Logo: to the web site of Uppsala University

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.

The interplay between membrane subregions and receptor tyrosine kinases (RTK) will influence signaling in both normal and pathological RTK conditions. In this study, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor β (PDGFR-β) internalizations were investigated by immunofluorescent microscopy following simultaneous treatment with EGF and PDGF-BB. We found that the two receptors utilize separate routes of internalization, which merges in a common perinuclear endosomal compartment after 45 min of stimulation. This is further strengthened when contrasting the recruitment of either EGFR or PDGFR-β to either clathrin or caveolin-1: PDGFR-β dissociates from caveolin-1 upon stimulation, and engages clathrin, whilst an increased recruitment of EGFR, to both clathrin and caveolin-1, was observed upon EGF stimulation. The association between EGFR and caveolin-1 is supported by the observation that EGFR was localized in lipid raft associated fractions, whereas PDGFR-β was not. We also found that disruption of lipid rafts using MβCD led to an increased EGFR dimerization and phosphorylation in response to ligand, as well as a dramatic decrease in AKT- and a smaller but robust decrease in ERK1/2 phosphorylation. This suggest that lipid rafts may be important to effectively connect the EGFR with downstream proteins to facilitate signaling. Our data implies that cholesterol depletion of the plasma membrane affect the signaling of EGFR and PDGFRβ differently.

Platelet-derived growth factor receptors (PDGFR) have been implicated in both pathological and physiological signaling and play a major role in the homeostasis of the tumor microenvironment. It has previously been shown that Cdc42, which belongs to the family of Rho GTPases, impairs the degradation of EGFR and VEGFR2. In the current work, we have investigated how Cdc42 as well as Rac1 and RhoA affect PDGFRα and -β signaling and downstream activation of AKT, ERK1/2, STAT1 and STAT3. In response to individual depletion of all Rho GTPases both PDGFRα and -β show a significant reduction in their phosphorylation. We could see a delayed internalization in response to Cdc42 or Rac1 depletion and an increased steady-state amount in response to RhoA depletion for both PDGFRα and -β. Downstream of both receptors, we saw a dramatic effect on STAT signaling, however, AKT and ERK1/2 signaling was unaffected. PDGF-BB-induced STAT1 and STAT3 phosphorylation was severely impaired in response to the depletion of either Cdc42, Rac1 or RhoA. Furthermore, STAT1 protein levels were also decreased in response to depletion of the Rho GTPases. In conclusion, we show that both PDGFRα and PDGFRβ rely on Cdc42, Rac1 and RhoA for proper signaling. Furthermore, we also show that STAT1 and STAT3 depend on Cdc42, Rac1 and RhoA for their signaling downstream of PDGFRs, and STAT1 for its protein stability.