(Received for publication, September 11, 1995)
From the
Granulocyte-macrophage colony-stimulating factor (GM-CSF) rapidly and transiently induces the transcriptional activation of the early growth response gene-1 (egr-1) in the human factor-dependent myeloid leukemic cell line, TF-1. We previously demonstrated that the cAMP response element (CRE) is required for GM-CSF-induced egr-1 expression and that phosphorylation of CREB on serine 133 plays a critical role during GM-CSF signal transduction. To determine whether GM-CSF activates signaling pathways through a protein kinase Adependent or -independent pathway, we measured cAMP levels following GM-CSF or forskolin treatment of TF-1 cells. Forskolin but not GM-CSF stimulation resulted in an increase in cAMP levels. Transient transfection assays with TF-1 cells were also performed with a -116-nucleotide egr-1 promoter construct and the protein kinase inhibitor, PKI. Although PKI inhibited forskolin induction of the -116-nucleotide construct, it did not affect GM-CSF stimulation of this construct. In the present study, we demonstrated that GM-CSF induces egr-1 expression through a protein kinase A-independent pathway.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) ()stimulates the proliferation and maturation of myeloid
progenitors and enhances the function of differentiated effector
cells(1, 2, 3) . The biological activities of
GM-CSF are mediated by a heterodimeric receptor which consists of an
and
subunit. The
subunit is critical for signal
transduction, but does not contain intrinsic tyrosine kinase
activity(4, 5) . The interaction of GM-CSF and its
receptor results in the activation of a number of signaling molecules,
including JAK2, Ras, Raf, and mitogen-activated protein
kinase(4, 6, 7) . Phosphorylation of several
of these kinases leads to induction of the growth-related genes,
c-fos, c-myc, and egr-1(4, 8) . The link between cytoplasmic
events and the activation of specific transcription factors in the
nucleus has not been studied extensively.
We previously demonstrated that the induction of the immediate early gene, egr-1, is rapid, transient, and independent of protein synthesis(9) . Transient transfections of egr-1 promoter constructs in the human factor-dependent myeloid leukemic cell line, TF-1, showed that the cAMP response element (CRE) located between nucleotides -57 and -76 was required for transcriptional activation in response to GM-CSF(9) . We also demonstrated that the CRE-binding protein, CREB, associates with the CRE in the -116-nt region of the promoter and is phosphorylated on serine 133 in GM-CSF-stimulated cells(10) . This phosphorylation of CREB is critical for GM-CSF-induced egr-1 expression.
The mechanism of CREB phosphorylation on serine 133 has been shown previously to be mediated through a protein kinase A-dependent pathway(11, 12, 13) . Recently, CREB has been demonstrated to be activated by a PKA-independent pathway(14) . To determine whether GM-CSF signaling results in activation of CREB through a PKA-dependent or -independent pathway in TF-1 cells, we measured cAMP levels in cells stimulated with GM-CSF. We further examined the effects of the protein kinase A inhibitor, PKI, on the transcriptional activation of egr-1. PKI is a potent inhibitor of the catalytic subunit of the cAMP-dependent protein kinase. Overexpression of the cloned human PKI has been shown to inhibit protein kinase A activity(15, 16, 17) . Transient co-transfection assays were performed in TF-1 cells with an expression plasmid containing PKI and a -116-nt construct, which contains the CRE site. Our results demonstrate that GM-CSF stimulation does not increase cAMP levels and that the transcriptional activation of egr-1 occurs through a PKA-independent pathway.
Figure 1:
Measurement of cAMP levels in TF-1 cells
stimulated with GM-CSF. TF-1 cells (10) were factor- and
serum-starved for 24 h and placed in RPMI and 0.5% BSA. Cells were
stimulated at 1, 2, 5, 10, and 15 min with rhGM-CSF (1 nM) or
forskolin (10 µM) and lysed with 60% ethanol. Supernatants
were analyzed for cAMP levels (picomoles/tube) using the cAMP Assay
System (Amersham). Solid curve, TF-1 cells stimulated with
forskolin; dashed curve, cells stimulated with GM-CSF. These
data represent the average of three separate experiments; each time
point was performed in duplicate.
Our experiments demonstrated that the -116-nt construct transfected with the vector control, pcDNA3, resulted in a 4.0-fold induction in response to GM-CSF stimulation (Fig. 2). When the -116-nt construct was co-transfected with the PKI construct, there was no difference in fold induction in response to GM-CSF in three independent experiments (p = 0.748). The difference in the fold induction between pCAT or the -116-nt construct co-transfected with the vector pcDNA3 was significant (p < 0.05). In response to forskolin stimulation, a 2.1-fold induction of the -116-nt construct was observed (Fig. 2). Although this fold induction was approximately half of that seen with GM-CSF, the value was found to be statistically significant (p < 0.05) compared to that of the vector pCAT. When the -116-nt construct was co-transfected with the PKI plasmid and stimulated with forskolin, a statistically significant decrease in fold induction was also observed compared to the pcDNA3 vector (p < 0.05, Fig. 2). These data represent an average of two to four separate experiments, with each transfection performed in duplicate. The low degree of stimulation by forskolin may be due to the fact that a single CRE in the -116-nt construct was not sufficient to activate transcription more than 2-fold. Our previous results demonstrated that the serum response element contained within the -116-nt region is required for GM-CSF-induced transcriptional activation of egr-1. Cooperation between CREB and serum response element-binding proteins may enhance interaction with the transcriptional machinery, explaining the increase in stimulation by GM-CSF.
Figure 2:
PKI inhibits the transcriptional
activation of egr-1 by forskolin but not by GM-CSF. TF-1 cells
(10) were factor- and serum-starved for 24 h and placed in
serum-free media. Twenty micrograms of reporter construct -116cat
and 22 µg of PKI were electroporated into TF-1 cells and stimulated
with diluent control (0.02% BSA in PBS), GM-CSF (1 nM), or
forskolin (10 µM) for 4 h. Three micrograms of CMV
-galactosidase plasmid was co-transfected as the internal control
for transfection efficiency. CAT or
-galactosidase assays were
performed. Fold induction represents the percent acetylation of
constructs stimulated with GM-CSF or forskolin divided by the percent
acetylation of constructs stimulated with diluent control. p values were determined by Student's t test
analysis. These data represent three independent experiments, with each
transfection performed in duplicate.
We also examined the effect
of PKI on the basal activity of the -116cat construct. The basal
activity represents the percent acetylation of cells treated with the
diluent control (PBS, 0.02% BSA). The presence of PKI affected the
basal activity of -116cat (data not shown). PKI, however, did not
affect the general transcriptional activity in TF-1 cells or produce
nonspecific inhibition of cellular processes, since the CMV
-galactosidase activity was not significantly different in cells
transfected with vector compared to PKI. Our results demonstrated that
GM-CSF does not increase cAMP levels in TF-1 cells and that the
presence of PKI does not affect the transcriptional activity of
-116cat in response to GM-CSF. Although PKI affected the basal
activity of -116cat, the fold induction and the
-galactosidase activity were not affected and were consistent with
our previously published data(9, 10) .
The present findings suggest that egr-1 transcriptional activation by CREB in response to GM-CSF is mediated by a protein kinase A-independent pathway. Previously published reports studying the role of adenylate cyclase and cAMP during GM-CSF signal transduction have been inconsistent and cell type-dependent. Studies with macrophages and bone marrow progenitor cells have shown that GM-CSF can elevate intracellular levels of cAMP and activate PKA(18, 19) . Furthermore, GM-CSF enhances the metastatic phenotype of lung carcinoma cells through a protein kinase A-dependent pathway(20) . However, in the mouse mast cell line, PT18, GM-CSF did not alter the activity of cAMP-dependent protein kinase or protein kinase C(21) . Interestingly, our results are consistent with studies that show a decrease in cellular cAMP levels, which is associated with stimulation of HL-60 proliferation, while elevations in cyclic nucleotides are related to an inhibition of HL-60 proliferation and potentiation of differentiation(22) . Since GM-CSF stimulates TF-1 cell proliferation, our findings are in accordance with this hypothesis.
Recently, a novel CREB kinase has
been identified as phosphorylating CREB on serine 133 through a protein
kinase A-independent pathway in nerve growth factor-stimulated
cells(14) . Nerve growth factor stimulates the differentiation
of PC12 cells by activating Ras-dependent pathways, resulting in CREB
phosphorylation and induction of c-fos. Furthermore,
p90 was shown to phosphorylate CREB on serine 133 in
melanoma cells treated with fibroblast growth factor(23) . Our
results suggest that a novel CREB kinase or a previously known
serine/threonine kinase such as p90
or p70s6 may be one
of the candidate kinases responsible for activation of CREB during
GM-CSF signal transduction. Further studies to identify the
kinase-activating CREB will provide a link between the signals from the
cytoplasm to the nucleus that induce growth-related genes.