Platelet-derived growth factor-BB (PDGF-BB) binds to its tyrosine kinase receptors (PDGFRs) and stimulates mitogenicity and survival of cells of mesenchymal origin. Activation of PDGFRs initiates a number of downstream signaling pathways, including phosphatidyl 3'-inositol kinase (PI3-kinase), phospholipase Cγ and MAP kinase pathways. In this report, we show that Erk5 MAP kinase is activated in response to PDGF-BB in the smooth muscle cell line MOVAS in a manner dependent on Mekk2, Mek1/2, Mek5, PI3-kinase and protein kinase C (PKC). The co-operation of Mek1/2 and Mekk2 in the activation of Erk5, suggests a close co-regulation between the Erk1/2 and Erk5 MAP kinase pathways. Furthermore, we found that classical PKCs are important for Erk5 activation. In addition, we found that PKCζ interacts with Erk5 and may exert a negative feed-back effect. We observed no nuclear accumulation of Erk5 in response to PDGF-BB stimulation, however, we identified a mechanism by which cytoplasmic Erk5 influences gene expression; Erk5 was essential for PDGF-BB-mediated Smad1/5/8 signaling by stimulating release and/or activation of bone morphogenetic protein(s) (BMPs). Thus, PDGF-BB-induced Erk5 activation involves parallel stimulatory and inhibitory pathways and promotes Smad1/5/8 signaling.

It has become clear that lipid rafts functions as signaling hotspots connecting cell surface receptors to intracellular signaling pathways. However, the exact involvement of lipid rafts in receptor tyrosine kinase signaling is still poorly understood. In this study, we have analyzed platelet-derived growth factor (PDGF) receptor β (PDGFR-β) signaling in two different cell lines depleted of cholesterol, and as a consequence, disruption of lipid rafts. Cholesterol depletion of BJ-hTERT fibroblasts using methyl-β-cyclodextrin (MβCD) did not affect PDGFR-β activation as measured by its tyrosine phosphorylation. However, we did observe a small reduction in AKT phosphorylation and a more robust decrease of ERK1/2 activation. In contrast, in the osteosarcoma cell line U2OS, we noticed a deficient receptor activation. Interestingly, in U2OS cells, the ERK1/2 pathway was unaffected, but instead AKT and SRC signaling was reduced. These results suggest that cell type specific wiring of signaling pathways can lead to differential sensitivity to cholesterol depletion. Furthermore, MβCD treatment had a much more pronounced morphological effect on U2OS compared to BJ-hTERT cells. This is consistent with a previous report claiming that cancer cells are more sensitive to cholesterol depletion than normal cells. Our data supports the possibility that cholesterol lowering drugs may impede tumor growth.

Mammalian target of rapamycin (mTOR) can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor) and mTORC2 (containing Rictor). Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF)-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictor-null cells the phosphorylation of phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC) was impaired, and the PKCα protein levels strongly reduced. Furthermore, interfering with the PLCγ/Ca2+/PKC pathway inhibited PDGF-BB-induced Akt phosphorylation. In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD). It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner and promotes phosphorylation of the S6 protein, whereas mTORC2, in concert with PLCγ signaling, promotes Akt phosphorylation.

Extracellular regulated kinase (Erk) 5 is a member of the mitogen activated protein (MAP) kinase family that has been implicated in both cell proliferation and survival. In the present study, we found that stimulation with platelet-derived growth factor (PDGF)-BB leads to a transient activation of Erk5, which was shown to be dependent on recruitment of both Src kinases and the tyrosine phosphatase Shp2 to the activated PDGF receptor beta (PDGFRbeta). We could also demonstrate that Shp2 docking to the receptor is critical for Src kinase activation, suggesting that Shp2 may contribute to Erk5 activation through its involvement in Src kinase activation. Under control conditions, PDGF-BB promoted a sustained Akt phosphorylation. However, reduction of the expression of Erk5 by siRNA resulted in only a transient Akt phosphorylation, and an inability of PDGF-BB to suppress caspase 3 activation and inhibit apoptotic nuclear morphological changes such as condensed or fragmented chromatin under serum-free conditions.

MAP kinase phosphatase-3 (MKP3), also known as DUSP6 or Pyst1, is a dual specificity phosphatase considered to selectively dephosphorylate extracellular-signal-regulated kinase 1/2 (Erk1/2). Here, we report that in NIH3T3 cells, MKP3 is induced in response to platelet-derived growth factor (PDGF)-BB treatment in an Erk1/2- and phosphatidylinositol 3-kinase (PI3K)-dependent manner, but independently of Erk5 expression. Silencing of MKP3 expression did not affect PDGF-BB-induced Erk1/2 or p38 phosphorylation; however, their basal level of phosphorylation was elevated. Furthermore, we found that PDGF-BB-mediated activation of Erk5 and Akt was enhanced when the MKP3 expression was reduced. Interfering with Mek1/2 or PI3K using the inhibitors CI-1040 and LY-294002, respectively, inhibited PDGF-BB-induced MKP3 expression. Functionally, we found that MKP3 silencing did not affect cell proliferation, but enhanced the chemotactic response toward PDGF-BB. Although both Akt and Erk5 have been linked to increased cell survival, downregulation of MKP3 did not alter the ability of PDGF-BB to protect NIH3T3 cells from starvation-induced apoptosis. However, we observed an increased apoptosis in untreated cells with reduced MKP3 expression. In summary, our data indicate that there is negative cross-talk between Erk1/2 and Erk5 that involves regulation of MKP3 expression, and that PI3K in addition to promoting Akt phosphorylation also negatively modulates Akt, through MKP3 expression.

Dual specificity phosphatase (DUSP) 4 has been described as a negative regulator of MAP kinase signaling, in particular for the ERK1/2 and JNK pathways. We found that DUSP4 expression was upregulated in response to prolonged platelet-derived growth factor (PDGF)-BB stimulation. The PDGF-BB-induced DUSP4 expression was dependent on ERK1/2, STAT3 and p53. We found that inhibition of ERK1/2 effectively reduced DUSP4 mRNA levels, whereas STAT3 was necessary for maintaining p53 expression. p53 has binding sites in the DUSP4 promoter and was found to promote DUSP4 expression.