5.3.9.1 GRA of MAP kinase activation in TF-1 cells
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Lane number 1 2 3 4 5 6 7 8 Time (min) 0 1 5 10 15 30 45 605.3.9 2 GRA of MAP kinase activation in neutrophils
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Lane number 1 2 3 4 5 6 Time (min) 0 1 5 15 30 605.4 DISCUSSION
The HL-60 cell line has been extensively used as a model to examine the expression and function of the GM -CSF receptor during myeloid differentiation. In this study undifferentiated and differentiated HL-60 cells were stimulation with GM -CSF and the dow n-stream signalling events from the GM -CSF R exam ined. G M -CSF stim ulation o f im m ature HL-60 cells resulted in both STAT 5 and M AP kinase activation, but no detectable JAK 2 phosphorylation.
The level of STAT 5 activation in the undifferentiated HL-60 cells was much lower than in the differentiated HL-60 cells, but similar kinetics of activation were observed for both cell types, with activation being rapid and persisting for up to 1 hour follow ing G M -CSF stim ulation. Analysis o f total cellular JAK 2 protein levels revealed a much lower level of JAK 2 expression in the undifferentiated cells than the differentiated cells, which might account for the inability to detect any tyrosine phosphorylation of JAK 2 and also the low levels of activated STAT 5 in the undifferentiated cells.
It is possible that these differentiation linked changes in GM-CSF signal transduction observed in H L-60 cells may be related to the higher level o f GM -CSF receptor expression in the more differentiated cells. HL-60 cells have been shown to display a differentiation-linked increase in GM-CSF receptors. In a previous study we found that the number of low and high affinity receptors per cell on undifferentiated cells was 1231±398 and 299±98 respectively, rising to 23661788 and 647+249 respectively after 6 days culture in DMSO (Roberts et al., 1994 ). A similar increase in GM-CSF receptor number has been reported after 5 days culture in RA (Budel et al., 1993). Stim ulation o f undifferentiated and differentiated H L-60 cells with saturating and non-saturating GM-CSF concentrations indicate that activation of STAT 5 and M AP kinase first occurs in both cell types with 1 ng/m l GM -CSF, a concentration calculated to occupy 60% of the receptors. Interestingly there was a difference in the magnitude of activation at this concentration, with only 28% of maximal STAT 5 activation and 16% of total Erk 2 protein phosphorylation occurring in the undifferentiated HL-60 cells, whereas in the differentiated cells 68% of maximal STAT 5 activation and 42% of total Erk 2 protein phosphorylation occurred, which may reflect the greater num ber of high affinity receptors occupied at this concentration. H ow ever in the differentiated H L-60 cells, this sub-optim al concentrations of GM-CSF, still resulted in sustained M AP kinase activation, but this increase in GM -CSF R occurring with differentiation did not lead to a change in the kinetics of STAT 5 activation indicating that these pathways are separately regulated. This prolongation of MAP kinase activation seen with differentiation in the HL-60 cells could have important downstream effects. In the phaeochromocytoma PC 12 cell
line, epidermal growth factor (EGF) results in transient MAP kinase (Erk 2) activation and is associated with cellular proliferation whereas nerve growth factor results in prolonged activation, translocation of MAP kinase to the nucleus and differentiation (M arshall et al., 1995). This difference in the kinetics of MAP kinase activation could reflect the different signal transduction pathways leading to Ras activation that are activated by the different receptors, but when the EGF R is overexpressed there is sustained M AP kinase activation and differentiation ensues. It is thus possible that the change in kinetics of GM-CSF mediated MAP kinase activation associated with differentiation that was observed, is at least in part due to the change in high affinity recepto r expression. However in the differentiated H L-60 cells, sub-optim al concentrations o f GM-CSF, calculated to occupy a broadly similar number o f high affinity receptors as optimal (10 ng/ml) levels of GM -CSF in the undifferentiated cells, still resulted in sustained MAP kinase activation. This is in accord with previous data on GM-CSF-mediated phosphorylation patterns in differentiated HL-60 cells (Roberts et al., 1994) and suggests that differentiation-linked changes other than up-regulation of GM-CSF receptors may also partake in the modulation of the kinetics of the M AP kinase response.
The kinetics o f M AP kinase activation was also investigated for TF-1 cells and neutrophils. TF-1 cells are immature myeloid leukaemic cells and the kinetics of activation were similar to the undifferentiated HL-60-cells with no phosphorylated M AP kinase detectable after 15 minutes whereas activation was still observed at 30 minutes in the neutrophils.
These data indicate that the differentiation status o f a cell can influence both the kinetics and magnitude of activation of a given pathway, and that the JAK 2 / STAT 5 pathway and the Ras / MAP kinase pathway are independently regulated.