The Stress Kinase Mitogen-Activated Protein Kinase Kinase (Mkk)7 Is a Negative Regulator of Antigen Receptor and Growth Factor Receptor–Induced Proliferation in Hematopoietic Cells

During the development of all multicellular organisms, cell fate decisions determine whether cells undergo proliferation and differentiation, or apoptosis. Developmental programs and environmental agents trigger distinct and evolutionarily conserved signal transduction cascades that relay signals mediating proliferation, survival, or death. The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases which transduce signals from the cell membrane to the nucleus in response to a wide range of stimuli 1,2. These molecules participate in several different intracellular signaling pathways that control a spectrum of cellular processes, including cell growth, differentiation, transformation, apoptosis, and stress responses 3,4. Important members of the MAPK family are the extracellular signal-regulated kinases ERK1 and ERK2, ERK5, p38 MAPK, and the stress-activated protein kinases (SAPKs, also known as the c-Jun N-terminal kinases [JNKs]) 5,6.

SAPKs/JNKs are activated in response to a variety of cellular stresses such as changes in osmolarity or metabolism, DNA damage, heat shock, ischemia, shear stress, inflammatory cytokines such as TNF and IL-1, and ceramide 3,4,7. Once activated, SAPKs/JNKs regulate gene transcription via phosphorylation of transcription factors, including c-Jun, JunD, activating transcription factor (ATF)-2, nuclear factor of activated T cells (NFAT)4, or ELK-1 8. Biochemical analyses have indicated that SAPK/JNKs and SAPK/JNK-regulated signaling pathways are involved in cell fate decisions resulting in apoptosis, oncogenic transformation, activation of T and B lymphocytes, induction of proinflammatory cytokines, regulation of proliferation and cell cycle arrest, and cardiovascular, renal, and hepatic stress responses 3,4. Genetic analyses have confirmed that SAPKs/JNKs regulate T cell activation 9, Th1/Th2 differentiation 10, neuronal cell death 11, UV-induced cell death in fibroblasts 12, and dorsal closure during Drosophila development 13.

The prototypical MAPK phosphorylation cascade consists of a MAPK kinase kinase (MAPKKK or MEKK), a MAPK kinase (MKK), and a MAPK 14,15. MAPKKK phosphorylate and activate MKK, dual-specificity protein kinases that in turn phosphorylate MAPK. Activation of MAPKs requires phosphorylation of both the threonine and tyrosine residues in the Thr-X-Tyr motif. Several mammalian MKKs have been identified to date, including MEK1, MEK2, MKK3, MKK4 (also known as SEK1 or JNKK1), MEK5, MKK6 16,17,18, and MKK7 (SEK2, JNKK2; references 19,20,21,22). SAPKs/JNKs are activated directly by MKK4 and MKK7, but by no other MKKs. Recent studies of murine embryonic stem (ES) cells lacking the mkk4 gene 23,24,25 have demonstrated that MKK4 is critical for the activation of SAPKs/JNKs in response to the protein synthesis inhibitor anisomycin or heat shock. However, SAPK/JNK activity was still inducible in mkk4-deficient ES cells subjected to UV-irradiation or changes in osmolarity. Loss of MKK7 in ES cells abrogates, but does not abolish, SAPK/JNK activation in response to osmolarity changes, UV-irradiation, heat shock, or anisomycin 10. In addition, D-MKK4 and D-MKK7 serve nonredundant functions in Drosophila 3. These results implied that the SAPK/JNK activators MKK7 and MKK4 operate independently and that different stresses utilize distinct MAPK signaling pathways to activate SAPKs/JNKs.

Recently is has been shown that mkk7-deficient and jnk1/jnk2 double mutant T cells hyperproliferate in response to antigen receptor crosslinking 10, suggesting that the MKK7-regulated stress kinase signaling pathway might negatively regulate T cell activation. Whether genetic inactivation of MKK7 expression has also a role in the activation and proliferation of other hematopoietic cells is not known. We generated mkk7^−/− chimeric mice using recombination activating gene (rag)1 blastocyst complementation and a gene targeting strategy that allowed us to analyze the function of MKK7 in T and B lymphocytes and non-lymphoid bone marrow mast cell lines (BMMCs). mkk7^−/−rag1^−/− chimeric mice have enlarged thymi, and thymocytes from these mice hyperproliferate in response to antigen receptor stimulation. Similarly, mkk7 deficiency leads to hyperproliferation of B cells and BMMCs after stimulation of antigen or growth factor receptors. Mutation of mkk7 in BMMCs results in loss of expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1. In BMMCs, MKK7 is required to relay signaling in response to IgE, anisomycin, UV-irradiation, or NaCl stimulation to activation of SAPKs/JNKs. Ectopic reexpression of p16INK4a in mkk7^−/− mast cells abrogates the hyperproliferative response. This study shows that a stress signaling kinase, i.e., MKK7, can negatively regulate the proliferation of multiple hematopoietic cell lineages.

A 12-kb genomic mkk7 fragment was isolated from a 129/Ola mouse library and inserted into the NotI site of pBluescript II. A targeting vector was constructed containing a 709-bp short arm and a 5.4-kb long arm of homology flanking a neomycin resistance cassette (Neo) inserted into the EcoRI/Bgl-II sites of the genomic clone in antisense orientation to mkk7 transcription. The linearized construct was electroporated into 10⁷ E14K ES cells derived from 129/Ola mice. ES cell colonies resistant to G418 [0.3 mg/ml] were screened for homologous recombination by PCR using primers specific for mkk7 genomic sequences and Neo. Recombinant colonies were confirmed by Southern blotting of XbaI-digested genomic DNA hybridized to a 530-bp 3′ flanking probe. E14K ES cells heterozygous for the mkk7 mutation were retargeted using a hygromycin-resistance cassette that replaced parts of exon 13 and exon 14 of the mkk7 gene. For construction of somatic chimeras, two independent mkk7^−/− ES cell clones (mkk7^neo/hyg) and a mkk7^+/− (mkk7^+/neo) E14K ES cell clone were injected into 3.5 day rag1^−/− blastocysts and transferred to pseudopregnant foster mothers as described 26. As E14 ES cells are derived from 129/Ola mice, age-matched 129/Ola mice were used as wild-type controls in some experiments. For Northern blotting, total RNA (20 μg) was isolated from lymph node cells, thymocytes, spleen cells, and mast cell lines, electrophoresed, transferred to a Hybond-N membrane (Amersham Pharmacia Biotech), and hybridized with probes specific for mkk7 22. All data presented in this study were obtained from two independently derived mkk7^−/− ES cell clones, and all results were comparable between them. If not otherwise stated, all mice used for experiments were between 6–10 wk of age. All mice were maintained at the animal facilities of the Ontario Cancer Institute under specific pathogen-free conditions according to institutional guidelines.

Single cell suspensions of thymi, lymph nodes, bone marrow, blood, and spleens from mkk7^+/+, mkk7^+/− rag1^−/−, and mkk7^−/−rag1^−/− chimeras were stained with FITC-, PE-, or biotin-conjugated Abs reactive to: Thy1.2, CD3ε, TCRα/β, TCRVβ6, TCRVβ8.1/8.2, TCRVβ14, TCRγ/δ, CD4, CD8, CD25, CTLA4, CD28, CD95 (FAS), CD5, CD44, CD45, CD69, CD11a (LFA-1), CD11b (Mac-1), MHC class II (I-Ab), CD23, B220, CD43, sIgM, sIgD, CD19, or Gr-1 (all Abs were from BD PharMingen). For the analysis of thymocyte precursors, single cell suspensions were stained with PE-conjugated anti-CD4, anti-CD8, anti-CD3ε, anti-B220, anti-CD11b, anti-Gr-1, and anti-TCRα/β; FITC-conjugated anti-CD25; and biotin-conjugated anti-CD44. PE-negative precursor cells (triple-negative thymocytes) were analyzed for expression of CD25 and CD44. Biotinylated Abs were visualized using streptavidin-RED670 (GIBCO BRL). All samples were analyzed by flow cytometry using a FACScan™ (Becton Dickinson).

For proliferation assays, purified lymph node T cells (10⁵ cells/well) were placed into round bottom 96-well plates (Nunc) in freshly prepared IMDM (10% FCS, 10 μM β-mercaptoethanol) and stimulated with PMA plus the Ca²⁺ ionophore A23617, soluble anti-CD3ε (clone 145-2C11, hamster IgG), soluble anti-CD28 (clone 37.51; BD PharMingen), or the mitogen Con A (Amersham Pharmacia Biotech). B cells (10⁵ cells/well) were stimulated with anti-IgM (61-6800; Zymed Laboratories), anti-IgM F(ab′)₂ fragment (61-5900; Zymed Laboratories), anti-CD40 (clone 145-2C11; BD PharMingen), or LPS (Sigma-Aldrich). Cells were stimulated in triplicate for different time periods and pulsed for the last 12 h with 1 μCi/well [³H]thymidine (Amersham Pharmacia Biotech). [³H]Thymidine incorporation was measured using a β-scintillation counter (Coulter). To determine cytokine production in these cultures, the culture supernatants were removed and assayed in triplicate for the production of IL-2 and IFN-γ using ELISA (R&D Systems). For the analysis of thymocyte apoptosis, single cell suspensions were cultured for various times in 24-well flat bottom tissue culture plates (Nunc) at a density of 2 × 10⁶/ml in IMDM (10% FCS, 10 μM β-mercaptoethanol). The following stimuli were administered: anisomycin (10 μg/ml; Sigma-Aldrich); LY294002 (50 μM; Sigma-Aldrich); dexamethasone (10 nM; Sigma-Aldrich); anti-CD95 (FAS) (Jo2; BD PharMingen); sorbitol (0.4 M; Sigma-Aldrich); NaCl (0.3 M); heat shock (43°C, 30 min); or anti-CD3ε (clone 145-2C11, hamster IgG; BD PharMingen). In the latter case, anti-CD3ε was immobilized on the plates with 10 μg/ml rabbit anti–hamster IgG (Jackson ImmunoResearch Laboratories). At various time points after stimulation, thymocytes were harvested, stained with the vital dye 7-amino-actinomycin D (7-AAD), anti-CD4, and anti-CD8, and subjected to flow cytometric analysis. Apoptosis was confirmed using Annexin V staining to visualize membrane changes, propidium iodide (PI) staining and trypan blue exclusion to test for membrane integrity, and DIOC6[3] staining to test for mitochondrial potential.

Bone marrow cells were flushed from femurs of mkk7^−/−rag1^−/− chimeric and control mice. Cells were washed twice with PBS and resuspended at 5 × 10⁵/ml in OptiMEM medium supplemented with 10% FCS, 50 μM β-mercaptoethanol, antibiotics (penicillin plus streptomycin; GIBCO/BRL), and 2 ng/ml IL3 (Genzyme). BMMCs were isolated by continuous transfer of cells growing in suspension as described 27. Proliferation of BMMCs was assessed by [³H]thymidine incorporation. In brief, cells were deprived of IL-3 for 12 h, cultured in the presence of various concentrations of IL-3 or stem cell factor (SCF) for 24 h, and pulse-labeled with [³H]thymidine for 12 h. For cell death assays, BMMCs were deprived of IL-3 and/or serum, or stimulated with NaCl, UV-irradiation, anisomycin, or heat shock as above. At various time points after stimulation, apoptosis was determined in triplicate cultures by 7-AAD and Annexin V staining. Cell cycle progression was monitored using PI staining. For infection experiments, pBabe-puro or pBabe-puro–expressing p16INK4a (gift of E. Wagner, Institute for Molecular Pathology, Vienna, Austria) were transfected into a phoenix-E packaging cell line. mkk7^−/− BMMCs were cultured in the culture supernatant of the packaging cells for 1 d and selected with 2.5 μg/ml puromycin for 1 wk. In vitro generation of B220⁺IgM⁺ B cells and FcεR⁺c-Kit⁺ mast cells from mkk7-mutant ES cell lines was as described 28,29.

BMMCs (2 × 10⁶/100 μl) were cultured in medium alone or stimulated with IL-3 (30 ng/ml), NaCl (0.5 M), anisomycin (10 μg/ml), UV-irradiation (500 mJ, using a DNA-Stratalinker; Stratagene), heat shock (45°C in a prewarmed waterbath), cycloheximide (50 μg/ml), or TNFα (100 ng/ml), for various time periods. For FcεR stimulation, cells were incubated with anti-DNP IgE mAb (7 μg/ml, clone SPE7; Sigma-Aldrich) for 1 h on ice, followed by the addition of 50 ng/ml DNP (Sigma-Aldrich) at 37°C to crosslink the FcεR. Cells were pelleted by centrifugation and resuspended in 100 μl ice cold lysis buffer (1% Triton X-100, 10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 30 mM Na₄P₂O₇, 5 mM EDTA, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na₃VO₄). Cell lysates were fractionated by SDS-PAGE and the proteins transferred to PVDF membranes and immunoblotted. Activation of p38 MAPK, ERK1/2, and PKB/Akt was detected using Abs specific for phospho-p38 MAPK (Thr180/Tyr182), phospho-ERK1/2 (Thr202/Tyr204), phospho-PKB/Akt (Ser473 and Thr308), or phospho-MKK4 (Thr223; New England Biolabs, Inc. or Upstate Biotechnology), because phosphorylation at these sites has been shown to induce activation of these kinases. To verify equivalent loading and to confirm the identity of the phosphorylated proteins, membranes were stripped and blotted with Abs to nonphosphorylated PKB/Akt, ERK1/ERK2, SAPKs/JNKs, p38 MAPK (all from Santa Cruz Biotechnology, Inc.), and MKK4 (Upstate Biotechnology) or actin (Sigma-Aldrich). Protein levels of p16INK4a, p27, actin, JunB, cyclinD1 (Santa Cruz Biotechnology, Inc.), and MKK7 (BD Transduction) were determined in total cell lysates by immunoblotting followed by visualization using enhanced chemoluminescence (ECL). For detection of SAPK/JNK kinase activity, total SAPKs/JNKs were immunoprecipitated (1 h, 4°C) from BMMC lysates using polyclonal rabbit anti-SAPK/JNK IgG reactive against all SAPK/JNK isoforms 5. Immune complexes were harvested on protein A-Sepharose beads. For kinase assays, immune complexes were washed three times with the lysis buffer. The beads were resuspended in 20 μl kinase buffer (10 mM MgCl₂, 50 mM Tris-Cl, pH 7.5, 1 mM EGTA, pH 7.5) and SAPK/JNK activity was assayed at 30°C for 30 min in the presence of 1 μCi [γ-³²P]ATP using 5 μg GST-c-Jun as the in vitro substrate. The reaction was stopped by the addition of 5× SDS sample buffer. GST-c-Jun phosphorylation was visualized by autoradiography as described previously 5.

To disrupt the murine mkk7 gene in E14K ES cells, a portion of exon 9, including the phosphorylation motif, was replaced with a PGK-Neo cassette (Fig. 1 A). Because homozygous inactivation of the mkk7 gene in mice leads to embryonic lethality (reference 10, and our own unpublished data), we targeted the second allele in mkk7^+/− PGK-Neo ES cells using a different targeting vector containing a hygromycin resistance cassette (Hygro; Fig. 1 B). This vector targeted a different region (parts of exons 13 and 14) of the mkk7 gene, allowing us to ascertain homologous recombination at both alleles (Fig. 1 C). Disruption of both mkk7 alleles in ES cells was confirmed by Southern blot analysis of genomic DNA (Fig. 1 D) and Western blotting (Fig. 1 F).

To study the development and activation of T and B cells in the absence of MKK7, two different mkk7^−/− (mkk7^Neo/Hyg) ES cell lines were used to generate mkk7^−/−rag1^−/− chimeric mice via rag1 blastocyst complementation 23. The capacity for hygromycin selection also permitted us to generate mkk7^−/− BMMCs from the chimeric mice. Loss of mkk7 mRNA expression in several different cell types from mkk7^−/−rag1^−/− mice was confirmed by Northern blot analysis. No detectable mkk7 mRNA expression could be found in RNA isolated from mutant ES cells (not shown), thymocytes, lymph nodes, or two different BMMC lines (Fig. 1 E). Absence of mkk7 mRNA expression was confirmed by reverse transcription (RT)-PCR (not shown). Thus, our gene targeting strategy provided us with a means of analyzing the role of MKK7 in multiple hematopoietic cell lineages, i.e., T and B lymphocytes and BMMCs.

Although mkk7^−/−rag1^−/− chimeric mice showed normal numbers and subpopulations of peripheral T cells, we were surprised to observe that these animals displayed thymi of significantly increased cellularity (Table). The proportions of CD4⁻CD8⁻ double negative (DN) progenitor cells, immature CD4⁺CD8⁺ double positive (DP) cells, and mature CD4⁺ and CD8⁺ single positive (SP) thymocytes were equal in mkk7^−/−rag1^−/− and mkk7^+/− rag1^−/− thymi (Fig. 2 A). Moreover, there were no differences in surface expression levels of TCRα/β, CD3, CD4, CD8, CD28, CD45, TCRVβ subclasses, or CD95 on SP or DP thymocytes (not shown). The maturation of DN precursor populations as defined by c-Kit, CD44, and CD25 surface expression, and the maturation of immature DP thymocytes to mature SP thymocytes as defined by CD69, CD44, HSA, CD5, and H2-K^b expression, were also similar in mkk7^−/−rag1^−/− and mkk7^+/−rag1^−/− thymocyte populations. Thus, MKK7 is not required for thymocyte development or thymic positive selection. However, loss of MKK7 leads to increased thymic cellularity.

Thymic cellularity is maintained by a balance between apoptosis and cellular proliferation, activities that in many cell types involve SAPK/JNK signaling. To examine the impact of MKK7 deficiency on thymocyte survival, we evaluated the responses in vitro of mkk7^−/−rag1^−/− thymocytes to treatment with anti-CD3ε or anti-FAS (CD95), stimuli known to induce the apoptotic death of DP thymocytes. No apparent differences in the kinetics or extents of cell death were observed between mutant and control thymocytes after stimulation of either CD3ε (Fig. 2 B) or FAS (Fig. 2 C). Furthermore, the susceptibility of mkk7^−/− thymocytes to apoptosis induced by anisomycin, the PI3K inhibitor LY294002, sorbitol (osmotic shock), or dexamethasone was comparable to that of mkk7^+/− chimeric thymocytes (Fig. 2 D). However, mkk7-deficient thymocytes exhibited hyperproliferation in response to antigen receptor engagement (Fig. 2 E). These data imply that antigen receptor–triggered hyperproliferation of thymocytes, and not reduced cell death, accounts for the increased thymic cellularity in mkk7^−/−rag1^−/− chimeric mice. However, as mainly single positive thymocyte proliferate in our assays, we cannot exclude the possibility that MKK7 controls thymocyte cell death in response to a yet unidentified signals that controls cellularity. Moreover, MKK7 could control proliferation of DN progenitor cells.

The SAPK/JNK signaling pathway has been shown to integrate antigen receptor and costimulatory signals in primary T cells and T cell lines 30 and to control Th1/Th2 differentiation 10,31. It has also been reported that T cells bearing mutations of either mkk4 or jnk2 display partial defects in cell proliferation and IL-2 production in response to suboptimal stimulation of the TCR plus CD28 9,32. In addition, transfection and pharmacological inhibitor studies have suggested that MKK7 might regulate production of IL-2 in T cell lines 33. On the other, it has been reported in mkk7 and jnk1/jnk2 double mutant T cells that the MKK7-SAPK/JNK signaling pathway has no role in T cell activation but rather influences effector functions 10. Moreover, the same group reported that mkk7 and jnk1/jnk2 double mutant T cells hyperproliferate and produce more IL-2 in in vitro stimulation assays 10. We therefore explored whether the absence of MKK7 had a similar effect on the numbers, activation, or function of peripheral T cells in our chimeric mice. Lymph nodes and spleens of mkk7^−/−rag1^−/− chimeric mice contained normal numbers and ratios of CD4⁺ and CD8⁺ T cells (Table and Fig. 3 A). Moreover, the surface levels of TCRα/β, CD3, CD4, CD8, CD28, CD45, CD44, LFA1, CD25, and CD69 on both splenic and lymph node CD4⁺ and CD8⁺ T cells were comparable in mkk7^−/−rag1^−/− and mkk7^+/+rag1^−/− mice (not shown). Thus, despite enhanced thymic cellularity, loss of MKK7 does not affect cell numbers or surface phenotypes of peripheral T cells.

To investigate the role of MKK7 in T cell function, we analyzed the activation and proliferation of peripheral T cells in response to different concentrations of anti-CD3ε Ab, anti-CD3ε plus anti-CD28 Abs, the mitogen Con A, or PMA/calcium-ionophore, stimuli that bypass the initial antigen receptor signal. At all doses and stimuli tested, the kinetics (not shown) and extents (Fig. 3 B) of proliferation were comparable between mkk7^+/−rag1^−/− and mkk7^−/− rag1^−/− lymph node T cells. Similarly, IL-2 and IFN-γ production by lymph node T cells was also normal in the absence of MKK7 (not shown). It should be noted that in some experiments we observed increased proliferation as well as increased IL-2 and IFN-γ production by mkk7^−/− T cells compared with mkk7^+/− T cells; however, this increased proliferation was never statistically significant. These results show that loss of MKK7 has no apparent effect on peripheral T cell homeostasis, activation or function.

SAPK/JNK activation has been implicated in signal transduction downstream of B cell stimulation via CD40 engagement or LPS treatment 34. However, no proliferative or functional defects were apparent in B cells from mice deficient for mkk4, jnk1, or jnk2 31,35,36. mkk7^−/−rag1^−/− chimeric mice displayed normal numbers and differentiation of B220⁺CD25⁺, B220⁺CD25⁻, B220⁺CD43⁺, B220⁺CD43⁻, B220⁺sIgM⁺, and CD19⁺sIgM⁺sIgD⁺ B cells in the bone marrow, and normal populations of B220⁺sIgM⁺sIgD⁺ B cells in peripheral lymphoid organs (Table, Fig. 4 A, and not shown). Basal serum levels of the Ig subclasses IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA were also comparable in mkk7^−/−rag1^−/− and mkk^+/−rag1^−/− chimeric mice (not shown). Thus, B cell development in the bone marrow and mature B cell populations in the periphery appear normal in the absence of MKK7.

To determine whether MKK7 is required for B cell activation, we measured B cell proliferation in response to LPS treatment, anti-CD40 Ab, IgM (Fab′)₂ crosslinking, and the engagement of IgM plus CD40. Intriguingly, the proliferation of mkk7^−/−rag1^−/− B cells was significantly enhanced in response to anti-IgM (Fab′)₂ crosslinking (Fig. 4 B). The increased proliferative response was still observed when mkk7-deficient cells were stimulated with the intact anti-IgM Ab, suggesting that the hyperproliferation is not due to impaired negative signals mediated via FcγRIIB (not shown). The proliferation of mkk7-deficient B cells was also markedly enhanced by treatment with LPS, anti-CD40, or anti-IgM (Fab′)₂ plus anti-CD40 (Fig. 4 B). Thus, as was true for thymocytes, loss of MMK7 in B cells results in hyperproliferation in response to growth stimuli or engagement of the antigen receptor.

SAPKs/JNKs and their activators MKK4 and MKK7 are expressed in all hematopoietic lineages. However, the role of MKK7 and SAPKs/JNKs in the function of non-lymphoid hematopoietic cells has not been explored using mutational analysis. Our targeting strategy of mutating one mkk7 allele with a Neo-cassette and the other allele with a hygromycin-resistance vector allowed us to study the role of MKK7 in a non-lymphoid hematopoietic lineage, BMMC lines. Because mast cells derived from mkk7^−/− ES cells in the chimeric mice (but not those derived from rag1^−/− ES cells) were resistant to hygromycin, several BMMC lines known to be mkk7^−/− could readily be established (Fig. 1 E). As controls, we generated mkk7^+/+ BMMCs from 129/Ola mice, which have the same genetic background as the mkk7^−/− E14 ES cells. BMMCs of both genotypes showed similar expression levels of c-Kit (SCF-R) and the IgE receptor (not shown), two markers characteristic of mature BMMCs 37. Thus, loss of MKK7 expression does not prevent the emergence and differentiation of BMMCs. However, the proliferation of mkk7^−/− BMMCs in response to the mast cell growth factor IL-3 (Fig. 5 A) or SCF (c-Kit-ligand; Fig. 5 B) was strikingly increased compared with that of wild-type BMMCs, paralleling the hyperproliferation observed in mkk7-deficient thymocytes and B cells. Again, no significant differences between mkk7^+/+ and mkk7^−/− BMMCs were observed in apoptosis induced by growth factor deprivation or osmotic stress (NaCl) (not shown), or by anisomycin, heat shock, or UV-irradiation (Fig. 5 C). Similar results were obtained using in vitro differentiated FcεR⁺c-Kit⁺ mast cell lines from mkk7^+/+, mkk7^+/−, and mkk7^−/− ES cells that were selected and differentiated under identical culture conditions (not shown) 28. These data indicate that loss of MKK7 results in the hyperproliferation of both lymphoid and non-lymphoid hematopoietic cells in response to multiple growth factor or antigen receptor stimulation.

At the molecular level, immunoblot analyses of proteins in BMMCs revealed that expression levels of p46 and p56 SAPK/JNK isoforms, p38 MAPK, ERK1/ERK2, PKB/Akt, and actin were comparable in mkk7^+/+ and mkk7^−/− BMMCs (Fig. 6 A). However, expression of the cell cycle inhibitory molecules, p16INK4a, which act principally on cyclinD1 and CDK4-6, was completely abrogated in mkk7^−/− BMMCs, leading to a concomitant increase in cyclinD1 expression (Fig. 6 A). Interestingly, whereas expression of c-Jun was comparable between mkk7^+/+ and mkk7^−/− BMMCs, expression of JunB, which has been shown to upregulate p16INK4a expression 38, was markedly decreased in mkk7^−/− BMMCs. Expression of p27, another cell cycle inhibitor, was not affected by the absence of MKK7 (Fig. 6 A). However, it should be noted that loss of MKK7 does not always result in downregulation of p16INK4a expression, as p16INK4a expression appeared normal in B220⁺IgM⁺ B cells after in vitro differentiation of mkk7^+/+ and mkk7^−/− ES cells into B cells (Fig. 6 B, and data not shown). Thus, mutation of mkk7 in BMMCs, but not in B cells, results in loss of expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1.

The previous findings that SAPKs/JNKs could be activated by both MKK4-dependent and MKK4-independent signaling cascades 23,24,25, suggested that cells can sense particular stresses and trigger distinct signaling paths in response. To investigate whether similar MKK7-dependent or -independent pathways might also control SAPK/JNK activation, mkk7^+/+ and mkk7^−/− BMMCs were either treated with stress stimuli or activated via engagement of membrane receptors, and the activation of components of stress signaling pathways was assessed.

In wild-type BMMCs, SAPKs/JNKs are strongly activated in response to Fcε receptor stimulation (Fig. 6 C). Moreover, SAPKs/JNKs are strongly activated after stimulation with anisomycin, UV-irradiation, or NaCl (Fig. 6 D). However, IL-3, anisomycin, heat shock, UV-irradiation, NaCl, cycloheximide, or TNFα used at various doses and in a series of activation protocols failed to activate SAPKs/JNKs in mkk7^−/− BMMCs (Fig. 6C and Fig. D, and not shown). FcεR stimulation or anisomycin treatment led to normal activation (phosphorylation) of p38 MAPK in mkk7^−/− BMMCs. Similarly, activation of ERK1/ERK2 and PKB/Akt induced by IgE plus antigen or IL-3 was comparable in mkk7^+/+ and mkk7^−/− BMMCs (not shown). Thus, MKK7 is not required for the activation of p38 MAPK, ERK1/ERK2, or PKB/Akt in BMMCs. Interestingly, whereas wild-type BMMCs expressed very low levels of MKK4 protein regardless of stress stimulus, MKK4 was strongly upregulated in mkk7^−/− BMMCs (Fig. 6 E). Furthermore, the stimuli NaCl, anisomycin, UV-irradiation, or heat shock, which did not activate SAPKs/JNKs in the absence of MKK7 (Fig. 6 D), were able to induce strong activation of MKK4, as detected by an Ab specific for the phosphorylated form of MKK4 (Fig. 6 E). Thus, at least in mast cells, MKK7 expression is essential for SAPK/JNK activation, and upregulated levels of activated MKK4 alone are insufficient.

The finding that MKK4 alone cannot activate SAPKS/JNKs in BMMCs may be explained in two ways. Either MKK4 is irrelevant for this function in BMMCs or MKK4 and MKK7 cooperate in vivo to achieve full SAPK/JNK activation. In an attempt to establish a biochemical basis for cooperation, we designed an in vitro kinase assay system in which purified recombinant MKK4, MKK7, and SAPKβ were mixed together followed by assay of SAPK/JNK activity. Addition of either recombinant wild-type MKK4 or wild-type MKK7 to SAPKβ/JNK3 induced only moderate or low, respectively, levels of SAPKβ/JNK3 kinase activity (Fig. 7). Even addition of a constitutively active form of MKK7 (MKK7DE) induced only moderate SAPKβ/JNK3 kinase activity. However, addition of both MKK4 and MKK7DE resulted in a dramatic and synergistic induction of SAPKβ/JNK3 activation, strong evidence of cooperativity between these two pathway intermediates. Thus, MKK7 is an essential activator of SAPKs/JNKs in mast cells and its function is not redundant with that of MKK4. Rather, at least in vitro, both MKK4 and MKK7 are necessary for complete SAPK/JNK activation.

Mutation of mkk7 in BMMCs results in hyperproliferation, loss of expression of JunB and the cell cycle inhibitor p16INK4a, and upregulation of cyclinD1 (Fig. 6 A). We therefore speculated that mutation of mkk7 in BMMCs may results in hyperproliferation because reduced JunB expression and a loss of p16INK4a expression leads to upregulated cyclinD1 and accelerated cell cycle progression. To address whether p16INKa has a role in hyperproliferation of mkk7^−/− BMMCs, we reexpressed p16INK4a in mkk7-deficient BMMCs using a retroviral expression vector (Fig. 8, inset). Importantly, reexpression of p16INK4a suppressed hyperproliferation of mkk7^−/− BMMCs (Fig. 8). This result suggests that mkk7 deficiency might cause downregulation of p16INK4a and subsequent hyperproliferation of BMMCs.

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 9,32, 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 3,4. 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 43,44. 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 23,24,25. 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^−/−rag2^−/− 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 47,48. 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^−/−rag1^−/− 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 52,53.

The results of our in vitro kinase assays (Fig. 6 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.

We thank M. Saunders for scientific editing; J. Woodgett, K. Bachmaier, Y. Kong, N. Joza, C. Krawczyk, I. Kozieradzki, K. Gowing, M. Cheng, J. Joza, L. Barra, E. Griffith, L. Weir, M. Crackower, B. Bolan, C. Dunstan, H. Hsu, L. Zhang, M. Nghiem, S. Simonet, B. Youngson, and J. Ho for discussion and reagents.

This work is supported by the National Cancer Institute of Canada.