Expression and function of NUMB in odontogenesis

Abstract

NUMB is a multifunctional protein implicated to function in self-renewal and differentiation of progenitors in several tissues. To characterize the transcripts and to analyze the expression pattern of NUMB in odontogenesis, we isolated 2 full-length clones for NUMB from mouse dental pulp mRNA. One novel sequence contained 200 bp insertion in the phosphotyrosine binding domain (PTB). Confocal microscopy analysis showed strong NUMB expression in human dental pulp stem cells (hDPSC) and preameloblasts. Western blot analysis indicated that NUMB isoforms were differentially expressed in various dental tissues. Immunohistochemical analysis showed that in postnatal mouse tooth germs, NUMB was differentially expressed in the preameloblasts, odontoblasts, cervical loop region, and in the dental pulp stem cells during development. Interestingly, overexpression of NUMB in HAT-7, a preameloblast cell line, had dramatic antagonizing effects on the protein expression level of activated Notch 1. Further analysis of the Notch signaling pathway showed that NUMB significantly downregulates sonic hedgehog (Shh) expression in preameloblasts. Therefore, we propose that NUMB maintains ameloblast progenitor phenotype at the cervical loop by downregulating the activated Notch1 protein and thereby inhibiting the mRNA expression of Shh.

Figure 1

(a) RT-PCR analysis of NUMB isoforms in day 7 dental pulp cells. RT-PCR analysis of mRNA extracted from day 7 dental pulp culture shows two PCR bands corresponding to 2 kb and 1.8 kb obtained using primers designed to amplify the longest NUMB reading frame. (b) The alignment of the sequence obtained from 1.8 kb and the 2 kb PCR fragments. The 2 kb PCR fragment contains 236 bp insertion in the phosphotyrosine binding domain (PTB) 201 bp downstream of the initiation start site.

Figure 2

Localization of NUMB in various odontogenic cells: immunocytochemistry staining of NUMB expression in T4-4 odontoblast cells (a), human dental pulp stem cell (b), and preameloblast cells (c). Membrane patches isolated from preameloblast HAT-7 cells stained using NUMB antibody (d).

Figure 3

Western blot analysis of NUMB isoforms in odontogenic cells: total protein lysates were obtained from odontoblasts (T4-4 cells); dental pulp stem cells (DPCs); preameloblasts (HAT-7); tooth germs isolated from postnatal day 4 molars (P4TG). Notice that NUMB isoforms are differentially expressed in these tissues.

Figure 4

Localization of NUMB in odontogenic tissues at postnatal day 3: localization of NUMB in P3 incisors (B) and molars (C and D). NUMB can be detected in the ameloblasts (Am); odontoblasts (Od); ameloblast progenitors (cervical loop); in the dental pulp cells (P) in close vicinity to the odontoblasts. (D1) shows the immature ameloblasts, immature odontoblasts, and dental pulp cells from incisor and molar, respectively. (a) is the negative control. Scale bar represents 100 μm for (a), (c), and (d) and 20 μm for C1 and D1, respectively.

Figure 5

Localization of NUMB in odontogenic tissues at postnatal day 5: NUMB expression can be detected in the ameloblasts and odontoblasts. Notice that in (a1), NUMB is positively stained in some ameloblasts but negatively stained in the adjacent cells (indicated by arrows). Scale bar represents 100 μm for (a); 50 μm for (b); 20 μm for (a1), (a2), and (b1), (b2), respectively.

Figure 6

Localization of NUMB in odontogenic tissues at postnatal day 7: NUMB expression is more defined in the immature ameloblasts, and very low expression levels can be detected in the immature odontoblasts. Scale bar represents 50 μm for (a), 100 μm for (b), and 50 and 20 μm for (a1), (b1), and (b2), respectively.

Figure 7

Overexpression of NUMB-GFP in HAT-7 cells: (a) Western blot analysis to detect overexpression of NUMB-GFP in HAT-7 cells. Total proteins were isolated from 2 clones, namely, clone 1 and 2. (b) Immunohistochemical analysis detected that Notch1 protein expression is dramatically reduced in the NUMB overexpressing cell line (stable cell line clone 2 was used). The green fluorescence represents activated Notch1 protein.

Figure 8

Notch signaling pathway PCR array. Genes from the Notch1 signaling pathway that were affected by NUMB overexpression.

Figure 9

Hypothetical model. NUMB regulates stem cell properties at two levels. NUMB downregulates the expression of the activated Notch 1 protein level in the progenitor cells at the cervical loop and thereby inhibits the Shh expression in the adjacent preameloblasts. Thus, NUMB influences the expression of downstream genes such as Notch1 and Shh and thus performs critical role in ameloblast differentiation.