1α,25-Dehydroxyvitamin D3 Synergism toward Transforming Growth Factor-β1-induced AP-1 Transcriptional Activity in Mouse Osteoblastic Cells via Its Nuclear Receptor

Abstract

TGF-␤11 is locally produced by osteoblasts and accumulates abundantly in bone matrix tissue (1-3). This local cytokine plays an important role as a "coupling factor" in bone remodeling (4 -7). We (8, 9) previously demonstrated that TGF-␤1 is a potent activator of AP-1, a transcriptional factor that is a heterodimer of FOS and JUN proteins, in mouse osteoblastic MC3T3-E1 cells. AP-1 activates the transcription of target genes by binding to specific promoter elements called TRE. In fact, several studies (10 -16) have suggested that AP-1 is a regulatory factor in bone metabolism. Therefore, it is of interest to investigate the mechanism by which AP-1 regulates the metabolism of TGF-␤1-treated bone cells, which regulation may occur in an autocrine and/or paracrine fashion.1␣-25-(OH) 2 D 3 also is an important systemic hormone in bone metabolism and regulates transcriptionally the expression of several genes involved in the differentiation of osteoblastic cells (17)(18)(19)(20)(21)(22)(23). Many studies (20 -26) have shown that the hormone binds to its receptor, VDR, in the cell nucleus and acts via binding of this complex to the VDR response element. This VDR-dependent action is referred to as the "genomic action" of the hormone. On the other hand, other recent studies (27-37) have suggested that the hormone is also able to induce several biological activities via protein kinase C, ceramide signaling pathways, and via an increase in the intracellular calcium concentration, which are called "nongenomic action."Several investigators (21-24) have demonstrated negative regulation between AP-1 and VDR of the transcriptional activity of osteocalcin and collagen genes, both of which are involved in bone formation. However, positive regulation by 1␣-25-(OH) 2 D 3 of AP-1 transcriptional activity in osteoblastic cells has not been demonstrated in detail. Therefore, it is of interest to explore the possibility of 1␣-25-(OH) 2 D 3 positive regulation of AP-1 transcriptional activity via "genomic" or "nongenomic" action.In this regard, we investigated in the present study the regulation by 1␣-25-(OH) 2 D 3 of TGF-␤1-induced AP-1 transcriptional activity in osteoblastic MC3T3-E1 cells. As a result, we demonstrated the presence of 1␣-25-(OH) 2 D 3 synergism toward TGF-␤1-induced AP-1 transcriptional activity via genomic action. This demonstration suggests the presence of a novel positive regulation by 1␣-25-(OH) 2 D 3 of AP-1 transcriptional activity in osteoblastic cells via the VDR-dependent pathway (genomic action). MATERIALS AND METHODSReagents-Human recombinant TGF-␤1 was purified to homogeneity (Ͼ98.9%, determined by SDS-polyacrylamide gel electrophoresis analysis: King Brewer, Kakogawa, Japan). 1␣-25-(OH) 2 D 3 , 24,25-(OH) 2 D 3 , and 22-oxa-1,25-dihydroxyvitamin D 3 (OCT) were kindly provided by Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). ␣-MEM was obtained from Flow Laboratories (McLean, VA). FCS was from HyClone (Logan, UT). 5Ј-[␣-32 P]dCTP megaprime DNA labeling system and [␥-32 P]ATP were purchased from Amersham...

UTP Induces Osteopontin Expression through a Coordinate Action of NFκB, Activator Protein-1, and Upstream Stimulatory Factor in Arterial Smooth Muscle Cells

“…Until now, two large regions of the OPN promoter have been involved in OPN regulation; the Ϫ1000 to Ϫ700 region is mainly a target for hormones (16,38), whereas the Ϫ270 to ϩ1 region has been shown to be activated by various transcription factors, including AP-1 (15). AP-1 seems to be a common element in the activation of OPN expression by growth factors, because it has been involved in FGF-2-and TGF␤-induced OPN expression in osteoblasts (39,40). Moreover, cis-regulating elements (Lef-1, Ets, and c-Jun) located upstream of Ϫ1000 were involved in the basal expression of OPN in breast cancer cells (22).…”

Sphingosine 1-phosphate acts as a signal molecule in ceramide signal transduction of TNF-.ALPHA.-induced activator protein-1 in osteoblastic cell line MC3T3-E1 cell

“…Many studies (11,19,20,(47)(48)(49)(50)(51) have shown that osteogenic and osteolytic factors are able to stimulate expression of the c-fos and c-jun genes in ostoblastic cells. We have also previously shown that TNF-α is able to stimulate expression of c-fos and c-jun and to activate AP-1 in mouse osteoblastic MC3T3-E1 cells (11,19,20).…”

Involvement of Nuclear Receptor Co-repressors in the Development of Human Breast Cancers

“…The phosphorylated form of SMADs forms stable complexes with SMAD4 and these complexes translocate into nucleus where they activate transcription. Recently, TGF-ß has been shown to act cooperatively with vitamin D 3 , indicating a cross-talk between these two signaling pathways (Yoshizawa et al, 1997;Takeshita et al, 1998). The mechanism of synergism between TGF-ß and vitamin D 3 appears to be mediated by SMAD3, but not SMAD2 (Yanagisawa et al, 1999).…”