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NF-B Activation in Human Dental Pulp Stem Cells by TNF and LPS

J. Chang^1,, C. Zhang^2,, N. Tani-Ishii³, S. Shi⁴, and C.-Y. Wang^1,*

¹ Laboratory of Molecular Signaling and Apoptosis, Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, 1011 N. University Ave., Ann Arbor, MI 48109-1078, USA;
² School of Stomatology, Peking University Health Science Center, Beijing, China;
³ Division of Operative Dentistry and Endodontics, Department of Oral Medicine, Kanagawa Dental College, Kanagawa, Japan; and
⁴ Section of Oral Biology, Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA;

View larger version (62K): [in a new window]   Figure 1. TNF or LPS activates IKK. (A) DPSCs were treated with TNF (10 ng/mL) and P. gingivalis LPS (1 µg/mL) for the indicated times. Fifty-µg aliquots of protein extracts were probed with polyclonal antibodies against IB (1:1000) or monoclonal antibodies against phospho-specific antibodies (1:1000). For loading control, the blots were stripped and re-examined with monoclonal antibodies against -tubulin (1:15, 000). (B) E. coli LPS activates IKK. DPSCs were treated with E. coli LPS (200 ng/mL) or TNF for the indicated time points. Western blot analysis was performed as described in (A).

View larger version (58K): [in a new window]   Figure 2. TNF or LPS induces the nuclear translocation of NF-B. (A) DPSCs were treated with TNF (10 ng/mL) for the indicated time. Five-µg aliquots of nuclear proteins were incubated with 32P-labeled NF-B oligonucleotide probes. For the supershift assay, nuclear proteins were pre-incubated with polyclonal antibodies against p65 for 10 min and then probed with32P-labeled NF-B oligonucleotide probes. (B) DPSCs were treated with P. gingivalis LPS (1 µg/mL) for the indicated times. EMSA was performed as described in (A).

View larger version (69K): [in a new window]   Figure 3. LPS induces IL-8 expression in DPSCs. (A) DPSCs were treated with TNF or P. gingivalis LPS for the indicated times. Fifty-µg aliquots of protein extracts were probed with polyclonal antibodies against phospho-specific p65. For loading control, the membrane was stripped and re-probed with polyclonal antibodies against p65. (B) DPSCs were treated with TNF for the indicated times. Ten-µg aliquots of total RNAs were hybridized with 32P-labeled IL-8 cDNA probes. For loading control, the blot was stripped and re-probed with 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes. (C) DPSCs were treated with P. gingivalis LPS for the indicated times. Northern blot analysis was performed as described in (B). (D) DPSCs were treated with TNF for the indicated times. Total RNAs were probed with 32P-labeled IL-6 cDNA probes. (E) DPSCs were treated with E. coli LPS for the indicated times. Total RNAs were probed with 32P-labeded IL-6 cDNA probes.

View larger version (68K): [in a new window]   Figure 4. P. endodontalis LPS activates IKK. (A) Cells were treated with P. endodontalis LPS (1 µg/mL) for the indicated times. Western blot analysis was performed as described in Fig. 1. (B) Western blot was performed as described in Fig. 3A.