Transforming growth factor-beta1 expression is up-regulated in maturation-stage enamel organ and may induce ameloblast apoptosis

Abstract

Transforming growth factor-beta1 (TGF-beta1) regulates a variety of cellular responses that are dependent on the developmental stage and on the origins of the cell or the tissue. In mature tissues, and especially in tissues of epithelial origin, TGF-beta1 is generally considered to be a growth inhibitor that may also promote apoptosis. The ameloblast cells of the enamel organ epithelium are adjacent to and responsible for the developing enamel layer on unerupted teeth. Once the enamel layer reaches its full thickness, the tall columnar secretory-stage ameloblasts shorten, and a portion of these maturation-stage ameloblasts become apoptotic. Here we investigate whether TGF-beta1 plays a role in apoptosis of the maturation-stage ameloblasts. We demonstrate in vitro that ameloblast lineage cells are highly susceptible to TGF-beta1-mediated growth arrest and are prone to TGF-beta1-mediated cell death/apoptosis. We also demonstrate in vivo that TGF-beta1 is expressed in the maturation-stage enamel organ at significantly higher levels than in the earlier secretory-stage enamel organ. This increased expression of TGF-beta1 correlates with an increase in expression of the enamel organ immediate-early stress-response gene and with a decrease in the anti-apoptotic Bcl2 : Bax expression ratio. We conclude that TGF-beta1 may play an important role in ameloblast apoptosis during the maturation stage of enamel development.

Fig. 1

Antiproliferative and toxic effects of TGF-β treatment on ALC cells. (A) To assess cell proliferation, ALC cells were seeded into 96 well plates and treated for 24 hr with the indicated concentrations of TGF-β. Reduction of MTT to an insoluble formazan dye by mitochondrial enzymes was quantified for each well by OD₅₅₀ measurements and the results were used to calculate percent cell proliferation (treated OD₅₅₀/untreated OD₅₅₀) X 100. Error bars represent the standard error of the mean. (B) To assess cell survival, limiting dilutions of ALC cells were seeded into culture flasks, allowed to adhere for 18 hr, and were treated for 24 hr with the indicated concentrations of TGF-β. After 12–14 days, the resulting colonies were stained, counted, and percent cell survival was calculated (number treated/untreated colonies) x100. Error bars represent the standard error of the mean.

Fig. 2

Quantification by qPCR of Bax/Bcl2 expression in ALC cells treated with TGF-β for 24 hr. Bax or Bcl2 expression was determined and each were normalized to the expression levels of the internal reference control gene (eEF1α1). The graph shows the Bax to Bcl2 expression ratio with and without 24 hr of 1 ng/ml TGF-β treatment. Six wells were assayed for each experimental treatment and error bars represent the standard error. *, indicates statistical significance (p< 0.01).

Fig. 3

Identification of TGF-β in ameloblast cells of the enamel organ as determined by immunohistochemistry. (A) Negative control that lacked the primary antisera treatment. Note that some background staining can be observed above the ameloblasts (am). (B) Staining for TGF-β1 in an adult mouse mandibular incisor [20X magnification]. (C) Magnified image of the indicated secretory stage ameloblasts from panel B. (D) Magnified image of the indicated maturation stage ameloblasts from panel B. Note that staining appeared to be enhanced for TGF-β1 in the ameloblast cytoplasm at the maturation stage as compared to the secretory stage of enamel development. Scale bars represent 120 µm.

Fig. 4

Quantification by qPCR of TGF-β1 expression in mouse enamel organ during the secretory and maturation stages of enamel development. (A) Total RNA from first molar enamel organs was extracted from 4 day-old mice (secretory stage) or was extracted from 11 day-old mice (maturation stage) and was assessed for TGF-β expression levels. (B) Total RNA was also extracted from the kidney of 4 and 11 day-old mice. The kidney served as an epithelial derived negative control for the enamel organ results. Each expression analysis result was normalized to the expression level of the internal reference control gene (eEF1α1). Six different samples were assayed for each tissue and error bars represent the standard error. *, indicates statistical significance (p< 0.01).

Fig. 5

Quantification by qPCR of immediate early stress response gene expression in mouse enamel organ during the secretory and maturation stages of enamel development. Total RNA extracted from first molar enamel organs from 4 day-old mice (secretory stage) and 11 day-old mice (maturation stage) was assessed for gene expression. (A) c-Jun, (B) c-Fos, (C) Egr-1, (D) c-Myc. Each expression analysis result was normalized to the expression level of the internal reference control gene (eEF1α1). Six samples were assayed for each tissue for each gene and error bars represent the standard error. Note that c-Jun expression increased at the maturation stage to almost six times over that observed in the secretory stage of enamel development. *, indicates statistical significance (p< 0.01).

Fig. 6

Identification of p-Smad2/3 and p-c-Jun in ameloblast cells of the enamel organ as determined by immunohistochemistry. (Top panels) Staining for p-Smad2/3 in an adult mouse mandibular incisor [40× magnification]. (Bottom panels) Staining for p-c-Jun in an adult mouse mandibular incisor [40× magnification]. Note that staining for Smad2/3 was dramatically increased in the maturation stage ameloblasts and that more p-c-jun nuclear staining was also present in the maturation stage ameloblasts when compared to secretory stage ameloblasts.

Fig. 7

Quantification by qPCR of Bcl2 and Bax gene expression in mouse enamel organ during the secretory and maturation stages of enamel development. Total RNA extracted from first molar enamel organs from 4 day-old mice (secretory stage) and 11 day-old mice (maturation stage) was assessed by for gene expression. (A) Bcl2, (B) Bax, (C) Bcl2/Bax ratio, (D) The kidney served as an epithelial derived negative control for the enamel organ results for Bcl2/Bax. Each expression analysis result was normalized to the expression level of the internal reference control gene (eEF1α1). Six samples were assayed for each tissue for each gene and error bars represent the standard error. Note that that the greatly decreased ratio of Bcl2 expression to Bax expression in the maturation stage enamel organ favors apoptosis. *, indicates statistical significance (p<0.01).