Two transactivation domains of hypoxia-inducible factor-1alpha regulated by the MEK-1/p42/p44 MAPK pathway

At a low-oxygen tension, cells increase the expression of several genes (such as erythropoietin, the vascular endothelial growth factor, and glycolytic enzymes) in order to adapt to hypoxic stress. A common transactivator, named the hypoxia-inducible factor 1 (HIF-1) activates these genes. HIF-1 is a heterodimeric transactivator that is composed of alpha and beta subunits. HIF-1 activity is primarily determined by the hypoxia-induced stabilization of the alpha subunit, whereas the HIF-1beta subunit is expressed constitutively. Our previous observation implied that the MEK-1/p42/p44 MAPK pathway is involved in the hypoxia-induced transactivation ability, but not in the stabilization and DNA binding of HIF-1alpha. In this paper, we dissected the transactivation domain of HIF-1alpha in more detail, and tested the correlation between specific domains of HIF-1alpha and specific signaling pathways. We designed several fusion proteins that contain deletion mutants of HIF-1alpha that is linked to the DNA binding domain of the yeast protein Gal4. By using the Gal4-driven reporter system, we tested the transactivation activities of the Gal4/HIF-1alpha fusion proteins in Hep3B cells. Our findings suggest that tyrosine kinases, the MEK-1/p42/p44 MAPK pathway, but not the PI-3 kinase/Akt pathway, are involved in the hypoxia-induced transactivation of HIF-1alpha. We have shown that the functional transactivation activities are located at both 522-649 and 650-822 amino acids of HIF-1alpha. Treatment of PD98059, a MEK-1 inhibitor, blocked the hypoxia-induced transactivation abilities of both the 522-649 and 650-822 amino acids of the C-terminal half of HIF-1alpha. This implies that the MEK-1/p42/p44 MAPK signaling pathway cannot distinguish between the two hypoxia-induced transactivation domains.     

Activation of the Bcl-2 promoter by nerve growth factor is mediated by the p42/p44 MAPK cascade

The Bcl-2 protein has an anti-apoptotic effect in neuronal and other cell types. We show for the first time that the Bcl-2 promoter is activated by the neuronal survival factor nerve growth factor (NGF) and that this effect is dependent on a region of the promoter from -1472 to -1414. This activation requires the Rap-1 G protein and the MEK-1 and p42/p44 MAPK enzymes but is independent of other NGF-activated signalling pathways involving protein kinase A or protein kinase C.     

Stimulation of DNA synthesis and cell proliferation of human mammary myoepithelial-like cells by hepatocyte growth factor/scatter factor depends on heparan sulfate proteoglycans and sustained phosphorylation of mitogen-activated protein kinases p42/44

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparan/dermatan sulfate-binding growth factor produced by stromal cells that acts as a paracrine effector on neighboring epithelia. HGF/SF stimulated DNA synthesis in human mammary (Huma) 109 myoepithelial-like cells grown on collagen I and fibronectin substrata but not when grown on plastic. Dual phosphorylation of mitogen-activated protein kinases (p42/44(MAPK)) was required for this stimulation of DNA synthesis. In Huma 109 cells cultured on plastic, HGF/SF stimulated a transient phosphorylation of p42/44(MAPK), which reached a maximum at 10 min after addition of the growth factor and returned to near basal levels after 20 min. In contrast, the phosphorylation of p42/44(MAPK) stimulated by HGF/SF in cells cultured on collagen I or fibronectin was sustained over 45 min. In Huma 109 cells deficient in sulfated glycosaminoglycans, HGF/SF failed to stimulate p42/44(MAPK) phosphorylation or DNA synthesis on any substratum, even when soluble heparan sulfate proteoglycans purified from the cells or from the culture medium were added. However, HGF/SF stimulated DNA synthesis and a sustained phosphorylation of p42/44(MAPK) in sulfated glycosaminoglycan-deficient Huma 109 cells plated on a substratum of medium HSPGs but not cell HSPGs. The HGF/SF-induced proliferation is thus highly dependent on heparan sulfate proteoglycans in myoepithelial-like cells.     

Proliferation of pulmonary interstitial fibroblasts is mediated by transforming growth factor-beta1-induced release of extracellular fibroblast growth factor-2 and phosphorylation of p38 MAPK and JNK

Idiopathic pulmonary fibrosis (IPF; a progressive lung disease) is characterized by parenchymal remodeling with enlarged air spaces called honeycomb cysts and palisades of fibroblasts called fibroblast foci. In IPF, lung epithelial cells covering honeycomb cysts and fibroblast foci aberrantly express the active conformation of the potent fibrogenic cytokine transforming growth factor-beta1 (TGF-beta1). Using explanted rat lung slices, we transfected alveolar epithelial cells with the retrovirus pMX containing a site-directed mutation in which Cys223 and Cys225 were substituted with serines, resulting in release of biologically active TGF-beta1 and fibroblast proliferation and remodeling that resembled IPF. Fibroblasts obtained from transfected explants and in culture for 6 weeks incorporated 6.59 +/- 1.55-fold more [3H]thymidine compared with control fibroblasts without transfection or fibroblasts obtained from transfected explants cultured with antibody to fibroblast growth factor-2 (FGF-2). Primary lung fibroblasts obtained from normal rat lungs cultured with TGF-beta1 expressed increased levels of phosphorylated p38 MAPK and JNK, but not ERK1/2. The presence of TGF-beta1 caused an immediate release of extracellular FGF-2 from primary pulmonary fibroblasts; and in the presence of anti-FGF-2 antibody, phosphorylated p38 MAPK and JNK were abrogated. TGF-beta inhibits cell proliferation by suppression of c-Myc and induction of p15INK46, p21CIP1, or p27KIP. Fibroblasts cultured with TGF-beta1 showed no regulation of c-Myc or induction of p15INK46, p21CIP1,or p27KIP. These findings suggest that pulmonary fibroblasts may not respond to the anti-proliferative effects of TGF-beta1, but proliferate in response to TGF-beta1 indirectly by the release of FGF-2, which induces phosphorylation of p38 MAPK and JNK.     

Chemokine CCL20 enhances the growth of HuH7 cells via phosphorylation of p44/42 MAPK in vitro

CCR6 is the receptor of chemokine CCL20. In the present study, we demonstrated that the surface expression of CCR6 was enhanced on the human HCC cell lines (HuH7, PLC/PRF/5, and HepG2) especially on HuH7 cells, but not on HLE or HLF cells. These HCC cell lines (HuH7, PLC/PRF/5, and HepG2) especially the HuH7 cells secreted a significant amount of CCL20 spontaneously, whereas HLE or HLF did not. Stimulation by CCL20 up-regulated the mRNA expression of CCR6 in HuH7 cells and significantly enhanced the growth of HuH7 cells. CCL20-stimulated growth of HuH7 cells was abrogated by the inhibition of downstream signal transduction pathway mediated by p44/42 MAPK, but not by p38 MAPK or SAPK/JNK. CCR6 expression in human HCC tissues was confirmed by RT-PCR. These results indicate that the growth of a proportion of human HCC cells may be mediated by CCL20-CCR6 axis, like HuH7 cells, in an autocrine or paracrine manner.     

Fibroblast growth factor-2 promotes keratan sulfate proteoglycan expression by keratocytes in vitro

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.     

TGF-beta-induced matrix proteins inhibit p42/44 MAPK and JNK activation and suppress TNF-mediated IkappaBalpha degradation and NF-kappaB nuclear translocation in L929 fibroblasts

The role of transforming growth factor beta1 (TGF-beta1)-induced extracellular matrix proteins in the modulation of cellular response to the cytotoxic effect of tumor necrosis factor (TNF) or Fas ligand was investigated. Murine L929 fibroblasts were prestimulated with or without TGF-beta1 for 1-24 h and the resulting extracellular protein matrices were prepared. Unstimulated control L929 cells were then cultured on these matrices. Compared to control matrix-stimulated L929 cells, the TGF-beta1 matrix-stimulated cells resisted TNF killing in the presence of actinomycin D (ActD), but became more susceptible to killing by anti-Fas antibodies/ActD. The induced TNF resistance is independent of the NF-kappaB antiapoptotic effect. For example, exposure of TGF-beta1 matrix-stimulated L929 cells to TNF failed to result in IkappaBalpha degradation and NF-kappaB nuclear translocation or activation. Also, control matrix stimulated the activation of p42/44 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in L929 cells, whereas TGF-beta1 matrix suppressed the activation. Nonetheless, in response to TNF, JNK activation was restored in the TGF-beta1 matrix-stimulated cells. By metabolic labeling, ammonium sulfate precipitation and N-terminal amino acid microsequencing, TGF-beta1 was shown to induce a novel matrix protein of 46 kDa (p46) from L929 cells. Adsorption of p46 by peptide antibodies against its N-terminus removed the TGF-beta1 matrix protein-mediated protection against TNF/ActD cytotoxicity and its enhancement of anti-Fas/ActD killing, indicating that p46 is responsible for these effects. Immunostaining of L929 cells revealed that the antibodies were bound to a membrane protein of 100 kDa (p100). Thus, the matrix p46 is likely derived from the released membrane p100.     

Extracellular cysteine/cystine redox regulates the p44/p42 MAPK pathway by metalloproteinase-dependent epidermal growth factor receptor signaling

Previous research shows that stimulation of proliferation of colon carcinoma (Caco-2) cells by a more reduced extracellular cysteine/cystine (Cys/CySS) redox state occurs with no apparent effect on intracellular glutathione and that this stimulation is lost on addition of epidermal growth factor. The purpose of the present study was to determine whether a more reduced extracellular Cys/CySS redox state activates the mitogenic p44/p42 mitogen-activated protein kinase (MAPK) pathway and whether this is signaled through the epidermal growth factor receptor (EGFR). Caco-2 cells were exposed to a range of physiological extracellular redox conditions from -150 to 0 mV. In the absence of added growth factors, the most reduced (-150 mV) redox state induced an 80% increase in EGFR phosphorylation, and this was followed by a marked increase in phosphorylation of p44/p42 MAPK. Inhibitors of EGFR (AG1478) and p44/p42 MAPK (U0126) phosphorylation blocked redox-dependent p44/p42 phosphorylation, indicating that signaling occurred by EGFR. These effects were inhibited by pretreatment with a nonpermeant alkylating agent, showing that signaling involved thiols accessible to the extracellular space. The EGFR ligand TGF-alpha was increased in culture medium at more reduced redox states. Redox-dependent phosphorylation of EGFR was completely prevented by a metalloproteinase inhibitor (GM6001), and an antibody to TGF-alpha partially inhibited the phosphorylation of p44/p42 MAPK by redox. Thus the data show that a redox-dependent activation of metalloproteinase can stimulate the mitogenic p44/p42 MAPK pathway by a TGF-alpha-dependent mechanism. Because Cys availability and Cys/CySS redox are dependent on nutrition, disease, and environmental exposures, the results suggest that cell proliferation could be influenced physiologically by Cys-dependent redox effects on growth factor signaling pathways.     

BK-induced COX-2 expression via PKC-delta-dependent activation of p42/p44 MAPK and NF-kappaB in astrocytes

Bradykinin (BK) is an inflammatory mediator, elevated levels in the region of several brain injury and inflammatory diseases. It has been shown to induce cyclooxygenase-2 (COX-2) expression implicating in inflammatory responses in various cell types. However, the signaling mechanisms underlying BK-induced COX-2 expression in astrocytes remain unclear. First, RT-PCR and Western blotting analysis showed that BK induced the expression of COX-2 mRNA and protein, which was inhibited by B(2) BK receptor antagonist Hoe140, suggesting the involvement of B(2) BK receptors. BK-induced COX-2 expression and translocation of PKC-delta from cytosol to membrane fraction were inhibited by rottlerin, suggesting that PKC-delta might be involved in these responses. This hypothesis was further supported by the transfection with a dominant negative plasmid of PKC-delta significantly blocked BK-induced COX-2 expression. BK-stimulated p42/p44 MAPK phosphorylation, COX-2 mRNA expression, and prostaglandin E(2) (PGE(2)) release were attenuated by PD98059, indicating the involvement of MEK/p42/p44 MAPK in this pathway. Accordingly, BK-stimulated phosphorylation of p42/p44 MAPK was attenuated by rottlerin, indicating that PKC-delta might be an upstream component of p42/p44 MAPK. Moreover, BK-induced COX-2 expression might be mediated through the translocation of NF-kappaB into nucleus which was blocked by helenalin, rottlerin and PD98059, implying the involvement of NF-kappaB. These results suggest that in RBA-1 cells, BK-induced COX-2 expression and PGE(2) release was sequentially mediated through PKC-delta-dependent activation of p42/p44 MAPK and NF-kappaB. Understanding the regulation of COX-2 expression and PGE(2) release induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.     

Internalization and degradation of heparin is not required for stimulus of heparan sulfate proteoglycan synthesis

In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombotic heparan sulfate proteoglycan (HSPG) produced by endothelial cells. The putative heparin binding site(s) that may be related to this phenomenon were investigated. In the preceding article, using various heparin probes, it was shown that the heparin does not bind to the endothelial cell surface, but only to the extracellular matrix. The present study demonstrated that, when the cells were exposed to heparin at 37 degrees C, the heparin was internalized and with time was localized in lysosomes. However, endocytosis of heparin was not required for the stimulation of HSPG synthesis. The requirement for heparin degradation in the stimulus of HSPG synthesis was also investigated. When the cells were incubated with chloroquine, a lysosomotropic amine that raises the lysosomal pH thus inhibiting enzymatic degradation of internalized compounds, stimulation of HSPG synthesis was still observed. These combined results indicate that neither internalization nor degradation of heparin is required for stimulation of HSPG synthesis, and suggests that its binding to the extracellular matrix could be responsible for this effect.     

Cellular specificity for the activation of fibroblast growth factor-2 by heparan sulfate proteoglycan

Heparan sulfate proteoglycans (HSPGs) promote cellular proliferation through interaction with FGF-2. To examine the role of cellular specificity of HSPG in FGF-2 function, a recombinant soluble isoform of CD44 (rsCD44v3,8-10) was expressed in various cell types; 293 T fibroblasts, the epithelial carcinoma cell lines A431 and HOTZ, the myelomonocytic cell line THP-1, and the Ig-secreting B lymphoblast IM9. The capacity of the recombinant HSPGs expressed in these cell lines to bind and present FGF-2 to the high-affinity receptor FGFR1 was addressed. This novel approach showed a minor difference in the binding and in the FGF-2 stimulating activity of rsCD44v3,8-10 HSPGs from fibroblasts and epithelial cells. However, FGF-2 binding of rsCD44v3,8-10 from IM9 and THP-1 cells was significantly lower, and stimulation of FGF-2 by rsCD44v3,8-10 from these two cell types could not be detected. We tested the possibility that the differences among cell types were related to the functional profile of endogenous HSPGs. The initial survey of a wider panel of cell types revealed high levels of HSPGs synthesis on the surface of 293 T, epithelial and IM9 cells, but low levels on the surface of other cells of hematopoietic origin. Surprisingly, native HSPGs from fibroblasts and epithelial cell lines promoted FGF-2 biological activity to vastly different extents, and cell surface HSPGs from IM9 cells induced an FGF-2 response. Altogether, the results suggested a role for cell-specific HS modification in addition to synthesis as regulatory mechanisms for the cellular specificity of proteoglycan function.     

Fibroblast growth factor receptors 1 and 2 interact differently with heparin/heparan sulfate. Implications for dynamic assembly of a ternary signaling complex

Heparan sulfate (HS) regulates the kinetics of fibroblast growth factor 2 (FGF2)-stimulated intracellular signaling and differentially activates cell proliferation of cells expressing different FGF receptors (FGFRs). Evidence suggests that HS interacts with both FGFs and FGFRs to form active ternary signaling complexes. Here we compare the interactions of two FGFRs with HS. We show that the ectodomains of FGFR1 IIIc and FGFR2 IIIc exhibit specific interactions with different characteristics for both heparin and porcine mucosal HS. These glycans are both known to activate FGF signaling via these receptors. FGFR2 interacts with a higher apparent affinity than FGFR1 despite both involving 6-O-, 2-O-, and N-sulfates. FGFR1 and FGFR2 bind heparin with mean association rate constants of 1.9 x 10(5) and 2.1 x 10(6) m(-1)s(-1), respectively, and dissociation rate constants of 1.2 x 10(-2) and 2.7 x 10(-2) s(-1), respectively. These produced calculated affinities of 63 and 13 nm, respectively. Hence, FGFR1 and FGFR2 bind to heparin chains with markedly different kinetics and affinities. We propose a mechanistic model where the kinetic parameters of the HS/FGFR interaction are a key element regulating the formation of ternary complexes and the resulting FGF signaling outcomes.     

Lipoteichoic acid-stimulated p42/p44 MAPK activation via Toll-like receptor 2 in tracheal smooth muscle cells

Lipoteichoic acid (LTA), the principal component of the cell wall of gram-positive bacteria, triggers several inflammatory responses. However, the mechanisms underlying its action on human tracheal smooth muscle cells (HTSMCs) were largely unknown. This study was to investigate the mechanisms underlying LTA-stimulated p42/p44 mitogen-activated protein kinase (MAPK) using Western blotting assay. LTA stimulated phosphorylation of p42/p44 MAPK via a Toll-like receptor 2 (TLR2). Pretreatment with pertussis toxin attenuated the LTA-induced responses. LTA-stimulated phosphorylation of p42/p44 MAPK was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (PLC; D609), phosphatidylinositol (PI)-PLC (U-73122), PKC (staurosporine, G?-6976, rottlerin, or Ro-318220), MEK1/2 (U-0126), PI 3-kinase (LY-294002 and wortmannin), and an intracellular Ca(2+) chelator (BAPTA-AM). LTA directly evoked initial transient peak of [Ca(2+)](i), supporting the involvement of Ca(2+) mobilization in LTA-induced responses. These results suggest that in HTSMCs, LTA-stimulated p42/p44 MAPK phosphorylation is mediated through a TLR2 receptor and involves tyrosine kinase, PLC, PKC, Ca(2+), MEK, and PI 3-kinase.     

Activation of cytosolic phospholipase A2 in Her14 fibroblasts by hydrogen peroxide: a p42/44(MAPK)-dependent and phosphorylation-independent mechanism

Reactive oxygen species (ROS) have been implicated in the pathogenesis of diseases as well as various normal cellular processes. It has been suggested that ROS function as mediators of signal transduction, given that they can mimic growth factor-induced signaling. The ROS H2O2 has been reported to activate phospholipase A2 (PLA2) and, therefore, we investigated if and through which pathway ROS activate cytosolic PLA2 (cPLA2) in Her14 fibroblasts. cPLA2 was activated concentration-dependently by H2O2 in a transient manner. In addition, the lipophilic cumene hydroperoxide was shown to induce cPLA2 activity in the same manner. H2O2-induced cPLA2 activity in Her14 cells was partially phosphorylation-dependent, which was mediated through the Raf-MEK-p42/44(MAPK) pathway and occurred partially through a phosphorylation-independent mechanism. ROS can lead to changes in the (micro) viscosity of membranes due to the presence oxidized lipids, thereby increasing the substrate availability for cPLA2. In support of this, treatment of Her14 cells with H2O2 induced lipid peroxidation time-dependently as determined from degradation of lipid arachidonate and linoleate and the formation of aldehydic degradation products. Furthermore, H2O2 induced translocation of cPLA2 to the membrane fraction in a calcium-independent fashion, with a concomitant increase in cPLA2 activity. Collectively, the results suggest that oxidative stress-induced cPLA2 activity is partially phosphorylation-dependent and is further increased due to increased substrate availability by the action of ROS on membranes.