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The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-κB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth

Regulation and Function of MAP Kinases in PDGF Signaling

Platelet-derived growth factor (PDGF) is a family of signaling molecules that stimulates cell growth, survival and migration. PDGF is recognized by specific transmembrane proteins, the PDGF receptors, which relay the signals to the cell activating the Mitogen-activated protein (MAP) kinases and other signaling pathways. Aberrant activation of these pathways is frequently detected in cancer. Hence, the study of these processes is essential for identifying potential drug targets or diagnostic markers. In paper I, we identified Receptor Subfamily 4 Group A Member 1 NR4A1 to be regulated by PDGF via MAP kinases, clarifying the role of Extracellular signal–regulated kinases (Erk) 1/2, Erk5 and Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) in its regulation. NR4A1 was found to be important for the tumorigenic potential, measured as anchorage-independent growth, of glioblastoma cells. Since the cellular responses elicited by PDGF result from the balance between phosphorylation and dephosphorylation events, we investigated the role of the dual specificity phosphatases DUSP4/MKP-2 and DUSP6/MKP-3. In paper II, we describe the crucial role of Erk1/2 and p53 in the expression of DUSP4/MKP2. Moreover, we observed that DUSP4/MKP-2 downregulation decreases Erk5 activation and accelerates PDGFRβ internalization and downregulation resulting in a specific inhibition of Signal transducers and activators of transcription (Stat) 3, Src and protein kinase C (PKC), and partially of p38, Stat1/5 and Phoshoplipase Cγ (PLCγ). In paper III, we report that DUSP6/MKP-3 creates a negative cross-talk between Erk1/2 and Erk5 and an auto-inhibitory feedback loop on the PI3-kinase/Akt pathway. In paper IV, we identify a new regulative mechanism of the PDGF pathway. PDGF induces Erk5 expression and activation that modulates the PDGFRβ activity. After Erk5 downregulation, the receptor undergoes to a faster and stronger activation that results in a faster internalization and degradation. In conclusion, we present a mechanism through which the PDGF/MAP kinases support tumor growth, and elucidate different regulatory pathways involved in PDGF signaling

NR4A1 Promotes PDGF-BB-Induced Cell Colony Formation in Soft Agar

The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-κB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth

NR4A1 expression does not decrease after inhibition of p38, JNK and Src pathways.

<p>NIH3T3 cells were serum-starved overnight in 0.1% BSA and then pretreated for 1 h with DMSO or inhibitors SB203580 10 µM (A), SP600125 10 µM (B) and SU6656 2 µM (C), and then stimulated for indicated time periods with PDGF-BB (20 ng/ml); total cell lysates (TCL) were prepared and subjected to immunoblotting (Ib).</p

PDGF-BB induces NR4A1 via Erk1/2 and Erk5.

<p>NIH3T3 cells were serum-starved overnight in 0.1% BSA and treated with inhibitors starting 1 h before stimulation with PDGF-BB (20 ng/ml) for the indicated time periods. Total cell lysate (TCL) (A and C) were analyzed by immunoblotting (Ib) using NR4A1, Erk2, phospho-Erk1/2 and Erk5 antibodies. NR4A1 mRNA was measured with quantitative RT-PCR and panel B shows one out of three independent experiments performed; error bars indicate the standard deviation between three replicates. An asterisk (*) indicate a p-value≤0.05; with two (**) when it is ≤0.01 and with three (***) when it is ≤0.001.</p

A small pool of the cytoplasmic NR4A1 translocates into the nucleus after long PDGF-BB stimulation.

<p>Cytoplasmic and nuclear fractions were prepared from NIH3T3 cells serum-starved overnight in 0.1% BSA and stimulated with PDGF-BB (20 ng/ml) for indicated periods of time and NR4A1 levels were analyzed by immunoblotting (Ib). The purity of the fraction were confirmed by immunoblotting for the nuclear marker Lamin and the cytoplasmic marker Tubulin.</p

NR4A1 promotes PDGF-BB-induced anchorage-independent growth.

<p>NR4A1 was overexpressed by stable transfection in NIH3T3 cells (A), and U-251MG (B) and U-105MG (C) glioblastoma cells; cells were cultured in soft agar in starvation medium (3% serum) with or without 50 ng/ml PDGF-BB. The number and size of the colony forming unit (CFU) were measured after 10 days. For each cell line a representative experiment is shown, out of at least two independently performed, and the standard deviation between three replicates is indicated in the error bar. A p-value≤0.05 is indicated with an asterisk (*).</p

PDGF-BB-induced NR4A1 expression is decreased after treatment with the NF-κB inhibitor BAY11-7082.

<p>NIH3T3 cells were serum-starved overnight in 0.1% BSA. Cells were pretreated for 4 h with DMSO or NF-κB inhibitor BAY11-7082 (10 µM) (A and B) and then stimulated for indicated time periods with PDGF-BB (20 ng/ml). mRNA was measured with quantitative RT-PCR (A) and total cell lysate (TCL) analyzed by immunoblotting (Ib) using NR4A1 antibody and tubulin, as loading control (B). Panel A shows the result of one out of three independent experiments performed; error bars indicate the standard deviation between three replicates. An asterisk (*) indicate a p-value≤0.05.</p