Immunolocalization of connective tissue growth factor, transforming growth factor-beta1 and phosphorylated-SMAD2/3 during the postnatal tooth development and formation of junctional epithelium

Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta 1 (TGF-β₁) and TGF-β₁-induced CTGF expression is regulated through SMAD pathway. However, there is no literature showing the expression of TGF-β₁-SMAD2/3-CTGF signaling pathway during postnatal tooth development and the formation of junctional epithelium (JE). Hence, we aimed to analyze the localization of TGF-β₁, CTGF and phosphorylated SMAD2/3 (p-SMAD2/3) in the developing postnatal rat molars. Wistar rats were killed at postnatal (PN) 0.5, 3.5, 7, 14 and 21days and the upper jaws were processed for immunohistochemistry. At PN0.5 and PN3.5, weak staining for TGF-β₁ and CTGF was evident in preameloblasts (PA), while moderate to strong staining was seen in odontoblasts (OD), dental papilla (DPL), secretary ameloblasts (SA), preodontoblasts (PO) and polarized odontoblasts (PoO). There was no staining for p-SMAD2/3 in PA, SA, PO and PoO, although strong staining was localized in DPL. OD was initially moderately positive and then negative for p-SMAD2/3. At PN7, intense staining for TGF-β₁ and CTGF was observed in SA, OD, dental pulp (DP) and predentin respectively. p-SMAD2/3 was strongly expressed in DP and moderately expressed in SA and OD. At PN14 and PN21, both reduced enamel epithelium (REE) and JE showed a strong reaction for TGF-β₁ and CTGF. p-SMAD2/3 was intensely and weakly expressed in REE and JE respectively. These data demonstrate that the expression of CTGF, TGF-β₁ and p-SNAD2/3 is tissue-specific and stage-specific, and indicate a regulatory role for a TGF-β₁-SMAD2/3-CTGF signaling pathway in amelogenesis, dentinogenesis and formation of JE.