Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells

Abstract

Background: CCN2/CTGF is known to be involved in tooth germ development and periodontal tissue remodeling, as well as in mesenchymal tissue development and regeneration. In this present study, we investigated the roles of CCN2/CTGF in the proliferation and differentiation of periodontal ligament cells (murine periodontal ligament-derived cell line: MPL) in vitro.

Results: In cell cultures of MPL, the mRNA expression of the CCN2/CTGF gene was stronger in sparse cultures than in confluent ones and was significantly enhanced by TGF-beta. The addition of recombinant CCN2/CTGF (rCCN2) to MPL cultures stimulated DNA synthesis and cell growth in a dose-dependent manner. Moreover, rCCN2 addition also enhanced the mRNA expression of alkaline phosphatase (ALPase), type I collagen, and periostin, the latter of which is considered to be a specific marker of the periosteum and periodontium; whereas it showed little effect on the mRNA expression of typical osteoblastic markers, e.g., osteopontin and osteocalcin. Finally, rCCN2/CTGF also stimulated ALPase activity and collagen synthesis.

Conclusion: These results taken together suggest important roles of CCN2/CTGF in the development and regeneration of periodontal tissue including the periodontal ligament.

Figure 1

Expression of CCN2/CTGF mRNA in sparse and confluent MPL cultures (in situ hybridization) with or without TGF-β stimulation. MPL cultures in the sparsity phase (A and B) and confluence phase (C and D) were treated with TGF-β (B and D) or PBS (control: A and C). The cells were then cultured for 12 hrs, fixed, and hybridized with an antisense riboprobe for ccn2/ctgf mRNA. CCN2/CTGF mRNA was distinctly expressed in the cells in the sparse (A), but not in those in the confluent (C) state. However, the addition of TGF-β enhanced the level of CCN2/CTGF mRNA strongly, not only in the sparse cultures (B), but also in the confluent ones (D).

Figure 2

RT-PCR analysis of the expression of CCN2/CTGF mRNA in sparse (S) and confluent (C) MPL cultures. Total RNA was purified from MPL cultures under sparse and confluent conditions, and 1 μg in each sample was reverse transcribed and used for PCR. The expression of CCN2/CTGF mRNA was stronger under the sparse than the confluent condition as evaluated in the exponential phase of the amplification (25 cycles). Levels of GAPDH mRNA, used as an internal control, were almost the same among these samples.

Figure 3

(A) Effect of rCCN2/CTGF on DNA synthesis in MPL cells. MPL cells were inoculated at a density of 5 × 10³/well in 96-multi-well plates and cultured in α-MEM with 10% FBS for 2 days. Then, the culture medium was changed to α-MEM with 0.5% FBS, and the cells were incubated with various concentrations of rCCN2/CTGF for 24 hrs and labeled with [³H] thymidine for the last 4 hrs. The incorporated radioactivity was determined by a liquid scintillation counter. Values represent the averages ± SD. Data were computed with the results of 2 independent series of experiments with multiple sample numbers. Asterisks denote statistically significant difference from the vehicle-treated control. (B) Effect of rCCN2/CTGF on the proliferation of MPL cells. MPL cells were inoculated at a density of 2 × 10³/well in 96-multi-well plates and cultured in α-MEM with 10% FBS for 24 hrs. Then, the medium was changed to α-MEM with 1% FBS, and the cells were cultured with various concentrations of rCCN2/CTGF for 3 days. The cell number was computed by using Tetra-Color One as specified by the manufacturer. Points and bars represent the averages and SD, respectively. Asterisks indicate significant differences between vehicle-treated control and rCCN2/CTGF-treated samples at the significance level of **p < 0.01 or *p < 0.05.

Figure 4

Effects of rCCN2/CTGF on mRNA expression of osteoblast and PDL-related markers in MPL cells. Confluent cultures of MPL cells were treated with rCCN2/CTGF (100 ng/ml, closed bars) or vehicle (dotted bars) for 24 hrs. Total RNA was purified, and 1 μg of each sample was reverse-transcribed and used for PCR. Alkaline phosphatase (ALPase), type I collagen, and periostin mRNAs were shown to be up-regulated clearly by the addition of rCCN2/CTGF under exponential conditions (25, 30, and 25 cycles, respectively) for amplification. The expression of GAPDH, used for an internal control, was almost the same among these samples.

Figure 5

Effects of rCCN2/CTGF on ALPase activity from MPL cells. When MPL cells reached confluence, the culture medium was replaced with α-MEM containing 1% FBS, and the cells were then cultured with various concentrations of rCCN2/CTGF. After 48 hrs, the cell layers were collected, and ALPase activity was determined as described in "Materials and Methods." Values represent the averages ± SD of 3 separate experiments. Asterisks denote statistically significant differences from the vehicle-treated control at the significance level of *p < 0.01.

Figure 6

Effect of rCCN2/CTGF on the synthesis of collagen and total protein in MPL cells. Various concentrations of rCCN2/CTGF were added to confluent cultures of MPL cells, and the cells were cultured for 12 hrs. Then, the cells were labeled with [³H] proline for another 12 hrs, after which their cell layers were collected for analysis. The radioactivities of [³H] proline incorporated into total nascent proteins and collagenase-digestable portions were measured as described in "Materials and Methods." Slashed and closed boxes indicate the collagen and total protein synthesis, respectively. Values represent the mean average ± SD (n = 2). Asterisks denote statistically significant differences (**p < 0.01, *p < 0.05) from the vehicle-treated control, except the double asterisks specifying a significant difference between the stimulation levels of total protein and collagen synthesis (as indicated by the bracket). Data were computed with the results of 2 independent series of experiments with multiple sample numbers.