Phosphorylation of CREB, a cyclic AMP responsive element binding protein, contributes partially to lysophosphatidic acid-induced fibroblast cell proliferation

Lysophospholipids regulate a wide array of biological processes including cell survival and proliferation. In our previous studies, we found that in addition to SRE, CRE is required for maximal c-fos promoter activation triggered by lysophosphatidic acid (LPA). c-fos is an early indicator of various cells into the cell cycle after mitogenic stimulation. However, role of CREB activation in LPA-stimulated proliferation has not been elucidated yet. Here, we investigate how LPA induces proliferation in Rat-2 fibroblast cell via CREB activation. We found that total cell number and BrdU-positive cells were increased by LPA. Moreover, levels of c-fos mRNA and cyclin D1 protein were increased via LPA-induced CREB phosphorylation. Furthermore, LPA-induced Rat-2 cell proliferation was decreased markedly by ERK inhibitor (U0126) and partially by MSK inhibitor (H89). Taken together, these results suggest that CREB activation could partially up-regulate accumulation of cyclin D1 protein level and proliferation of LPA-stimulated Rat-2 fibroblast cells.     

Osteogenic differentiation of C2C12 myogenic progenitor cells requires the Fos-related antigen Fra-1 – A novel target of Runx2

Bone formation is a developmental process requiring the differentiation of mesenchymal stem cells into osteoblasts. It is established that Runx2 tightly regulates osteoblast differentiation and bone formation. Fos-related antigen Fra-1 is an essential factor for bone formation. Current evidence does not support a relationship between Fra-1 and Runx2 in osteogenesis. Here, we explored the possibility that Runx2 regulates Fra-1 expression during osteogenic differentiation of C2C12 myogenic progenitor cells. Expression of Fra-1 was induced rapidly after activation of Runx2 in a Tet-on stable C2C12 cell-line (C2C12/Runx2^Dox sub-line). Transient transfection assay showed that Runx2 activates Fra-1 promoter-reporter activity, suggesting that Fra-1 may be a direct target of Runx2. To determine the minimal region of the Fra-1 promoter that was activated by Runx2, a series of Fra-1 promoter deletion constructs were made. By transient transfection assay, we defined the minimal region to the proximal 342 bp (?84 to +258). Two potential Runx2-binding sites (at positions +139 and +208) were predicted within this region. Mutation of the Runx2 motif at position +208 significantly decreased Fra-1 promoter activity compared to wild type, whereas mutation of Runx2 at position +139 had no effect. Electrophoretic mobility shift assay (EMSA) demonstrated the existence of one atypical Runx2-binding element at position +208, and chromatin immunoprecipitation (ChIP) assay revealed that Runx2 bound to the native Fra-1 promoter in vivo via this site. Finally, forced expression of Fra-1 resulted in upregulation of alkaline phosphatase (ALP), a marker of early osteoblast differentiation. Together, these results indicate that Fra-1 is a direct target of Runx2 during osteogenic differentiation of C2C12 myogenic progenitor cells.     

Activated MEK Stimulates Expression of AP-1 Components Independently of Phosphatidylinositol 3-Kinase (PI3-Kinase) but Requires a PI3-Kinase Signal To Stimulate DNA Synthesis

To investigate the contribution that ERK/mitogen-activated protein kinase signalling makes to cell cycle progression and gene expression, we have constructed cell lines to express an inducible version of activated MEK1. Using these cells, we show that activation of MEK leads to the expression of Fra-1 and Fra-2 but not c-Fos. Treatment of Ras-transformed cells with the MEK inhibitor PD098059 blocks expression of Fra-1 and Fra-2, showing that in Ras transformation ERK signalling is responsible for Fra-1 and Fra-2 expression. Activation of MEK1 in growth-arrested cells leads to DNA synthesis; however, ERK activation alone is insufficient because the induction of DNA synthesis is blocked by inhibition of phosphatidylinositol 3-kinase (PI3-kinase). Activation of PI3-kinase is indirect, perhaps through autocrine growth factors, and is required for the induction of cyclin D1.