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IGF-1 Signaling Enhances Cell Survival in Periodontal Ligament Fibroblasts vs. Gingival Fibroblasts

Department of Periodontology & Oral Biology, Goldman School of Dental Medicine, Boston University, 700 Albany Street, W-201E, Boston, MA, 02118, USA;

View larger version (52K): [in a new window]   Figure 1. In situ detection of apoptosis. (A,B) Formaldehyde-fixed, paraffin-embedded healthy periodontal tissues of 4 molar teeth from 2 monkeys were analyzed by means of a TUNEL detection kit (Boehringer Mannheim). TUNEL-positive cells are arrowed. (C) The apoptotic cell percentage was obtained as the ratio of TUNEL-positive cells relative to the total number of counted fibroblasts. (D) The apoptotic cell density was expressed as TUNEL-positive cells per mm2. Mean ± SD (n = 12). **p < 0.01, t test, difference between PDLF and GF. Ce, cementum; PDL, periodontal ligament; AvB, alveolar bone; DG, deep gingiva.

View larger version (68K): [in a new window]   Figure 2. The role of IGF-1 signaling on PDLF vs. GF. Human PDLF and GF were cultured in six-well plates and were treated with IGF-1 and LY294002 as described in MATERIALS & METHODS. (A-D) We used flow cytometry to detect the annexin V-FITC-generated signal at 488 nm (FL1-H). The shadowed area represents fluorescent intensity distribution of PDLF cells. The area under the bold line represents fluorescent intensity distribution of GF cells. (E) The extent of DNA fragmentation was assessed by the diphenylamine (DPA) assay as described in MATERIALS & METHODS. (F) Cell lysate was analyzed by Western blot. Phosphorylated proteins were detected by the use of antibodies against phospho-PKB (Ser473) and phospho-Bad (Ser112). Antibodies against PKB or Bad were also used for detection of the total amount of PKB or Bad in the lysate. (G) We used an ELISA system (R&D Systems, Minneapolis, MN, USA) to determine the concentration of active caspase 3 by measuring the optical density at 450 nm. All bar graphs represent Mean ± SD, n = 4. *p < 0.05, t test, difference between PDLF and GF. D, serum-free DMEM. F, DMEM containing 10% fetal bovine serum. I, 10-8 M IGF-1. LY, 10-6 M LY294002.

View larger version (31K): [in a new window]   Figure 3. Expression of bcl-2 family genes between PDLF and GF. Human PDLF and GF were cultured, and apoptosis was induced by serum deprivation as described in MATERIALS & METHODS. The mRNA levels of bcl-2 family members were measured by RNase protection assay for anti-apoptotic genes (A,B) and pro-apoptotic genes (C,D). Radioactive signals were quantified with a PhosphorImager equipped with IMAGEQUANT software (Molecular Dynamics). A GAPDH probe was used as an internal control. Mean ± SD, n = 3. *P < 0.05, **P < 0.01, t test, difference between PDLF and GF.

View larger version (66K): [in a new window]   Figure 4. Up-regulation of IGFBP-5 in PDLF. (A) Human PDLF and GF were cultured in DMEM containing 10% FBS until sub-confluent. Total RNA (0.2 µg each) was extracted from cells of passages 4 and 7, and gene-specific primers (1 µM) were used for amplification of IGFBP-5 by RT-PCR. Human ß-actin control primers (0.2 µM) were used as internal standard. (B) The cell samples from passage 4 were analyzed by 10% SDS-PAGE with Western blotting and human IGFBP-5 antibody. (C,D) Sections of healthy periodontal tissues from Macaca mulatta monkeys were analyzed by immunohistochemistry as described in MATERIALS & METHODS. Cells immunoreactive for IGFBP-5 are indicated by arrows. Ce, cementum; PDL, periodontal ligament; AvB, alveolar bone; DG, deep gingiva.