Synthesis and intracellular transportation of type I procollagen during functional differentiation of odontoblasts.

The expression of type I collagen, the most component of dentin extracellular matrix proteins (ECMs) in odontoblast is correlated with the activity of dentin formation. Since odontoblast possesses a distinct cellular process for protein transport into the dentinal tubule, it is important to examine the intracellular protein localization. However, a study focusing on odontoblast processes has not been performed. Type I collagen is synthesized as procollagen, which is immediately converted to collagen upon secretion. After characterization of antiserum to rat type I procollagen, we investigated the intracellular localization of type I procollagen in odontoblasts during and after dentinogenesis, using immunohistochemistry and in situ hybridization. The level of mRNA expression decreased during dentinogenesis, whereas the intracellular localization of type I procollagen in odontoblast processes become more distinct. The percentage of dentinal tubules with type I procollagen increased significantly with aging. Odontoblasts in pulp horn, in particular, showed moderate expression of type I procollagen after dentinogenesis. Since loss of occlusion also caused a significant decrease in type I procollagen, we concluded that occlusal stimulation activated type I procollagen synthesis in odontoblasts. We also suggest that analysis of intracellular transport of type I procollagen via odontoblast processes may be a new approach to evaluation of odontoblast function.

Molecular cloning of PP2Ceta, a novel member of the protein phosphatase 2C family.

We have cloned a novel member of the mouse protein phosphatase 2C family, PP2Ceta. Sequence analysis suggests that PP2Ceta, PP2Czeta and NERPP-2C constitute a unique subgroup of the PP2C family. PP2Ceta had extremely low activity against alpha-casein compared with PP2Calpha and was localized mainly in cell nuclei, suggesting that PP2Ceta dephosphorylates a unique nuclear protein(s) in the cells.

Regulation of the interleukin-1-induced signaling pathways by a novel member of the protein phosphatase 2C family (PP2Cepsilon).

Although TAK1 signaling plays essential roles in eliciting cellular responses to interleukin-1 (IL-1), a proinflammatory cytokine, how the IL-1-TAK1 signaling pathway is positively and negatively regulated remains poorly understood. In this study, we investigated the possible role of a novel protein phosphatase 2C (PP2C) family member, PP2Cepsilon, in the regulation of the IL-1-TAK1 signaling pathway. PP2Cepsilon was composed of 303 amino acids, and the overall similarity of amino acid sequence between PP2Cepsilon and PP2Calpha was found to be 26%. Ectopic expression of PP2Cepsilon inhibited the IL-1- and TAK1-induced activation of mitogen-activated protein kinase kinase 4 (MKK4)-c-Jun N-terminal kinase or MKK3-p38 signaling pathway. PP2Cepsilon dephosphorylated TAK1 in vitro. Co-immunoprecipitation experiments indicated that PP2Cepsilon associates stably with TAK1 and attenuates the binding of TAK1 to MKK4 or MKK6. Ectopic expression of a phosphatase-negative mutant of PP2Cepsilon, PP2Cepsilon(D/A), which acted as a dominant negative form, enhanced both the association between TAK1 and MKK4 or MKK6 and the TAK1-induced activation of an AP-1 reporter gene. The association between PP2Cepsilon and TAK1 was transiently suppressed by IL-1 treatment of the cells. Taken together, these results suggest that, in the absence of IL-1-induced signal, PP2Cepsilon contributes to keeping the TAK1 signaling pathway in an inactive state by associating with and dephosphorylating TAK1.