INTRODUCTION: Porphyromonas gingivalis (P.g), a gram-negative anaerobe, is the major bacterium in the red complex (Socransky et al. 1998) and responsible for the onset and progression of severe periodontal disease. P. gingivalis is currently considered the ‘keystone’ pathogen of periodontal disease. It can produce several virulence factors, such as cysteine proteinases (gingipains), lipopolysaccharide (LPS), capsule and fimbriae. The LPS plays an important role in periodontal disease by inducing inflammation via stimulation of some cytokines such as TNF-ɑ. TNF-ɑ can activate expression of early response genes in macrophages, including Tumor Necrosis Factor-?-Induced Protein 1 (TNFAIP1). However, the role of TNFAIP1 in LPS-induced inflammation is largely unknown.
OBJECTIVE:
1. Identification of TNFAIP1 biological functions in response to LPS/TNF-ɑ;
2. Identification of the TNFAIP1 mediated signaling pathway;
3. Determination of factors involved in the TNFAIP-dependent signaling pathway;
4. Analysis of TNFAIP1 promoter activity.
MATERIALS AND METHODS: Mouse RAW cells, human THP-1 cells or MC3T3 cells were cultured in RPMI or ɑ-MEM media with 10% FBS at 37°C in 5% CO2. For DNA construction of TNFAIP1 cDNA or its promoter, DNAs were generated by polymerase chain reaction (PCR) with specific primers and templates. The cloned DNA sequences were confirmed by sequencing. Experiments to identify the biological function of TNFAIP1 and its promoter activity, utilized ELISA, DNA recovery, western blot, protein array, and promoter assay.
RESULTS:
1. LPS-induced the activation of p-MARK or p-PI3K (but not p-JAK), the production of TNF-ɑ, NFĸB or TNFAIP1 was confirmed by ELISA and western blot analysis;
2. Transfection of TNFAIP1 cDNA for 1-10 hours stimulated TNF-ɑ production in macrophage cells but not after longer exposure;
3. Caspase 1 and 3 were induced by TNFAIP1 after transfection of TNFAIP1 for 20 hours;
4. Overexpression of TNFAIP1 induced apoptotic proteins, such as Bcl-x, Caspase 3, Catalase, Claspin, Cytochromic, HO-1/HMOX1/HSP32, MCL-1, P27/Kip1, or SMAC/Diablo;
5. TNFAIP1 promoter DNA was cloned into pGL3 basic plasmid DNA to determine promoter activity. TNFAIP1 promoter activity was tested via its potential protein-protein interaction using luciferase gene expression. With a MAPK inhibitor, TNFAIP1 promoter activity was increased. In contrast, with an ATK inhibitor, TNFAIP1 promoter activity was reduced.
CONCLUSIONS:
1. TNFAIP1 is an important factor of the LPS/TNF-ɑ-dependent pathway;
2. MAPK or PI3K functions as an upstream factor of TNFAIP1, and LITAF is downstream factor of TNFAIP1-mediated signaling pathway in response to LPS;
3. Transfection of TNFAIP1 cDNA stimulated TNF-ɑ production for 1-10 hours exposure but reduced it for 10 - 20 hours exposure;
4. Overexpression of TNFAIP1 can increase expression of apoptotic proteins, Bcl-x, Caspase 3, Catalase, Claspin, Cytochromic, HO-1/HMOX1/HSP32, MCL-1, P27/Kip1, or SMAC/Diablo;
5. AKT and MAPK may act as transcriptional regulators of TNFAIP1 gene by binding to the promoter region. AKT upregulates TNFAIP1 gene expression and MAPK downregulates TNFAIP1 gene expression.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/49032 |
| Date | 20 June 2024 |
| Creators | Tangkham, Thanarut |
| Contributors | Tang, Xiaoren |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
Page generated in 0.0031 seconds