SAPKs/JNKs are activated in response to a variety of cellular and environmental cues. To determine the role of the SAPK/JNK activator MKK7 in hematopoietic cells, we mutated both mkk7 alleles in ES cells using two selectable markers and generated mkk7-deficient chimeric mice via rag1 complementation. Surprisingly, mkk7−/−rag1−/− chimeric mice exhibit markedly enlarged thymi and thymocyte hyperproliferation. mkk7-deficient mature B cells and mast cell lines also hyperproliferate in response to cytokine and antigen receptor stimulation, but respond normally to death stimuli. In mast cells, the absence of MKK7 results in enhanced cell cycle progression, increased expression of cyclinD1, and significantly reduced expression of the cyclin-dependent kinase inhibitor p16INK4a. In contrast, a deficiency of MKK7 does not affect the expression of p27 nor the activation of ERK1/2 or p38 MAPK. Reexpression of p16INK4a reduced the hyperproliferative phenotype in mkk7−/− mast cells. Intriguingly, although MKK4, the second direct SAPK/JNK activator, is strongly upregulated in mkk7−/− mast cell lines and activated in response to multiple stimuli, SAPK/JNK activation was still completely abolished in response to these same stimuli in the absence of MKK7. Thus, MKK7 is essential for SAPK/JNK activation in mast cells and MKK7 acts as a negative regulator of growth factor– and antigen receptor–induced proliferation of different hematopoietic cell lineages.
The engagement of growth factor receptors or stimulation by mitogens induces the activation of ERK1 and ERK2 in many cell types and these kinases regulate the activation of transcription factors governing cellular proliferation 39
. SAPKs/JNKs are activated in response to a variety of environmental and cellular stresses such as metabolic changes or DNA damage. These stresses can result in cell cycle arrest to allow for repair. However, other than a positive regulatory role for MKK4 and JNK2 in antigen receptor and CD28 costimulation-dependent proliferation of mature T cells 932
, there is little in vivo data to suggest that components of the stress signaling pathway can in fact negatively regulate cell growth. Our results show that loss of a stress signaling kinase, MKK7, results in hyperproliferation of thymocytes, mature B cells, mast cells (our results), and possibly T cells 10
. This hyperproliferation of mkk7−/
− hematopoietic cells appears to be due, not to impaired cell death, but rather to enhanced cell cycle progression.
SAPKs/JNKs can associate with all three members of the Jun-family of transcription factors, c-Jun, JunB, and JunD. These molecules probably have specific and distinct functions in cellular proliferation and depending on the stimulus and cell type, can also mediate differentiation, cell death, and/or growth arrest 40
. In fibroblasts, expression of c-Jun has a positive effect on proliferation 41
, whereas Jun-D 42
and JunB 38
negatively regulate growth. It has been recently shown that increased JunB expression in 3T3 fibroblasts induces high levels of p16INK4a but that other cell cycle inhibitors are not affected 38
. On the other hand, c-Jun overexpression inhibits p16INK4a transcription. Our data show that mkk7−/
− BMMCs display reduced expression of JunB, completely lack expression of p16INK4a, leading to dramatic upregulation of cyclinD1 expression. Thus, in the absence of MKK7, impaired c-Jun and JunD phosphorylation and/or lower expression of and deregulated JunB activity might explain the observed loss of p16INKa expression and increase in cell growth. The competing regulatory influences of JunB and c-Jun on p16INK4a expression provide a molecular framework within which SAPK/JNKs could conceivably control cell growth 38
. Consistent with this hypothesis, reexpression of p16INK4a suppressed hyperproliferation of mkk7−/
− mast cells. However, we observed normal p16INK4a expression in in vitro differentiated B cells. Thus, it needs to be determined whether, similar to BMMCs, hyperproliferation of mkk7−/
− B cells and thymocytes might be also regulated by p16INK4a. Moreover, our data in mkk7−/
− B cells suggest that other molecular targets than p16INK4a exist that control negative regulation of cell cycle progression downstream of MKK7. Identification of such targets should be of interest to the understanding of development and function of hematopoeitic lineages as well as the understanding of cellular transformation in leukemias.
It has been proposed that SAPK/JNK activation triggers apoptosis in response to many types of stress, including UV and γ-irradiation, protein synthesis inhibitors, high osmolarity, toxins, ischemia/reperfusion injury in heart attacks, heat shock, anti-cancer drugs, ceramide, peroxide, and inflammatory cytokines 34
. Several lines of evidence support this view. The overexpression of dominant negative MKK4 can block the induction of cell death by heat shock, irradiation, anti-cancer drugs, peroxide, ceramide, or cytokine deprivation 4344
. In addition, overexpression of inactive c-Jun or dominant negative MEKK1 inhibits the induction of apoptosis by irradiation, ceramide, or heat shock in U937 and BAE cells 43
, and protects PC12 cells from apoptosis triggered by nerve growth factor (NGF) withdrawal 45
. These results suggested that the MKK → SAPK/JNK → c-Jun signaling cascade can transduce proapoptotic signals.
However, recent studies of genetic “knockouts” of SAPK/JNK isoforms and MKK4 have demonstrated that MKK4 and SAPK/JNK activation are not essential for the induction of cell death in response to all apoptotic stimuli. For example, SAPKβ/JNK3 knockout mice are viable but display a specific defect in kainate-induced apoptosis of hippocampal neurons 11
. Similarly, double mutation of jnk1/jnk2
in primary murine fibroblasts protects them against UV- and anisomycin-induced apoptosis 12
. In contrast, we 46
and others 25
have previously reported defective liver formation and massive hepatocyte apoptosis in mouse embryos lacking MKK4. In this case, MKK4 provides a crucial and specific survival signal for hepatocytes during embryonic morphogenesis. Additional genetic analyses of mkk4
-deficient ES cell clones and mouse embryonic fibroblasts have confirmed that both MKK4-dependent and MKK4-independent pathways for SAPK/JNK activation exist 232425
. MKK4 is the critical activator of SAPKs/JNKs in response to anisomycin and heat shock, whereas SAPK/JNK activation in response to osmolarity changes, UV-irradiation, γ-irradiation, or ceramide is independent of MKK4, at least in these cells. Our experiments in mkk7−/
− BMMCs show that MKK7 is required for UV-, anisomycin-, and NaCl-induced SAPK/JNK activation. However, the kinetics and extent of UV-, anisomycin-, and NaCl-induced apoptosis were comparable in mkk7−/
− and mkk7+/+
BMMCs and thymocytes. Thus, although JNK1/2 might be required to mediate UV- and anisomycin-triggered cell death in fibroblasts 12
, MKK7-controlled SAPK/JNK activation does not have any apparent role in the apoptotic response of mast cells and thymocytes to the same stimuli. We conclude that, rather than being essential for apoptosis, the MKK7-SAPK/JNK pathway modulates the death response in a stimulus- and cell type–specific manner.
Signaling pathways for SAPK activation may also be developmentally regulated during lymphopoiesis. PMA/Ca2+
-ionophore stimulation can induce SAPK/JNK activation in mature T cells from mkk4−/
− chimeric mice, but not in immature thymocytes 32
. Perhaps not coincidentally, immature thymocytes express high levels of MKK7 and low levels of MKK4, whereas mature T cells express high levels of MKK4 and low levels of MKK7 4748
. Cell type–specific variation in expression of MKK7 and MKK4 in thymocytes versus mature T cells could explain the normal activation of mature lymph node T cells but enhanced thymic cellularity and thymocyte hyperproliferation observed in our mkk7−/
− chimeric mice.
The finding that different types of stress or different stages of development trigger distinct signaling pathways for SAPK/JNK activation has been explained by differential activation of MKK4 and MKK7 via upstream kinases, and/or differential scaffolding of the MKK4 and MKK7 signaling pathways via adaptor molecules 49
. It has further been proposed that cells can sense different types of endogenous or environmental stress signals and that MKK4- and MKK7-mediated pathways of SAPK/JNK activation are controlled by distinct “transducisomes”; that is, they are structurally and/or biochemically separated 49
. However, it has also been reported that JNK1 50
and JNK3 51
are synergistically activated in vitro by the presence of both MKK4 and MKK7, suggesting that complete activation of SAPK/JNK enzymatic activity may sometimes require phosphorylation by two different MKKs. This situation bears resemblance to recent findings that two separate binding and phosphorylation events by MEK to its substrate ERK may be required for complete ERK activation 5253
The results of our in vitro kinase assays ( D) provide further evidence that MKK4 and MKK7 must cooperate to fully activate SAPKs/JNKs. Functional synergy between MKK4 and MKK7 could explain why overexpression of dominant inhibitory MKK4 or dominant-negative MKK7 inhibits activation of SAPK/JNK in response to multiple stimuli. Whether the synergy between MKK4 and MKK7 in SAPK/JNK activation is a universal mechanism used by other MKK isoforms needs to be tested. Importantly, our results in mkk7−/
− mast cells show that MKK7 expression is required for SAPK/JNK activation in response to all stimuli tested despite the fact that MKK4 expression is upregulated and MKK4 is strongly phosphorylated. Impaired SAPK/JNK activation in mkk7−/
− mast cells despite increased phosphorylation of MKK4 could be explained by functional synergy between MKK4 and MKK7 in these cells in vivo. Alternatively, MKK7 itself and/or an MKK7-associated molecule could provide a scaffold required for the interaction between MKK4 and SAPK/JNKs. Such a mechanism has been reported for Jun-B and c-Jun in which Jun-B can recruit c-Jun to SAPKs/JNKs 54
In conclusion, our data provide evidence that the stress signaling kinase MKK7 is a negative regulator of growth factor and antigen receptor–driven proliferation in hematopoietic cells. We have also demonstrated that MKK7 is essential for SAPK/JNK activation in mast cells.