Wha Dokter

A dual function for p38 MAP kinase in hematopoietic cells: involvement in apoptosis and cell activation

In the present study we examined in more detail the dual role of the c-JUN N-terminal kinase (JNK) and p38 stress-activated protein kinase pathways in mediating apoptosis or cellular activation in hematopoietic cells. Growth factor deprivation of the erythroleukemic cell line TF-1 led to apoptosis which was associated with an enhanced activity of JNK and p38 and immediate dephosphorylation of the extracellular signal-regulated kinases (ERKs). Enhanced activity of p38 and JNK was not only observed during apoptosis but also in TF-1 cells stimulated with IL-1. IL-1 rescued TF-1 cells from apoptosis. In this case, the upregulation of p38 and JNK was associated with an enhanced activity of ERK. By using SB203580, a specific inhibitor of the p38 signaling pathway, it was demonstrated that p38 plays a pivotal role in the apoptotic process. SB203580 repressed the apoptotic process to a large extent. In contrast, PD98059, a specific inhibitor of the ERK pathway, counteracted the suppressive effects of SB203580 and IL-1 on the apoptotic process indicating that the protective effect of SB203580 and IL-1 might be the result of a shift in the balance between the ERK1/2 and p38/JNK route. This was also supported by experiments with TF-1 cells overexpressing the Shc protein that demonstrated a significantly lower percentage of apoptotic cells, which coincided with higher ERK activity. Finally, the IL-1 and SB203580-mediated effects were associated with an enhanced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) binding activity, which could also be blocked by PD98059. These data demonstrate a dual function of the p38 pathway whereby other factors, such as ERK kinases, AP-1 and NF-κB, might determine the final cellular response.

Regulation of p100 (NFKB2) expression in human monocytes in response to inflammatory mediators and lymphokines.

The transcription factor NF-κB plays an important role in the regulated expression of cytokines in human monocytes. A p100 subunit of NF-κB has IκB-like properties by sequestering the p65 transactivating subunit in the cytosol of cells. In transient transfection assays we demonstrated that p100 has an inhibitory effect on the NF-κB-dependent IL-6 promoter activity. In view of this finding, we studied the regulation of the p100 subunit in human monocytes in response to LPS, the inflammatory cytokines IL-1β and TNF-α and lymphokines. The results demonstrate that LPS, IL-1β, and TNF-α induce p100 expression at mRNA and protein level while IFN-γ, IL-3 and IL-4/IL-10 have no effect. The induction of p100 expression was shown to be mediated by a two-fold increase in the p100 transcription rate and a two-fold increase in p100 mRNA stability. Furthermore the p100 mediated upregulation was dependent on a tyrosine kinase dependent pathway rather than the protein kinase C pathway. NF-κB is a complex of either p50 homodimers or a p50/p65 heterodimer. The latter is known to strongly autoregulate p100 transcription. We therefore examined the composition of NF-κB induced by LPS vs the different lymphokines. LPS-induced NF-κB showed a distinct p65 supershift whereas the composition of NF-κB induced by different lymphokines did not show a change in p65. We conclude that the p100 subunit of the transcription factor NF-κB is induced by different inflammatory mediators while lymphokines fail to induce p100 expression which may be caused by the induction of NF-κB predominantly consisting of p50 homodimers.

Interleukin-4 prevents the induction of G-CSF mRNA in human adherent monocytes in response to endotoxin and IL-1 stimulation

Human recombinant interleukin‐4 (IL‐4) was studied for its effects on the expression of granulocyte‐colony stimulating factor (G‐CSF) mRNA in human adherent monocytes in the absence and presence of endotoxin and interleukin 1 (IL‐1), IL‐4 (15 ng/ml) did not induce G‐CSF transcripts in monocytes but suppressed the endotoxin‐induced G‐CSF expression when added simultaneously. Sequential treatment of monocytes with IL‐4 followed by endotoxin suppressed G‐CSF mRNA induction totally. This effect was independent of the presence of fetal bovine serum but dependent of the IL‐4 dose. Comparable results were obtained with IL‐1, IL‐1 (50 U/ml) induced G‐CSF expression in human adherent monocytes which could be counteracted by IL‐4 pretreatment. In addition, it was shown that the induction of G‐CSF mRNA by the calcium‐ionophore A23187 or by c‐AMP elevating agents could be blocked by IL‐4. These suppressive effects of IL‐4 were not related to changes in the half‐life of G‐CSF mRNA and were independent of protein synthesis. Finally it was demonstrated that IL‐4 had comparable effects on the G‐CSF secretion of endotoxin and IL‐1 stimulated human monocytes by using a murine bone marrow assay. These results indicate that IL‐4 down‐regulates the expression of G‐CSF gene and secretion of proteins in human activated monocytes.

Use of the EGP-2/Ep-CAM promoter for targeted expression of heterologous genes in carcinoma derived cell lines

EGP-2, also known as Ep-CAM, is expressed at high levels on the surface of most carcinomas and is therefore considered an attractive target for anticancer strategies. To explore the mechanisms regulating the expression of EGP-2, sequences 3.4 kb upstream of the transcription start site were isolated and assayed for their ability to control the expression of the EGP-2 cDNA, the green fluorescent protein, the luciferase reporter gene and the thymidine kinase and cytosine deaminase suicide genes. Expression of these chimeric constructs as assessed in a range of different cell lines was restricted to cell lines expressing EGP-2. In addition, only cells expressing EGP-2 were sensitive for gancyclovir after being transiently transfected with EGP-2 promoter-driven thymidine kinase. Deletion analyses defined 687 bp upstream as the basic proximal promoter region, which could confer epithelial-specific expression to the GFP reporter gene in vitro. As these EGP-2 sequences can confer promoter activity to reporter and suicide genes in an EGP-2 restricted manner, they may be useful for gene therapy of EGP-2 expressing carcinomas.

Molecular Mechanisms Involved in the Control of Differential Cytokine Expression in Human Monocytes

During the last few years a tremendous increase has been obtained in our knowledge of cytokines. Apart from the identification of new cytokines we have reached a better understanding of cytokine gene regulation as well as of signal transduction pathways leading to cytokine gene expression. One of the intriguing phenomena of the regulation of cytokines is the specificity in effects of stimulatory agents which has been observed in different haematopoietic cells. This differential regulation of cytokines is most extensively studied in human monocytes.