In this study, we present evidence that supports a pre-requisite role for GSK3β in KIT-mediated mast cell chemotaxis and KIT/FcεRI-mediated enhanced gene expression leading to cytokine production in HuMCs. As reported in other cell types (14
), in quiescent HuMCs, GSK3β was determined to be constitutively activated. This conclusion was supported by the observed basal phosphorylation of the activating tyrosine residue (Y216
) in GSK3β, the phosphorylation of its substrate GS at S641
and the reduction of these phosphorylation states in the GSK3β knockdown cells (). Although we did not observe a consistent increase in phosphorylation of GSK3β at the Y216
position in response to SCF and/or SA, under the conditions utilized to examine chemotaxis and cytokine production, there was an apparent increase in the phosphorylation of GS at S641
under these conditions, which was reduced in the GSK3β knockdown. The constitutive Y216
phosphorylation of GSK3β may be due to the cells being maintained in SCF. Indeed, when the cells were starved of SCF for a prolonged period of time (overnight) prior to stimulation, we were able to observe an SCF-dependent increase in the phosphorylation of this residue. Thus the phosphorylation of this residue may be directly dependent on Kit. Regardless, our results suggest that triggering of mast cells through KIT and/or FcεRI facilitates the ability of GSK3β to phosphorylate its substrate(s) without necessarily increasing its constitutive activity; a potential mechanism of action that is elaborated upon below.
In addition to the phosphorylation of (Y216
) in GSK3β, however, we observed that the inhibitory S9
residue GSK3β was also phosphorylated in a PI3K-dependent manner following SCF/ SA challenge ( and ). This phenomenon has also been reported in monocytes, dendritic cells, and T cells, following exposure to TLR2-, TLR4-, TLR5-, and TLR9-agonists, E coli
, and viral peptide respectively (36
). In our study, however, the observed increased phosphorylation of GS, at least at early time points, would suggest that downregulation of GSK3β activation may occur latently to the constitutive activation. We have previously demonstrated that PI3K, and signals dependent upon PI3K activity, are delayed responses compared to other signals initiated upon FcεRI or Kit activation (12
). Thus it is likely that any response due to downregulating GSK3β activity would be chronologically secondary to those regulated by GSK3β activation. Nevertheless, these data do suggest that the ability of GSK3β to phosphorylate its substrates may depend upon the net balance between positive and negative regulation of GSK3β activity.
The marked reduction in the ability of SCF/SA to enhance IL-8, IL-13, and GM-CSF mRNA levels and IL-8 and GM-CSF secretion, associated with the diminution of GSK3β activity in the GSK3β knockdown-HuMCs (), strongly supports a requirement for GSK3β activity in the regulation of KIT/FcεRI-mediated cytokine production. This conclusion is further supported by the close statistical correlation between the degree of GSK3β knockdown and IL-8 secretion. Similarly, the close correlation between GSK3β knockdown and reduction in SCF-induced chemotaxis in the GSK3β knockdown-HuMCs, also provides evidence for a pre-requisite role for GSK3β in the SCF-induced chemotactic response.
There are conflicting reports regarding the role of GSK3β in cytokine production in other cells of hematopoietic lineage. Treatment of monocytes with GSK3β inhibitors such as LiCl and/or SB216763, or with GSK3β–targeted siRNA, has been reported to inhibit TLR2-, 4-, 5-, and 9-dependent release of IL-1β, IL-6, TNF-α, IL-12, and IFN-γ but to enhance TLR-dependent production of IL-10 (36
). GSK3β inhibitors were also reported to inhibit E coli-induced IL-12, IL-6 and TNF-α, but not IL-10, release from dendritic cells (37
). In contrast, in T cells, GSK3β inhibitors were observed to enhance viral peptide-induced IL-2 production, whereas over-expression of GSK3β in T cells down-regulated the response (38
). This apparent dichotomy in the GSK3β-dependent regulation of cytokine generation in the various cell types may reflect the potential for GSK3β to both negatively and positively regulate transcriptional signaling pathways for cytokine production. Indeed, it is possible that, in addition to regulating positive signals, negative signaling pathways may also be regulated by GSK3β in mast cells. In this respect, it has been suggested that the ability of AKT to enhance cytokine generation through NF-AT activation in mouse mast cells may be due to downregulation of GSK3β activity (39
). Whether this may also be true for HuMCs is unclear from the present study, however the induced phosphorylation of the inhibitory GSK3β S9
residue in response to SCF/SA in HuMCs was reduced by the PI3K inhibitor wortmannin ().
There has emerged no common mechanistic explanation as to how GSK3β may be exerting its regulatory influence on cytokine generation and other process in hematopoietic cells. As we have previously demonstrated that the mTORC1 cascade contributes to KIT/FcεRI-mediated cytokine production and KIT-mediated mast cell chemotaxis; (12
) and as GSK3β has been proposed to regulate the mTOR pathway through phosphorylation of tuberin (26
), the scenario existed that, in the HuMCs, GSK3β may be acting via regulation of mTOR pathways. However, the observations that the KIT/FcεRI-mediated phosphorylation of components of the mTORC1 and mTORC2 cascades was not reduced in the GSK3β knockdown-HuMC, (), argues against this possibility. It has been proposed, that the contrasting roles for GSK3β in TLR cytokine production in monocytes may be explained by opposing regulation of the transcription factors CREB and NF-κB through competition for binding to a common co-activator protein CBP (CREB binding protein) (36
). According to this model, GSK3β inhibition would increase CREB activation allowing CREB to compete with the p65 subunit of NF-κB for binding to CBP. In our present study, however, although SCF/SA-induced phosphorylation of the p65 subunit of NF-κB was observed to be significantly reduced in the GSK3β knockdown-HuMCs (), we did not consistently observe an increase in CREB activity in these cells (data not shown). Regardless, these data are in agreement with other studies showing that GSK3β is required for NF-κB activation (19
The most remarkable defects that we observed however in the GSK3β knockdown-HuMCs were in the p38 and JNK MAPK pathways and, particularly, in the respective downstream transcription factors ATF2 and c-Jun. JNK activity has previously been shown to regulate cytokine production mediated by AP1 transcription factors in both mouse bone marrow-derived mast cells (32
) and HuMCs (6
) Similarly, both JNK and p38 have been previously described to regulate mast cell chemotaxis (33
). Therefore, the reduced SCF/SA-induced cytokine production and SCF-induced chemotaxis observed in the GSK3β knockdown-HuMCs may be explained by defective JNK and p38 signaling in these cells ().
How GSK3β may act as a pre-requisite signal for the regulation of these pathways may be explained by the unique manner in which GSKβ phosphorylates its substrates. As discussed, GSK3β substrates require prior phosphorylation by a secondary kinase at amino acids 4–5 COOH-termini to the GSK3β phosphorylation sites for optimal GSK3β-mediated phosphorylation. Thus, although, GSK3β is active in resting conditions, it cannot optimally phosphorylate its substrates, until upon FcεRI or KIT kinase activation, the GSK3β substrates become phosphorylated as a consequence of the activation of one of the kinases downstream of these receptors. This would then allow GSK3β to optimally phosphorylate its target signaling proteins and hence transduce the signals required for gene expression leading to cytokine production, and the processes required for chemotaxis (). Of potential relevance may be the presence of two highly conserved SxxxS/T sequences in MKK3 and MKK6 which are responsible for the phosphorylation and activation of p38, and in MKK4 and MKK7 which are responsible for the phosphorylation and activation of JNK. Multiple such sequences are also found in MEKK1 and MEKK4, upstream kinases of MKK4 and MKK7. Thus, phosphorylation of these sites by GSK3β following initial phosphorylation by a “priming” serine/threonine kinase may provide a mechanism by which constitutive activation of GSK3β may regulate the activation of p38 and JNK and subsequent downstream transcription factors. In support of this conclusion, we observed that SCF and SA/SCF-mediated MKK3/6 phosphorylation was markedly reduced in the GSK3β knockdown-HuMCs ().
Potential model by which constitutively activated GSK3β may regulate HuMC cytokine production and chemotaxis
In summary, here we have presented evidence to support the conclusion that GSK3β is a pre-requisite signal for KIT-mediated chemotaxis and KIT/FcεRI-mediated cytokine production in human mast cells. The regulation of cytokine generation by GSK3β could be explained by the differential regulation of transcriptional control downstream of JNK and p38, as well as transcriptional control of NF-κB p65 subunit, whereas the regulation of the chemotactic response by GSK3β may be explained by its modulation of JNK- and p38-dependent pathways. As with other cells types, it is however possible, that, as yet undefined, inhibitory pathways both regulating GSK3β activation and regulated by GSK3β activity may play a role in human mast cell biology. Thus GSK3β may be act as a central regulator for the precise control of the signaling processes required for mast cell chemotaxis and cytokine production.