Differential expression of mitogen activating protein kinases in periodontitis.

AIM: Following toll-like receptor (TLR) engagement, lipopolysaccharide (LPS) can stimulate the expression of pro-inflammatory cytokines thus activating the innate immune response. The production of inflammatory cytokines results, in part, from the activation of kinase-induced signalling cascades and transcriptional factors. Of the four distinct classes of mitogen-activated protein kinases (MAPK) described in mammals, p38, c-Jun N-terminal activated kinases (JNK1-3) and extracellular activated kinases (ERK1,2) are the best studied. Previous data have established that p38 MAPK signalling is required for inflammation and bone loss in periodontal disease pre-clinical animal models. MATERIALS & METHODS: In this study, we obtained healthy and diseased periodontal tissues along with clinical parameters and microbiological parameters. Excised fixed tissues were immunostained with total and phospho-specific antibodies against p38, JNK and ERK kinases. RESULTS: Intensity scoring from immunostained tissues was correlated with clinical periodontal parameters. Rank correlations with clinical indices were statistically significantly positive (p-value < 0.05) for total p38 (correlations ranging 0.49-0.68), phospho-p38 (range 0.44-0.56), and total ERK (range 0.52-0.59) levels, and correlations with JNK levels also supported association (range 0.42-0.59). Phospho-JNK and phospho-ERK showed no significant positive correlation with clinical parameters of disease. CONCLUSION: These data strongly implicate p38 MAPK as a major MAPK involved in human periodontal inflammation and severity.

Detection of phosphorylated mitogen-activated protein kinase in the developing spinal cord of the mouse embryo.

Global understanding of the proteome is a major research topic. The comprehensive visualization of the distribution of proteins in vivo or the construction of in situ protein atlases may be a valuable strategy for proteomic researchers. Information about the distribution of various proteins under physiological and pathological conditions should be extremely valuable for the basic and clinical sciences. The mitogen-activated protein kinase (MAPK) cascade plays an essential role in intracellular signaling in organisms. This cascade also regulates biological processes involving development, differentiation, and proliferation. Phosphorylation and dephosphorylation are integral reactions in regulating the activity of MAPKs. Changes in the phosphorylation state of MAPKs are rapid and reversible; therefore, the localizations of physiologically phosphorylated MAPKs in vivo are difficult to accurately detect. Furthermore, phosphorylated MAPKs are likely to change phosphorylated states through commonly used experimental manipulations. In the present study, as a step toward the construction of in situ phosphoprotein atlases, we attempted to detect physiologically phosphorylated MAPKs in vivo in developing spinal cords of mice. We previously reported an improved immunohistochemical method for detecting unstable phosphorylated MAPKs. The distribution patterns of phosphorylated MAPKs in the spinal cords of embryonic mice from embryonic day 13 (E13) to E17 were observed with an improved immunohistochemical method. Phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated c-Jun N-terminal kinase 1/2 (p-JNK1/2) were strongly observed in the marginal layer and the dorsal horn from E13 to E17. Our results suggest that p-ERK1/2 and p-JNK1/2 play critical roles in the developing spinal cord. Constructing phosphoprotein atlases will be possible in the future if this work is systematically developed on a larger scale than we presented here.

The role of the c-Jun N-terminal kinase 2-α-isoform in non-small cell lung carcinoma tumorigenesis.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase family and have been implicated in tumorigenesis. One isoform in particular, JNK2α, has been shown to be frequently activated in primary brain tumors, to enhance several tumorigenic phenotypes and to increase tumor formation in mice. As JNK is frequently activated in non-small cell lung carcinoma (NSCLC), we investigated the role of the JNK2α isoform in NSCLC formation by examining its expression in primary tumors and by modulating its expression in cultured cell lines. We discovered that 60% of the tested primary NSCLC tumors had three-fold higher JNK2 protein and two- to three-fold higher JNK2α mRNA expression than normal lung control tissue. To determine the importance of JNK2α in NSCLC progression, we reduced JNK2α expression in multiple NSCLC cell lines using short hairpin RNA. Cell lines deficient in JNK2α had decreased cellular growth and anchorage-independent growth, and the tumors were four-fold smaller in mass. To elucidate the mechanism by which JNK2α induces NSCLC growth, we analyzed the JNK substrate, signal transducer and activator of transcription 3 (STAT3). Our data demonstrates for the first time that JNK2α can regulate the transcriptional activity of STAT3 by phosphorylating the Ser727 residue, thereby regulating the expression of oncogenic genes, such as c-Myc. Furthermore, reintroduction of JNK2α2 or STAT3 restored the tumorigenicity of the NSCLC cells, demonstrating that JNK2α is important for NSCLC progression. Our studies reveal a novel mechanism in which phosphorylation of STAT3 is mediated by a constitutively active JNK2 isoform, JNK2α.

An optimized immunohistochemical method for detection of phosphorylated mitogen-activated protein kinases.

Study of the in vivo spatio-temporal localization of modified proteins is likely to become a major focus of proteomics research in the near future. In this study we optimized and tested an immunohistochemical procedure for detecting unstable phosphorylated mitogen-activated protein (MAP) kinases. Using our method, phosphorylated MAP kinases can be sensitively and reproducibly localized in the developing white matter of murine spinal cord on embryonic day 15. Our method is simple and effective, and so may be useful in future proteomics research.

c-JUN N-terminal kinase-1 (JNK1) but not JNK2 or JNK3 is involved in UV signal transduction in human epidermis.

BACKGROUND: c-Jun N-terminal kinase (JNK) plays a critical role in UV-induced apoptotic cell death. Although three isoforms are known in mammals, physiological roles of each isoform are still obscure. Furthermore, our recent findings show that serpin squamous cell carcinoma antigen (SCCA1) binds to JNK. OBJECTIVE: To determine which isoform is responsible for the UV signal transduction in human epidermis and whether SCCA1 is capable to regulate kinase activity of a specific isoform. METHODS: Immunohistochemical localization of each JNK isoform was investigated after UV irradiation in vivo and in vitro. Effect of recombinant SCCA1 on JNK kinase activity was also analyzed. RESULTS: Immunostaining for JNK1, 2 and 3 demonstrated marked elevation of JNK1 in spinous to granular cells of UV-irradiated skin, whereas they were expressed weakly in upper epidermis of the sun-protected, buttock skin. In cultured keratinocytes, only JNK1 is translocated into nucleus after UV irradiation. JNK2, which localized in the cytoplasm, or JNK3, which was confined in nucleus, remained in the same compartment after UV irradiation. We confirmed that only JNK1 mRNA was up-regulated after UV irradiation in cultured keratinocytes. In addition, recombinant SCCA1 suppressed kinase activity of JNK1 but did not affect JNK2 or JNK3 kinase activity. CONCLUSION: JNK1 is associated with UV signal transduction in human epidermis and SCCA1 is a suppressor of this process.

Signal transduction pathways in Burkitt's lymphoma cell lines BL41 and DG75 with different sensitivity to doxorubicin.

AIM: To understand the biochemical basis of cell sensitivity to cytotoxic effect of doxorubicine (DOX), we investigated signaling cascades mediated by c-Jun N-terminal protein kinases (JNK1/2), p38 mitogen-activated protein kinases (MAPK), extracellular signal-regulated protein kinase (ERK1/2) and protein kinase B/Akt in both DOX-sensitive BL41 and the DOX-resistant DG75 Burkitt's lymphoma (BL) cell lines. METHODS: To test the effect of DOX on different signaling cascades, BL41 and DG75 cells were treated with DOX for varying lengths of time. Cytotoxic effect of DOX was analyzed by Hoechst 33342 staining. Total amount of JNK1/2, ERK1/2, p38 MARK, Akt proteins, and also phosphorylated/activated forms of these enzymes were detected using Western blot analysis with specific antibodies. Immunophenotypic analysis of BL41 and DG75 cells was performed by indirect immunofluorescence staining. RESULTS: Our findings demonstrated that DOX treatment of the BL41 cells led to sustained activation of JNK1/2 and p38 MAPK. This activation/phosphorylation did not result from increased expression of either JNK1/2 or p38 MAPK since protein levels of JNK1/2 and p38 MAPK in DOX-treated and untreated cells were unaltered. Apoptotic signaling cascade induced by DOX in BL41 cell was accompanied by Akt dephosphorylation. The effect of DOX in drug-resistant cell line DG75 convoyed by dephosphorylation of JNK1/2, p38 MAPK and activation of Akt. Fate of BL cells did not depend from ERK activity. CONCLUSION: The outcome of cellular response to DOX in BL cell lines is determined by interference of at least three signaling pathways: JNK1/2, p38 MAPK and PKB/Akt. The balance between Akt/PKB and MAPK pathways is important in determining whether BL cells survive or undergo apoptosis in response to DOX treatment.