Abstract

Cells have ingenious mechanisms for interpreting complex signals from their external microenvironment. Previously, we have shown that phosphophoryn (PP) regulates the expression of bone/dentin marker genes via the integrin/MAPK signaling pathway (Jadlowiec, J., Koch, H., Zhang, X., Campbell, P. G., Seyedain, M., and Sfeir, C. (2004) J. Biol. Chem. 279, 53323-53330). We hypothesize that other signaling pathways important for mineralized tissue morphogenesis such as the Smad pathway could be involved in PP signaling. We determined activation of the Smad pathway in human adult mesenchymal stem cells following treatment with recombinant PP (rPP). We observed that PP enhanced phosphorylation of Smad1 within 30 min and Smad1 translocation to the nucleus within 1 h. PP up-regulated the expression of Smad1 target genes, Smad6, Dlx5, and Runx2. The timing of PP activation of Smad1 implies this is a direct effect; however, we also investigated the possible involvement of bone morphogenetic proteins in PP stimulation of the Smad pathway. PP was shown to up-regulate Bmp-2 gene expression 12 h post-treatment with PP, which is much later than initial detection of Smad1 phosphorylation at 30 min. Furthermore, addition of Noggin did not block Smad1 phosphorylation by PP. We propose that PP could signal via the Smad pathway by either directly stimulating the phosphorylation of Smad1 via integrins or other mechanisms. These might include integrin/bone morphogenetic protein receptor interactions or involvement of PP with other growth factors leading to the modulation of intracellular signaling. It is noteworthy that a non-transforming growth factor-beta family member activates the Smad pathway. The role of PP in regulating the Smad pathway raises very interesting questions regarding the role of PP during bone and tooth development.

Highlights

  • Phosphophoryn (PP)2 in cell signaling and differentiation

  • We examined the activation of the Smad pathway by analysis of the phosphorylation of the bone morphogenetic proteins (BMPs)-2 site on Smad1 (Ser463/Ser465), nuclear translocation, and transcriptional activity following treatment of human adult mesenchymal stem cells (hMSC) with PP

  • We detected an increase in phosphorylated Smad1 within 30 min of treatment (Fig. 1A)

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Summary

Introduction

Phosphophoryn (PP) in cell signaling and differentiation. We have determined the involvement of the MAPK pathway in PP’s signaling [1]. Our hypothesis is that the Smad pathway might be involved since many of the genes up-regulated by PP are critical to bone differentiation. Smads are the initial responders to receptor activation of the TGF-␤ family and have been studied as transcriptional activators of cell differentiation. BMPs are potent morphogens that exhibit a variety of roles across tissue and cell types (8 –15). Their functions have been studied in many organisms from Drosophila to humans and their role in development is highly specialized and critical. We exposed human adult mesenchymal stem cells (hMSC) to recombinant PP (rPP) and determined the levels of Smad phosphorylation, its subsequent nuclear translocation, and transcriptional activity. We found that PP regulates the Smad pathway and this may be independent of BMPs

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