Josiane Pierre

Interleukin-4 Induces Activation of Mitogen-activated Protein Kinase and Phosphorylation of Shc in Human Keratinocytes*

Most cytokines stimulate the p21pathway, leading to MAP kinase activation. One exception is interleukin-4 (IL-4), which has been shown not to activate this pathway in hematopoietic cells. However, IL-4 acts on a broad range of cells, including keratinocytes, in which it induces IL-6 production. We report here that IL-4 stimulation of human keratinocytic cell lines or primary cultures activates MAP kinase. In these cells, IL-4 stimulation induces the tyrosine phosphorylation of p42/44 MAP kinase as well as its catalytic activity. We also observed an increased phosphorylation of p46, an SH2-containing protein involved in the Ras pathway, as a result of IL-4 stimulation in human keratinocytic cell lines but not in T lymphocytes.

Binding of IL-4 to the IL-13Rα1/IL-4Rα receptor complex leads to STAT3 phosphorylation but not to its nuclear translocation

Interleukin‐4 (IL‐4) is a pleiotropic cytokine, which acts on both hematopoietic and non‐hematopoietic cells, through different types of receptor complexes. In this study, we report that in human B cells, IL‐4 caused rapid phosphorylation of Janus kinase (JAK) 1 and JAK3 tyrosine kinases. In keratinocytes, the hematopoietic‐specific receptor common γc chain is not expressed and the IL‐13 receptor α1 (IL‐13Rα1) participates in IL‐4 signal transduction. In keratinocytes, IL‐4 induced JAK1 and JAK2 phosphorylation but, unlike in immune cells, IL‐4 did not involve JAK3 activation for its signaling. In both cell types, IL‐4 induced phosphorylation and DNA binding activation of the signal transducer and activator of transcription (STAT) 6 protein. Furthermore, IL‐4 stimulation of keratinocytes also induced tyrosine phosphorylation of STAT3 which was found to bind to the phosphorylated IL‐13Rα1. STAT3 however did not significantly translocate to the nucleus, nor did it bind with high affinity to target DNA sequences.

STAT6 and Ets-1 Form a Stable Complex That Modulates Socs-1 Expression by Interleukin-4 in Keratinocytes

Supressor of cytokine signaling (SOCS)-1 is selectively and rapidly induced by appropriate agonists and modulates cytokine responses by interfering with the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway. On the basis of the observation that interleukin (IL)-4 up-regulates Socs-1 in the keratinocyte HaCaT cell line, we investigated which sequences of the 5′-Socs-1 gene are responsive to IL-4. We therefore have cloned the 5′-flanking region of this gene, and by promoter analysis we identified a functional IL-4-responsive element located at nucleotide (–684/–570) upstream from the transcription initiation site, whose presence and integrity are necessary to ensure IL-4 responsiveness. This element contains three STAT6 and one Ets consensus binding sequences of which specific mutations abolished IL-4 responsiveness either partially or totally. We also report that Ets-1 physically interacted with STAT6. Exogenous expression of Ets-1 in conjunction with STAT6 activation strongly inhibited expression of a Socs-1 promoter-luciferase reporter. Collectively, our data demonstrated the involvement of STAT6 and Ets, via a composite DNA element, in the IL-4 regulation of Socs-1 gene expression in keratinocytes.

Interleukin‐4 downregulates TNFα‐induced IL‐8 production in keratinocytes

Interleukin (IL)‐8 is a CXC chemokine induced by pro‐inflammatory cytokines such as TNFα, IL‐1β and IL‐6 in different cell types including keratinocytes. IL‐4 regulation of TNFα‐induced IL‐8 expression is cell‐type specific. In this study, we show that in the keratinocyte cell line HaCaT, IL‐4 decreases TNFα‐induced IL‐8 mRNA expression. We then investigated the mechanism of IL‐4 effect and showed that IL‐4 downregulates TNFα‐induced IL‐8 promoter activity in luciferase reporter assays. Moreover, overexpression of either the endogenous JAK inhibitor SOCS‐1 or a dominant negative form of the STAT6 transcription factor (STAT6ΔC) interferes with the IL‐4 inhibitory effect on IL‐8 promoter. Finally we demonstrate, using a NF‐κB‐dependent promoter luciferase construct that IL‐4 interferes, at least in part, with NF‐κB transcriptional activity. Overall our results suggest that IL‐4 regulates TNFα‐induced IL‐8 expression at a transcriptional level and this mechanism involves STAT6 and NF‐κB transcription factors.

Genistein analogues: effects on epidermal growth factor receptor tyrosine kinase and on stress-activated pathways

Two genistein analogues (MD831 and MD833) have been synthesized and analyzed for their biological properties and their mechanism of action im comparison to genistein either in vitro or in intact cells. We showed that, in vitro, one of these compounds (MD831) inhibits the tyrosine kinase activity associated with the epidermal growth factor receptor (EGFR) as efficiently as genistein. However, treatment of A431 cells with these compounds did not result in any significant modification of EGFR tyrosine phosphorylation. Extracellular-signal regulated kinase (ERK) phosphorylation in cells stimulated by EGF was enhanced in the presence of MD831, whereas the other compounds, genistein and MD833, were able to activate the c-jun N-terminal kinase (JNK). This study showed that two structurally related compounds could elicit markedly different pharmacological effects on two signalling pathways, one involved in the mitogenic response and the other in the stress response. Such compounds may be useful to characterize signalling events involved in cell response to physiological stimuli.

Epidermal growth factor receptor signaling cascade as target for tyrphostin (RG 50864) in epithelial cells: Paradoxical effects on mitogen-activated protein kinase kinase and mitogen-activated protein kinase activities☆

Tyrphostins are synthetic compounds that have been described as in vitro inhibitors of epidermal growth factor receptor (EGF-R) tyrosine kinase activity. The inhibitory effect of tyrphostins in intact cells has been shown only after prolonged treatment. However, these compounds appear to be readily incorporated, which suggests that tyrphostin acts indirectly on EGF-R. We studied the effects of a tyrphostin derivative, RG 50864, without preincubation in intact epithelial cells. We selected two human cell lines differing in degree of expression of the p185erbB2 protein, which is closely related to EGF-R. We showed that tyrphostin (RG 50864) had no effect on EGF-dependent EGF-R tyrosine phosphorylation in the parental cell line. On the contrary, it prolonged the EGF-dependent EGF-R and p185erbB2(V-E) tyrosine phosphorylation in p185erbB2(V-E)-expressing cells. Because tyrphostin has been shown to be an inhibitor of p185erbB2 and EGF-R in vitro, this finding indicates that the tyrphostin effect on p185erbB2(V-E) and EGF-R was the result of an indirect mechanism in transfected cells. Tyrphostin treatment alone led to the activation of mitogen-activated protein (MAP) kinase kinase or MAP kinase or extracellular signal-regulated kinase kinase (MEK), suggesting that one of the tyrphostin targets was upstream of MEK1. MAP kinase, however, was not activated after tyrphostin treatment. This finding indicates that tyrphostin had another target in intact cells because MEK1 activation by tyrphostin alone did not correlate with MAP kinase activation. In the two cell lines, tyrphostin modified the time course of EGF-dependent MEK and MAP kinase activation. We conclude that whereas tyrphostins were designed to inhibit EGF-R tyrosine kinase activity, under our conditions EGF-R is not a physiological target for tyrphostin, nor is one of its related protein tyrosine kinases, p185erbB2(V-E). On the contrary, our results show that tyrphostin targets are multiple, leading to complex effects on receptor signaling in these epithelial cells.

Sp2 regulates interferon-γ-mediated socs1 gene expression

Suppressor of cytokine signalling (SOCS) proteins are inducible feedback inhibitors of Janus kinase (JAK) and signal transducers and activators of transcription signalling (STAT) pathways. Interferon (IFN)-gamma induces the expression of the socs1 gene in several cell types through several cis elements present in its promoter and their binding proteins. Socs1 expression is induced in the human keratinocytes HaCaT cell line through sequential activation of STAT1 and IRF-1. Comparison of the 5-upstream sequences of the mouse and human socs1 genes identified conserved binding sites for IRF-1 regulatory elements. Although this response element is able to bind IRF-1 in human cells, no IFN-gamma responsiveness was observed with human socs1 promoter reporter constructs containing this element. In contrast the mouse socs1 promoter was fully responsive. The mouse promoter contains two cis-acting elements which modulate its expression and are recognized by IRF-1 and Sp2. Despite the absence of Sp2 in the 5-upstream sequence of the human promoter, silencing of Sp2 by RNA interference clearly demonstrated that Sp2 is required for IFN-gamma-induced regulation of socs1 mRNA both in human and mouse.

Early effect of BCNU on rat astrocytes: Inhibition of S6 kinase activation by growth factors

In primary cultures of astrocytes, methylmethane, 2-N-methyl 9-hydroxy-ellepticinium acetate, ditercalinium, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1,3 bis (2-chloroethyl)-1-nitrosourea (BCNU) blocked to various extents the activation of S6 kinase by acidic fibroblast growth factor and insulin [or insulin-like growth factor 1 (IGF1)]. The effects of the most active agent, BCNU, were time and concentration dependent. Pretreatment of cells with 50 microM BCNU for 1 hr completely prevented S6 kinase activation by growth factors for at least 2 days. The S6 kinase activity of unstimulated cells was slightly affected. S6 kinase activation by 12-O-tetradecanoylphorbol 13 acetate was also strongly impaired by treating cells with BCNU whereas activation by 8-bromo-cyclic AMP was slightly reduced. Cyclic AMP-dependent protein kinase and phospholipid and Ca(2+)-dependent protein kinase were unaffected. BCNU had no direct effect on IGF1 binding to cell surface receptors or on the S6 kinase activity of cell cytosols.

Mechanism of Action of M. luteus Apurinic/Apyrimidinic Endonucleases

Apurinic/apyrimidinic sites (AP-sites) occur in DNA. They could have different origin : spontaneous depurination, excision by specific DNA glycosylases of damaged bases. Such lesions are repaired by two independant mechanisms1.In the repair by reinsertion, specific insertase inserts the missing purine and regenerates the integrity of DNA. In the repair by base excision, a specific endonuclease for AP-sites (AP-endonuclease) recognises the lesion and incises the DNA backbone near the lesion. The termini generated by AP-endonucleases are of importance as they determine the polarity of the excision of the lesion. Furthermore there is an increasing interest for AP-endonucleases since pyrimidine dimers are excised in M. luteus by base excision2. In this paper we report the determination of the termini generated by the two different AP-endonucleases extracted from M. luteus. We also report experiments showing that these enzymes could be used to detect AP-sites introduced in DNA by γ-rays or by UV irradiation.