JNK1 is required for T cell-mediated immunity against Leishmania major infection.

c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase that plays important regulatory roles in helper T cell differentiation. In the current study, we used Jnk1-deficient mice to examine the function of JNK during an in vivo pathogenic infection, leishmaniasis, which is strongly influenced by Th1/Th2 effector mechanisms. The data show that Jnk1-deficient mice, despite their usually genetically resistant background, were unable to resolve Leishmania infections. Jnk1-/- mice displayed reduced delayed-type hypersensitivity in response to the pathogen, which was associated with a T cell defect. We found that, although these mice can direct an apparent Th1-response, there is also simultaneous generation of Leishmania-specific Th2 responses, which possibly down-modulate protective Th1-mediated immune function. These findings demonstrate that the negative regulation of Th2 cytokine production by the JNK1 signaling pathway is essential for generating Th1-polarized immunity against intracellular pathogens, such as Leishmania major.

Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway.

The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress. The anti-inflammatory properties of heme oxygenase-1 may serve as a basis for this cytoprotection. We demonstrate here that carbon monoxide, a by-product of heme catabolism by heme oxygenase, mediates potent anti-inflammatory effects. Both in vivo and in vitro, carbon monoxide at low concentrations differentially and selectively inhibited the expression of lipopolysaccharide-induced pro-inflammatory cytokines tumor necrosis factor-alpha, interleukin-1beta, and macrophage inflammatory protein-1beta and increased the lipopolysaccharide-induced expression of the anti-inflammatory cytokine interleukin-10. Carbon monoxide mediated these anti-inflammatory effects not through a guanylyl cyclase-cGMP or nitric oxide pathway, but instead through a pathway involving the mitogen-activated protein kinases. These data indicate the possibility that carbon monoxide may have an important protective function in inflammatory disease states and thus has potential therapeutic uses.

Defective IL-12 production in mitogen-activated protein (MAP) kinase kinase 3 (Mkk3)-deficient mice.

The p38 mitogen-activated protein kinase (MAPK) pathway, like the c-Jun N-terminal kinase (JNK) MAPK pathway, is activated in response to cellular stress and inflammation and is involved in many fundamental biological processes. To study the role of the p38 MAPK pathway in vivo, we have used homologous recombination in mice to inactivate the Mkk3 gene, one of the two specific MAPK kinases (MAPKKs) that activate p38 MAPK. Mkk3(-/-) mice were viable and fertile; however, they were defective in interleukin-12 (IL-12) production by macrophages and dendritic cells. Interferon-gamma production following immunization with protein antigens and in vitro differentiation of naive T cells is greatly reduced, suggesting an impaired type I cytokine immune response. The effect of the p38 MAPK pathway on IL-12 expression is at least partly transcriptional, since inhibition of this pathway blocks IL-12 p40 promoter activity in macrophage cell lines and IL-12 p40 mRNA is reduced in MKK3-deficient mice. We conclude that the p38 MAP kinase, activated through MKK3, is required for the production of inflammatory cytokines by both antigen-presenting cells and CD4(+) T cells.

Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for tumor necrosis factor-induced cytokine expression.

The p38 mitogen-activated protein kinase is activated by treatment of cells with cytokines and by exposure to environmental stress. The effects of these stimuli on p38 MAP kinase are mediated by the MAP kinase kinases (MKKs) MKK3, MKK4, and MKK6. We have examined the function of the p38 MAP kinase signaling pathway by investigating the effect of targeted disruption of the Mkk3 gene. Here we report that Mkk3 gene disruption caused a selective defect in the response of fibroblasts to the proinflammatory cytokine tumor necrosis factor, including reduced p38 MAP kinase activation and cytokine expression. These data demonstrate that the MKK3 protein kinase is a critical component of a tumor necrosis factor-stimulated signaling pathway that causes increased expression of inflammatory cytokines.

Molecular basis of T-cell differentiation.

In summary, a multitude of regulatory systems are employed to cause the selective activation of target cytokine genes in Th1 and Th2 effector cells. These mechanisms involve both positive and negative regulation and employ at least three kinds of mechanisms. In the first, selective expression of transcription factors such as GATA3 in Th2 cells and the homeobox gene HLX in Th1 cells occurs, and appears in both cases to play a causal role. Another example of this would be c-maf, discovered by the Glimcher laboratory. A second mechanism is by the selective accumulation of protein through posttranscriptional mechanisms. Thus, junB accumulates in Th2 cells despite the fact that the junB mRNA levels are not different between Th1 and Th2 cells. Finally, the selective use of signaling pathways, in the case studied here MAP kinase pathways, leads to the selective activation of target genes. We believe that transcriptional up-regulation of rac2 leads to the coupling of both the p38 and JNK MAP kinase pathways to the T-cell receptor and/or costimulatory receptors, thereby providing a lineage-specific signal.

Defective T cell differentiation in the absence of Jnk1.

The c-Jun NH2-terminal kinase (JNK) signaling pathway has been implicated in the immune response that is mediated by the activation and differentiation of CD4 helper T (TH) cells into TH1 and TH2 effector cells. JNK activity observed in wild-type activated TH cells was severely reduced in TH cells from Jnk1-/- mice. The Jnk1-/- T cells hyperproliferated, exhibited decreased activation-induced cell death, and preferentially differentiated to TH2 cells. The enhanced production of TH2 cytokines by Jnk1-/- cells was associated with increased nuclear accumulation of the transcription factor NFATc. Thus, the JNK1 signaling pathway plays a key role in T cell receptor-initiated TH cell proliferation, apoptosis, and differentiation.

Targeted disruption of the MKK4 gene causes embryonic death, inhibition of c-Jun NH2-terminal kinase activation, and defects in AP-1 transcriptional activity.

MKK4 is a member of the mitogen-activated protein kinase kinase group of dual specificity protein kinases that functions as an activator of the c-Jun NH2-terminal kinase (JNK) in vitro. To examine the function of MKK4 in vivo, we investigated the effect of targeted disruption of the MKK4 gene. Crosses of heterozygous MKK4 (+/-) mice demonstrated that homozygous knockout (-/-) animals die before embryonic day 14, indicating that the MKK4 gene is required for viability. The role of MKK4 in JNK activation was examined by investigation of cultured MKK4 (+/+) and MKK4 (-/-) cells. Disruption of the MKK4 gene blocked JNK activation caused by: (i) the mitogen-activated protein kinase kinase kinase MEKK1, and (ii) treatment with anisomycin or heat shock. In contrast, JNK activation caused by other forms of environmental stress (UV-C radiation and osmotic shock) was partially inhibited in MKK4 (-/-) cells. Regulated AP-1 transcriptional activity, a target of the JNK signal transduction pathway, was also selectively blocked in MKK4 (-/-) cells. Complementation studies demonstrated that the defective AP-1 transcriptional activity was restored by transfection of MKK4 (-/-) cells with an MKK4 expression vector. These data establish that MKK4 is a JNK activator in vivo and demonstrate that MKK4 is an essential component of the JNK signal transduction pathway.

Molecular cloning of the epidermal growth factor-stimulated protein kinase p56 KKIAMRE.

A 56 kDa protein kinase was molecularly cloned from human fetal brain. This protein kinase (p56 KKIAMRE) shares homology with p42 KKIALRE (Meyerson et al., 1992) and is related to the proline-directed protein kinase group of signal transducing enzymes. The p56 KKIAMRE and p42 KKIALRE protein kinases exhibit mutually exclusive expression in reproductive tissues; p56 KKIAMRE in testis and p42 KKIALRE in ovary. p56 KKIAMRE and p42 KKIALRE may therefore contribute to signal transduction within these highly differentiated tissues. p56 KKIAMRE and p42 KKIALRE are activated by treatment of cells with epidermal growth factor (EGF). Although p56 KKIAMRE and p42 KKIALRE contain the MAP kinase dual phosphorylation motif Thr-Xaa-Tyr (Thr-Asp-Tyr), phosphorylation on Thr and Tyr within this motif is not required for EGF-stimulated protein kinase activity.

Genomic loci of human mitogen-activated protein kinases.

Mitogen-activated protein (MAP) kinases [also known as Erks] have been established to function as important mediators of signal transduction by growth factor receptors. Several components of the MAP kinase signal transduction pathway have been demonstrated to be oncogenically activated in malignant tumors. These include growth factor receptors, the GTP-binding protein Ras, and the protein kinase Raf. The genes that encode MAP kinases therefore represent potential targets of carcinogenic insults. Here, we report the genomic loci of three MAP kinase genes are widely distributed within the human genome: p41mapk (Erk2) at 22q11.2; p44mapk (Erk1) at 16p11.2; and p63mapk (Erk3-related) at 18q12-21.

Isolation of the rat gene for Mullerian inhibiting substance.

Mullerian inhibiting substance (MIS), a testicular glycoprotein also known as anti-Mullerian hormone, plays a key role in male sexual development by causing regression of the Mullerian duct, the anlagen of the uterus, the Fallopian tubes, and part of the vagina. MIS is also expressed in the postnatal ovary, but its precise function is still not known. We report here the complete nucleotide sequence of the rat MIS gene. Rat MIS is encoded in five exons and is synthesized as a precursor of 553 amino acids, containing a 24-amino-acid leader. Based on homology with human MIS, we predict that the rat protein undergoes proteolytic processing at a site 108 amino acids from the C-terminus. Expression of the rat MIS mRNA is high in the 1-day-postnatal testis and decreases to a low level in the adult testis. In contrast, expression is not detected in the 1-day ovary, but increases to an intermediate level in the adult ovary. The rat gene should provide a good model for studying transcriptional regulation of MIS in the testis and ovary.

Cloning of murine and rat vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen 4 (VLA4). We have cloned the cDNAs for both murine and rat VCAM1 from endotoxin-treated lung libraries. Both sequences encode proteins with seven extracellular Ig-like domains, which show 75.9% and 76.9% identity, respectively, with human VCAM1. Both murine and human cell lines show VLA4-dependent binding to COS cells transiently expressing murine and rat VCAM1. Two mAbs, M-K/1 and M-K/2, which recognize an antigen on murine bone marrow stromal cell lines, bind to murine VCAM1 expressed in COS cells and block VCAM1-dependent adhesion, confirming that these mAbs recognize murine VCAM1.

Genomic Southern analysis with alkaline-phosphatase-conjugated oligonucleotide probes and the chemiluminescent substrate AMPPD.

We have used a chemiluminescent detection method to improve both the sensitivity and the speed of detection of human genes with oligonucleotide probes. A direct chemiluminescent substrate (AMPPD) was used in combination with an alkaline-phosphatase-labeled oligonucleotide probe to detect the human tissue of plasminogen activator gene by Southern blot analysis. X-ray exposures obtained after 4 h were comparable to those obtained after 7 days with a 32P-labeled oligomer. After 16 h, the signal was 12 times greater than the 32P signal. The detection of the single-copy tissue plasminogen activator gene in 0.25 micrograms of human genomic DNA (76,000 molecules) was achieved. The improved sensitivity obtained by chemiluminescent detection should increase the usefulness of oligonucleotide probes in the direct Southern analysis of human genetic disorders.

Imaging of DNA sequences with chemiluminescence.

We have coupled a chemiluminescent detection method that uses an alkaline phosphatase label to the genomic DNA sequencing protocol of Church and Gilbert [Church, G. M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991-1995]. Images of sequence ladders are obtained on x-ray film with exposure times of less than 30 min, as compared to 40 h required for a similar exposure with a 32P-labeled oligomer. Chemically cleaved DNA from a sequencing gel is transferred to a nylon membrane, and specific sequence ladders are selected by hybridization to DNA oligonucleotides labeled with alkaline phosphatase or with biotin, leading directly or indirectly to deposition of enzyme. If a biotinylated probe is used, an incubation with avidin-alkaline phosphatase conjugate follows. The membrane is soaked in the chemiluminescent substrate (AMPPD) and is exposed to film. Dephosphorylation of AMPPD leads in a two-step pathway to a highly localized emission of visible light. The demonstrated shorter exposure times may improve the efficiency of a serial reprobing strategy such as the multiplex sequencing approach of Church and Kieffer-Higgins [Church, G. M. & Kieffer-Higgins, S. (1988) Science 240, 185-188].