotting for expression of several major autoantigen candidates. EGCG inhibited the transcription and translation of major autoantigens, including SS-B/La, SS-A/Ro, coilin, DNA topoisomerase I, and alpha-fodrin. These findings, taken together with green tea's anti-inflammatory and antiapoptotic effects, suggest that green tea polyphenols could serve as an important component in novel approaches to combat autoimmune disorders in humans.

Analysis of interleukin-activated human gingival fibroblasts: modulation of chemokine responses by female hormones.

BACKGROUND: The female sex hormones are known to affect the response of numerous tissues to an immune challenge. Because such hormones normally fluctuate during puberty, pregnancy, and the menstrual cycle, more information about the hormonal modulation of such responses in the oral cavity is needed. Gingival fibroblasts (GF), major components of the oral tissues, are potentially sources for inflammatory mediators. METHODS: Macroarrays specific for cytokines and related proteins were used to examine the regulation of gene expression in GF under serum-free, resting conditions, after immune challenge with interleukin-1beta (IL-1beta), and in the presence of IL-1beta plus a progestin, +/-17beta-estradiol. Additional studies used enzymelinked immunosorbent assays (ELISAs) to test for secreted chemokines after the same treatments. RESULTS: Of the 392 genes on the macroarray, 66 were up- or downregulated at least 2-fold relative to the unstimulated control in an average of six different sub-lines. Chemokines represented the largest group (18%) of these regulated genes. Numerous genes whose expression was upregulated by IL-1beta were modulated downward by IL-1beta plus progestin, +/-17beta-estradiol. Measurements of the secretion of IL-8, a CXC chemokine, and MCP-1, a CC chemokine, confirmed the inhibitory effect of a progestin on these genes. CONCLUSIONS: Gingival fibroblasts are active participants in the immune response in the oral cavity, and may potentially produce many chemokine signals after exposure to IL-1beta. GF can attract neutrophils, monocytes, eosinophils, and fibroblasts to the area of injury, and aid in the wound repair process. The concentration of female sex hormones, especially progestin, may significantly affect these signaling systems.

Role of p21WAF1 in green tea polyphenol-induced growth arrest and apoptosis of oral carcinoma cells.

The cyclin-dependent kinase inhibitor p21WAF1 participates in cell growth, differentiation, and apoptosis. p21WAF1 can be induced by green tea polyphenol EGCG in several cancer cell types, but its role in the oral cancer cell response to EGCG is not known. We found that EGCG upregulates p21WAF1 in an oral carcinoma cell line, OSC2, by cDNA microarray. The current study determined the impact of siRNA-suppressed p21WAF1 and its response to EGCG on cell growth, DNA synthesis and apoptosis by RT-PCR, Western blot, BrdU incorporation, MTT and caspase 3 activity assays. Suppression of p21WAF1 by siRNA resulted in an accelerated cell growth and DNA synthesis, and increased cell viability. However, caspase 3 activity was not significantly inhibited. The evidence showed that p21WAF1 is involved in EGCG-induced growth arrest of OSC2 cells, which may facilitate caspase 3-mediated apoptosis. Thus, expression of functional p21WAF1 may promote phytochemical-mediated growth arrest and apoptosis in oral carcinoma cells.

Green tea polyphenol-induced epidermal keratinocyte differentiation is associated with coordinated expression of p57/KIP2 and caspase 14.

Epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea, exerts chemopreventive effects by selectively inducing apoptosis in tumor cells. In contrast, EGCG accelerates terminal differentiation in normal human epidermal keratinocytes (NHEK) mediated partially by up-regulation of p57/KIP2, a cyclin-dependent kinase inhibitor that confers growth arrest and differentiation. However, it is unclear if EGCG modulates caspase 14, a unique regulator of epithelial cell terminal differentiation associated with cornification. Here, we examined the effect of EGCG on caspase 14 expression in NHEK and correlated the protein and mRNA expression of p57/KIP2 with those of caspase 14 in either normal keratinocytes or p57/KIP2-expressing tumor cells (OSC2, an oral squamous cell carcinoma cell line). Additionally, paraffin-embedded normal and untreated psoriatic (aberrant keratinization) skin sections from humans were assessed for caspase 14 by immunohistochemistry. In NHEK, EGCG induced the expression of caspase 14 mRNA and protein levels within a 24-h period. The expression of p57/KIP2 in OSC2 cells was adequate to induce caspase 14 in the absence of EGCG; this induction of caspase 14 was down-regulated by transforming growth factor-beta1. In human psoriatic skin samples, caspase 14 staining in the upper epidermis was reduced, especially in nuclear areas. These results suggest that, in addition to p57/KIP2, EGCG-induced terminal differentiation of epidermal keratinocytes involves up-regulation of caspase 14. Further understanding of how EGCG modulates cellular differentiation may be useful in developing green tea preparations for selected clinical applications.

The effects of progesterone on matrix metalloproteinases in cultured human gingival fibroblasts.

BACKGROUND: Although pregnancy gingivitis is widely believed to result from elevated hormone concentrations, the mechanism(s) involved in the etiology of this condition remain unknown. Paradoxically, despite the apparent inflammation for a prolonged period, pregnancy gingivitis rarely progresses to periodontitis and usually resolves postpartum. We used several methods to test in vitro the hypothesis that the elevated progesterone levels of pregnancy might inhibit the production of some of the matrix metalloproteinases (MMPs) that are responsible for periodontal destruction. METHODS: Cultured human gingival fibroblasts (GF) were tested in phenol red-free, serum-free medium with or without the progestogen, medroxyprogesterone acetate (MPA; 10(-6) M), using interleukin-1beta (IL-1beta) to initiate immune responses and MMP production. These MMP responses were examined by macroarrays, reverse transcription-polymerase chain reaction (RT-PCR), zymograms, and enzyme-linked immunosorbent assay (ELISA). RESULTS: Array analysis showed that pretreatment of GF with MPA reduced mRNA induction for MMPs-1, -3, and -10 in response to 6 to 8 hours incubation with IL-1beta. RT-PCR confirmed, that after 24 hours with IL-1beta , GF pretreated with MPA had undetectable levels of mRNA for MMPs-1, -2, -3, -7, -10, and -13. Zymograms of culture media from this 24-hour period showed reduction in several proteolytic activities. Examination of such 24-hour media using ELISA for MMP-3 and pro-MMP-13 confirmed that secretion of these enzymes was upregulated by IL-1beta and modulated downward by pretreatment with MPA. CONCLUSIONS: Production by GF of numerous MMPs in response to IL-1beta was significantly reduced by progesterone. This steroidal modulation of proteolytic enzymes could help to explain why pregnancy gingivitis typically is not characterized by progression to periodontitis.