Pleiotrophin expression during odontogenesis

Abstract

Pleiotrophin (PTN) is an extracellular matrix-associated growth factor and chemokine expressed in mesodermal and ectodermal cells. It plays an important role in osteoblast recruitment and differentiation. There is limited information currently available about PTN expression during odontoblast differentiation and tooth formation, and thus the authors aimed to establish the spatiotemporal expression pattern of PTN during mouse odontogenesis. Immortalized mouse dental pulp (MD10-D3, MD10-A11) and odontoblast-like (M06-G3) and ameloblast-like (EOE-3M) cell lines were grown and samples prepared for immunocytochemistry, Western blot, and conventional and quantitative PCR analysis. Effects of BMP2, BMP4, and BMP7 treatment on PTN expression in odontoblast-like M06-G3 cells were tested by quantitative PCR. Finally, immunohistochemistry of sectioned mice mandibles and maxillaries at developmental stages E16, E18, P1, P6, P10, and P28 was performed. The experiments showed that PTN, at both the mRNA and protein level, was expressed in all tested epithelial and mesenchymal dental cell lines and that the level of PTN mRNA was influenced differentially by the bone morphogenetic proteins. The authors observed initial expression of PTN in the inner enamel epithelium with prolonged expression in the ameloblasts and odontoblasts throughout their stages of maturation and strong expression in the terminally differentiated and enamel matrix-secreting ameloblasts and odontoblasts of the adult mouse incisors and molars.

Figure 1.

(A) (Top) Conventional RT-PCR using pleiotrophin (PTN) mouse primers and cDNA from M06-G3 (lane 2), MD10-A11 (lane 3), MD10-D3 (lane 4), and mouse brain cDNA (PTN positive control, lane 5). (Bottom) Positive control with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers. (B) Quantitative PCR (q-PCR) showing relative expression of PTN and another extracellular, secreted molecule, collagen A1 (C01A1), in M06-G3, MD10-A11, and MD10-D3 cells, normalized against GAPDH mRNA levels. Standard deviation was calculated with three experiments. (C) q-PCR showing relative mRNA levels normalized against GAPDH mRNA levels for M06-G3 cells treated for 14 days with 200 ng BMP2, BMP4, or BMP7 (control is non-treated M06-G3 cells). Standard error was calculated with a minimum of three experiments performed. (D) Western blot of cell lysates from M06-G3, MD10-D3, MD10-A11, and EOE-3M cells. Positive control is shown in the right lane; mouse brain extract (Santa Cruz Biotechnology). (E–H) Immunocytochemistry of cells grown in culture until 80% to 90% confluent and stained with an antibody for PTN (Abcam; ab14025). The boxed areas are enlarged in the top left of the pictures. (E) U87MG cells. (F) M06-G3 cells. (G) EOE-3M cells. (H) Negative control M06-G3 cells (10% BSA used instead of primary antibody). Scale bars = 50 µm.

Figure 2.

Immunofluorescence of mouse M06-G3, MD10-A11, and MD10-D3 cells stained with the pleiotrophin (PTN) antibody (Abcam; ab10849) at a 1:50 dilution. The secondary antibody was tagged with Alexa Fluor 388, followed by DAPI counterstaining. The same exposure time was used for all images. The negative control is with BSA instead of primary antibody, and only the M06-G3 control is shown as an example. Scale bars = 50 µm.

Figure 3.

Immunohistochemistry of mouse tissue sections at developing stages E16, E18, P1, P6, and P10. Pleiotrophin (PTN) antibody (Abcam; ab14025) dilutions were 1:50. Positive PTN controls for brain and bone as well as representative negative controls (using BSA instead of primary antibody) are shown. IEE, inner enamel epithelium; OEE, outer enamel epithelium; DPM, dental pulp mesenchyme; SR, stellate reticulum; MC, Meckel’s cartilage; M1 and M2, molar 1 and 2; Dp, dental pulp; Od, odontoblasts; Am, ameloblasts; Ob, osteoblasts; Oc, osteocytes. Scale bars = 50 µm.

Figure 4.

Immunohistochemistry of mouse tissue sections in adult mouse incisor (P28). Pleiotrophin (PTN) antibody (Abcam; ab14025) dilutions were 1:50. Positive PTN controls for brain and bone as well as representative negative controls (using BSA instead of primary antibody) are shown. P, pulp; En, enamel; Am, ameloblasts; Od, odontoblasts; D, dentin; DEJ, dentino-enamel junction; SI, stratum intermedium. Scale bars = 50 µm.

Figure 5.

Immunohistochemistry of mouse molar and root tissue sections in the adult mouse (P28). Pleiotrophin (PTN) antibody (Abcam; ab14025) dilutions were 1:50. Negative controls (using BSA instead of primary antibody) are shown. C, cementum; Od, odontoblasts; PDL, periodontal ligament. Scale bars = 50 µm.