Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for activation of p38alpha and p38delta MAPK isoforms by TGF-beta 1 in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-beta1 in rat mesangial cells and its role in the stimulation of pro-alpha1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-beta1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-beta1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF-beta1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-beta1-induced p38 MAPK activation. TGF-beta1 selectively activated the p38 MAPK isoforms p38alpha and p38delta in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/-) mesangial cells. Thus, activation of p38alpha and p38delta is dependent on the activation of upstream MKK3 by TGF-beta1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-beta1-stimulated up-regulation of pro-alpha1(I) collagen expression but not TGF-beta1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-beta1 signaling pathway, and its activation is required for subsequent p38alpha and p38delta MAPK activation and collagen stimulation by TGF-beta1.     

The interaction of p38 with its upstream kinase MKK6

Mitogen‐activated protein kinase (MAPK; p38, ERK, and JNK) cascades are evolutionarily conserved signaling pathways that regulate the cellular response to a variety of extracellular stimuli, such as growth factors and interleukins. The MAPK p38 is activated by its specific upstream MAPK kinases, MKK6 and MKK3. However, a comprehensive molecular understanding of how these cognate upstream kinases bind and activate p38 is still missing. Here, we combine NMR spectroscopy and isothermal titration calorimetry to define the binding interface between full‐length MKK6 and p38. It was shown that p38 engages MKK6 not only via its hydrophobic docking groove, but also influences helix αF, a secondary structural element that plays a key role in organizing the kinase core. It was also shown that, unlike MAPK phosphatases, the p38 conserved docking (CD) site is much less affected by MKK6 binding. Finally, it was demonstrated that these interactions with p38 are conserved independent of the MKK6 activation state. Together, the results revealed differences between specificity markers of p38 regulation by upstream kinases, which do not effectively engage the CD site, and downstream phosphatases, which require the CD site for productive binding.     

MKK6-p38 MAPK signaling pathway enhances survival but not bone-resorbing activity of osteoclasts

Phosphorylated p38 mitogen-activating kinase (MAPK) is observed in osteoclasts under in vivo inflammatory situations. However, the role of p38 MAPK in osteoclast function has not been elucidated, because all external stimuli tested hitherto failed to induce the phosphorylation of p38 MAPK in osteoclasts in culture. In this study, a constitutively active form of MKK6 (MKK6CA) was expressed in osteoclasts using adenoviral gene transfer in vitro. MKK6CA expressed in osteoclasts phosphorylated p38 MAPK and enhanced the survival of osteoclasts. Dentine-resorbing activity of osteoclasts was not enhanced by the MKK6CA expression. These results suggest that p38 MAPK signaling plays a critical role in the survival of osteoclasts in inflammatory diseases.     

Differential activation of p38MAPK isoforms by MKK6 and MKK3

All four members of the mammalian p38 mitogen-activated protein kinase (MAPK) family (p38α, p38β, p38γ and p38δ) are activated by dual phosphorylation in the TGY motif in the activation loop. This phosphorylation is mediated by three kinases, MKK3, MKK6 and MKK4, at least in vitro. The role of these MKK in the activation of p38α has been demonstrated in studies using fibroblasts that lack MKK3 and/or MKK6. Nonetheless, the physiological upstream activators of the other p38MAPK isoforms have not yet been reported using MKK knockout cells. In this study, we examined p38β, γ and δ activation by MKK3 and MKK6, in cells lacking MKK3, MKK6 or both. We show that MKK3 and MKK6 are both essential for the activation of p38γ and p38β induced by environmental stress, whereas MKK6 is the major p38γ activator in response to TNFα. In contrast, p38δ activation by ultraviolet radiation, hyperosmotic shock, anisomycin or by TNFα is mediated by MKK3. Moreover, in response to osmotic stress, MKK3 and MKK6 are crucial in regulating the phosphorylation of the p38γ substrate hDlg and its activity as scaffold protein. These data indicate that activation of distinct p38MAPK isoforms is regulated by the selective and synchronized action of two kinases, MKK3 and MKK6, in response to cell stress.     

MKK3 Was Involved in Larval Settlement of the Barnacle Amphibalanus amphitrite through Activating the Kinase Activity of p38MAPK

The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway.     

p38α MAPK Is Required for Tooth Morphogenesis and Enamel Secretion

An improved understanding of the molecular pathways that drive tooth morphogenesis and enamel secretion is needed to generate teeth from organ cultures for therapeutic implantation or to determine the pathogenesis of primary disorders of dentition (Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678–27685). Here we present a novel ectodermal dysplasia phenotype associated with conditional deletion of p38α MAPK in ectodermal appendages using K14-cre mice (p38α^K14 mice). These mice display impaired patterning of dental cusps and a profound defect in the production and biomechanical strength of dental enamel because of defects in ameloblast differentiation and activity. In the absence of p38α, expression of amelogenin and β₄-integrin in ameloblasts and p21 in the enamel knot was significantly reduced. Mice lacking the MAP2K MKK6, but not mice lacking MAP2K MKK3, also show the enamel defects, implying that MKK6 functions as an upstream kinase of p38α in ectodermal appendages. Lastly, stimulation with BMP2/7 in both explant culture and an ameloblast cell line confirm that p38α functions downstream of BMPs in this context. Thus, BMP-induced activation of the p38α MAPK pathway is critical for the morphogenesis of tooth cusps and the secretion of dental enamel.     

Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed ‘classic’ features of apoptosis following exposure to pneumococci. Conversely, purified CD3⁺ T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3⁺ T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3⁺ T-cells in PBMC cultures required ‘classical’ CD14⁺ monocytes, which enhanced T-cell activation. CD3⁺ T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3⁺ T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.     

A Protoberberine Derivative HWY336 Selectively Inhibits MKK4 and MKK7 in Mammalian Cells: The Importance of Activation Loop on Selectivity

A protoberberine derivative library was used to search for selective inhibitors against kinases of the mitogen-activated protein kinase (MAPK) cascades in mammalian cells. Among kinases in mammalian MAPK pathways, we identified a compound (HWY336) that selectively inhibits kinase activity of mitogen-activated protein kinase kinase 4 and 7 (MKK4 and MKK7). The IC₅₀ of HWY336 was 6 µM for MKK4 and 10 µM for MKK7 in vitro. HWY336 bound to both kinases reversibly via noncovalent interactions, and inhibited their activity by interfering with access of a protein substrate to its binding site. The binding affinity of HWY336 to MKK4 was measured by surface plasmon resonance to determine a dissociation constant (K_d) of 3.2 µM. When mammalian cells were treated with HWY336, MKK4 and MKK7 were selectively inhibited, resulting in inhibition of c-Jun NH₂-terminal protein kinases in vivo. The structural model of HWY336 bound to either MKK4 or MKK7 predicted that HWY336 was docked to the activation loop, which is adjacent to the substrate binding site. This model suggested the importance of the activation loop of MKKs in HWY336 selectivity. We verified this model by mutating three critical residues within this loop of MKK4 to the corresponding residues in MKK3. The mutant MKK4 displayed similar kinase activity as wild-type kinase, but its activity was not inhibited by HWY336 compared to wild-type MKK4. We propose that the specific association of HWY336 to the activation loop of MKK4/MKK7 is responsible for its selective inhibition.     

Allogeneic differences in the dependence on CD4+ T-cell help for virus-specific CD8+ T-cell differentiation

CD4⁺ T-cell help enables antiviral CD8⁺ T cells to differentiate into fully competent memory cells and sustains CD8⁺ T-cell-mediated immunity during persistent virus infection. We recently reported that mice of C57BL/6 and C3H strains differ in their dependence on CD28 and CD40L costimulation for long-term control of infection by polyoma virus, a persistent mouse pathogen. In this study, we asked whether mice of these inbred strains also vary in their requirement for CD4⁺ T-cell help for generating and maintaining polyoma virus-specific CD8⁺ T cells. CD4⁺ T-cell-depleted C57BL/6 mice mounted a robust antiviral CD8⁺ T-cell response during acute infection, whereas unhelped CD8⁺ T-cell effectors in C3H mice were functionally impaired during acute infection and failed to expand upon antigenic challenge during persistent infection. Using (C57BL/6 × C3H)F₁ mice, we found that the dispensability for CD4⁺ T-cell help for the H-2^b-restricted polyoma virus-specific CD8⁺ T-cell response during acute infection extends to the H-2^k-restricted antiviral CD8⁺ T cells. Our findings demonstrate that dependence on CD4⁺ T-cell help for antiviral CD8⁺ T-cell effector differentiation can vary among allogeneic strains of inbred mice.     

Role of 4-1BB Receptor in the Control Played by CD8+ T Cells on IFN-γ Production by Mycobacterium tuberculosis Antigen-Specific CD4+ T Cells

Background

Antigen-specific IFN-γ producing CD4⁺ T cells are the main mediators of protection against Mycobacterium tuberculosis infection both under natural conditions and following vaccination. However these cells are responsible for lung damage and poor vaccine efficacy when not tightly controlled. Discovering new tools to control nonprotective antigen-specific IFN-γ production without affecting protective IFN-γ is a challenge in tuberculosis research.

Methods and Findings

Immunization with DNA encoding Ag85B, a candidate vaccine antigen of Mycobacterium tuberculosis, elicited in mice a low but protective CD4⁺ T cell-mediated IFN-γ response, while in mice primed with DNA and boosted with Ag85B protein a massive increase in IFN-γ response was associated with loss of protection. Both protective and non-protective Ag85B-immunization generated antigen-specific CD8⁺ T cells which suppressed IFN-γ-secreting CD4⁺ T cells. However, ex vivo ligation of 4-1BB, a member of TNF-receptor super-family, reduced the massive, non-protective IFN-γ responses by CD4⁺ T cells in protein-boosted mice without affecting the low protective IFN-γ-secretion in mice immunized with DNA. This selective inhibition was due to the induction of 4-1BB exclusively on CD8⁺ T cells of DNA-primed and protein-boosted mice following Ag85B protein stimulation. The 4-1BB-mediated IFN-γ inhibition did not require soluble IL-10, TGF-β, XCL-1 and MIP-1β. In vivo Ag85B stimulation induced 4-1BB expression on CD8⁺ T cells and in vivo 4-1BB ligation reduced the activation, IFN-γ production and expansion of Ag85B-specific CD4⁺ T cells of DNA-primed and protein-boosted mice.

Conclusion/Significance

Antigen-specific suppressor CD8⁺ T cells are elicited through immunization with the mycobacterial antigen Ag85B. Ligation of 4-1BB receptor further enhanced their suppressive activity on IFN-γ-secreting CD4⁺ T cells. The selective expression of 4-1BB only on CD8⁺ T cells in mice developing a massive, non-protective IFN-γ response opens novel strategies for intervention in tuberculosis pathology and vaccination through T-cell co-stimulatory-based molecular targeting.     

Balance between MKK6 and MKK3 mediates p38 MAPK associated resistance to cisplatin in NSCLC

The p38 MAPK signaling pathway has been proposed as a critical mediator of the therapeutic effect of several antitumor agents, including cisplatin. Here, we found that sensitivity to cisplatin, in a system of 7 non-small cell lung carcinoma derived cell lines, correlated with high levels of MKK6 and marked activation of p38 MAPK. However, knockdown of MKK6 modified neither the response to cisplatin nor the activation of p38 MAPK. Deeper studies showed that resistant cell lines also displayed higher basal levels of MKK3. Interestingly, MKK3 knockdown significantly decreased p38 phosphorylation upon cisplatin exposure and consequently reduced the response to the drug. Indeed, cisplatin poorly activated MKK3 in resistant cells, while in sensitive cell lines MKK3 showed the opposite pattern in response to the drug. Our data also demonstrate that the low levels of MKK6 expressed in resistant cell lines are the consequence of high basal activity of p38 MAPK mediated by the elevated levels of MKK3. This finding supports the existence of a regulatory mechanism between both MAPK kinases through their MAPK. Furthermore, our results were also mirrored in head and neck carcinoma derived cell lines, suggesting our observations boast a potential universal characteristic in cancer resistance of cisplatin. Altogether, our work provides evidence that MKK3 is the major determinant of p38 MAPK activation in response to cisplatin and, hence, the resistance associated with this MAPK. Therefore, these data suggest that the balance between both MKK3 and MKK6 could be a novel mechanism which explains the cellular response to cisplatin.     

CD4+ T Cell-Derived IL-2 Signals during Early Priming Advances Primary CD8+ T Cell Responses

Stimulating naïve CD8⁺ T cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. In this study, we show that the activation and differentiation of CD8⁺ T cells require IL-2 provided by activated CD4⁺ T cells at the initial priming stage within 0–2.5 hours after stimulation. This critical IL-2 signal from CD4⁺ cells is mediated through the IL-2Rβγ of CD8⁺ cells, which is independent of IL-2Rα. The activation of IL-2 signaling advances the restriction point of the cell cycle, and thereby expedites the entry of antigen-stimulated CD8⁺ T-cell into the S phase. Besides promoting cell proliferation, IL-2 stimulation increases the amount of IFNγ and granzyme B produced by CD8⁺ T cells. Furthermore, IL-2 at priming enhances the ability of P14 effector cells generated by antigen activation to eradicate B16.gp33 tumors in vivo. Therefore, our studies demonstrate that a full CD8⁺ T-cell response is elicited by a critical temporal function of IL-2 released from CD4⁺ T cells, providing mechanistic insights into the regulation of CD8⁺ T cell activation and differentiation.     

Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade

Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4. In addition, Galpha(q) and Gbetagamma stimulated MKK3 and MKK6 activities. The MKK3 and MKK6 activations by Galpha(q), but not by Gbetagamma, were dependent on phospholipase C and c-Src. Galpha(q) stimulated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner. On the other hand, Gbetagamma activated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-, Rac-, and Cdc42-dependent manner. Gbetagamma-induced MKK3 and MKK6 activations were dependent on a tyrosine kinase other than c-Src. These results suggest that Galpha(q) and Gbetagamma stimulate the activity of p38 MAPK by regulating MKK3 and MKK6 through parallel signaling pathways.