Regulation of human immunodeficiency virus type 1 infectivity by the ERK mitogen-activated protein kinase signaling pathway

ERK1 and ERK2 mitogen-activated protein kinases (MAPK) play a critical role in regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli. Mitogens and cytokines that activate MAPK in T cells have been shown to activate human immunodeficiency virus type 1 (HIV-1) replication. Little is known about the signal transduction pathways that activate HIV-1 replication in T cells upon activation by extracellular stimulation. Here, we report that activation of MAPK through the Ras/Raf/MEK signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity was enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK (MAPK kinase) in the absence of extracellular stimulation. Treatment of cells with PD 098059, a specific inhibitor of MAPK activation, or with a MAPK antisense oligonucleotide reduced the infectivity of HIV-1 virions without significantly affecting virus production or the levels of virion-associated Gag and Env proteins. MAPK has been shown to regulate HIV-1 infectivity by phosphorylating Vif (X. Yang and D. Gabuzda, J. Biol. Chem. 273:29879-29887, 1998). However, MAPK activation enhanced virus infectivity in some cells lines that do not require Vif function. The HIV-1 Rev, Tat, p17(Gag), and Nef proteins were directly phosphorylated by MAPK in vitro, suggesting that other HIV-1 proteins are potential substrates for MAPK phosphorylation. These results suggest that activation of the ERK MAPK pathway plays a role in HIV-1 replication by enhancing the infectivity of HIV-1 virions through Vif-dependent as well as Vif-independent mechanisms. MAPK activation in producer cells may contribute to the activation of HIV-1 replication when T cells are activated by mitogens and other extracellular stimuli.     

EGFR signaling pathway negatively regulates PSA expression and secretion via the PI3K-Akt pathway in LNCaP prostate cancer cells

Epidermal growth factor (EGF) and its receptor (EGFR) are involved in hormone-refractory growth and poor prognosis of a subgroup of human prostate cancer. In this communication, we investigated the regulation of PSA by the EGFR signaling pathway using LNCaP C-81 prostate cancer cells. Administration of EGF stimulated the growth of LNCaP C-81 cells, however, PSA expression and secretion were suppressed. An EGFR inhibitor, AG1478, abrogated the PSA suppression effect by EGF, in concurrence with the suppression of tyro-phosphorylation levels of EGFR. Interestingly, the AR level was also decreased in EGF-treated LNCaP C-81 cells. Moreover, LY294002, but not PD98059, inhibited the PSA and AR suppression effect by EGF in concurrence with the suppression of phosphorylation levels of Akt. In conclusion, our results strongly suggest the existence of a novel androgen-independent PSA regulatory mechanism, i.e., the EGFR signaling pathway negatively regulates PSA expression which may be induced by the alteration of AR expression via the PI3K-Akt pathway in LNCaP C-81 cells.     

Opposing effects of protein kinase Calpha and protein kinase Cepsilon on collagen expression by human lung fibroblasts are mediated via MEK/ERK and caveolin-1 signaling

The roles of MEK, ERK, the epsilon and alpha isoforms of protein kinase C (PKC), and caveolin-1 in regulating collagen expression were studied in normal lung fibroblasts. Knocking down caveolin-1 gave particularly striking results. A 70% decrease caused a 5-fold increase in MEK/ERK activation and collagen expression. The combined data reveal a branched signaling pathway. In its central portion MEK activates ERK, leading to increased collagen expression. Two branches converge on MEK/ERK. In one, increased PKCepsilon leads to MEK/ERK activation. In another, increased PKCalpha induces caveolin-1 expression, which in turn inhibits MEK/ERK activation and collagen expression. Lung fibroblasts from scleroderma patients with pulmonary fibrosis showed altered signaling. Consistent with their overexpression of collagen, scleroderma lung fibroblasts contain more activated MEK/ERK and less caveolin-1 than normal lung fibroblasts. Because cutaneous fibrosis is the hallmark of scleroderma, we also studied dermal fibroblasts. As in lung, there was more activated MEK/ERK in cells from scleroderma patients than in control cells, and MEK inhibition decreased collagen expression. However, the distinctive levels of PKCepsilon, PKCalpha, and caveolin-1 in lung and dermal fibroblasts from scleroderma patients and control subjects indicate that the links between these signaling proteins and MEK/ERK must function differently in the four cell types. Finally, we confirmed the relevance of these signaling cascades in vivo. The combined results demonstrate that a branched signaling pathway involving MEK, ERK, PKCepsilon, PKCalpha, and caveolin-1 regulates collagen expression in normal lung tissue and is perturbed during fibrosis.     

Regulation of the mitogen-activated protein kinase signaling pathway by SHP2.

Gab1-SHP2 association is required for Erk mitogen-activated protein kinase activation by several growth factors. Gab1-SHP2 interaction activates SHP2. However, an activated SHP2 still needs to associate with Gab1 to mediate Erk activation. It was unclear whether SHP2 is required to dephosphorylate a negative phosphorylation site on Gab1 or whether SHP2 needs the Gab1 pleckstrin homology (PH) domain to target it to the plasma membrane. We found that expression of a fusion protein consisting of the Gab1 PH domain and an active SHP2 (Gab1PH-SHP2DeltaN) induced constitutive Mek1 and Erk2 activation. Linking the active SHP2DeltaN to the PDK1 PH domain or the FRS2beta myristoylation sequence also induced Mek1 activation. Mek1 activation by Gab1PH-SHP2DeltaN was inhibited by an Src inhibitor and by Csk. Significantly, Gab1PH-SHP2DeltaN induced Src activation. Gab1PH-SHP2DeltaN expression activated Ras, and the Gab1PH-SHP2DeltaN-induced Mek1 activation was blocked by RasN17. These findings suggest that Gab1PH-SHP2DeltaN activated a signaling step upstream of Src and Ras. The SHP2 tyrosine phosphatase activity is essential for the function of the fusion protein. Together, these data show that the Gab1 sequence, besides the PH domain and SHP2 binding sites, is dispensable for Erk activation, suggesting that the primary role of Gab1 association with an activated SHP2 is to target it to the membrane.     

Inhibition of a signaling pathway in cardiac muscle cells by active mitogen-activated protein kinase kinase.

Signaling via the Ras pathway involves sequential activation of Ras, Raf-1, mitogen-activated protein kinase kinase (MKK), and the extracellular signal-regulated (ERK) group of mitogen-activated protein (MAP) kinases. Expression from the c-Fos, atrial natriuretic factor (ANF), and myosin light chain-2 (MLC-2) promoters during phenylephrine-induced cardiac muscle cell hypertrophy requires activation of this pathway. Furthermore, constitutively active Ras or Raf-1 can mimic the action of phenylephrine in inducing expression from these promoters. In this study, we tested whether constitutively active MKK, the molecule immediately downstream of Raf, was sufficient to induce expression. Expression of constitutively active MKK induce ERK2 kinase activity and caused expression from the c-Fos promoter, but did not significantly activate expression of reporter genes under the control of either the ANF or MLC-2 promoters. Expression of CL100, a phosphatase that inactivates ERKs, prevented expression from all of the promoters. Taken together, these data suggest that ERK activation is required for expression from the Fos, ANF, and MLC-2 promoters but MKK and ERK activation is sufficient for expression only from the Fos promoter. Constitutively active MKK synergized with phenylephrine to increase expression from a c-Fos- or an AP1-driven reporter. However, active MKK inhibited phenylephrine- and Raf-1-induced expression from the ANF and MLC-2 promoters. A DNA sequence in the MLC-2 promoter that is a target for inhibition by active MKK, but not CL100, was mapped to a previously characterized DNA element (HF1) that is responsible for cardiac specificity. Thus, activation of cardiac gene expression during phenylephrine-induced hypertrophy requires ERK activation but constitutive activation by MKK can inhibit expression by targeting a DNA element that controls the cardiac specificity of gene expression.     

Integrin-linked kinase complexes with caveolin-1 in human neuroblastoma cells

Integrin-linked kinase (ILK) and caveolin-1 (cav-1) are implicated in the pathogenesis of cancer. Overexpression of ILK leads to altered expression of cell cycle regulators, a decreased level of cell adhesion to the extracellular matrix, a decreased level of apoptosis, in vitro phosphorylation of Akt, and tumor formation in nude mice. Conversely, cav-1 expression is frequently downregulated in many forms of cancer. We examined whether ILK and cav-1 interact in SHEP human neuroblastoma cells because ILK is present in caveolae-enriched membranes and contains a putative cav-binding domain. SHEP cells were stably transfected with vector, wild-type ILK (ILK-wt), kinase-deficient ILK (ILK-kd), or mutant cav-binding domain ILK (ILK-mutCavbd). Control SHEP cells and ILK transfectants express high levels of ILK and cav-1. Immunoprecipitation with anti-cav-1 co-immunoprecipitates a 59 kDa protein that is immunoreactive with the anti-ILK antibody, and this interaction is partially prevented in cells expressing ILK-mutCavbd. Cav-1 and ILK partially colocalize in SHEP cells, also supporting these data. Last, affinity chromatography with a biotinylated cav-scaffolding domain peptide precipitates ILK-wt but not ILK-mutCavbd. These data suggest that the cav-binding domain of ILK and the cav-scaffolding domain of cav-1 mediate complex formation in human neuroblastoma cells.     

Regulation of Hex gene expression by a Smads-dependent signaling pathway

The homeobox gene Hex is expressed in multiple cell types during embryogenesis and is required for liver and monocyte development. Hex is expressed in the foregut region of late gastrula avian and mammalian embryos in a pattern that overlaps with expression of bone morphogenetic proteins (BMPs). Here we investigate the relationship between BMP signaling and Hex gene expression. We find that Hex expression in avian anterior lateral endoderm is regulated by autocrine BMP signaling. Characterization of the mouse Hex gene promoter identified a 71-nucleotide BMP-responsive element (BRE) that is required for up-regulation of Hex by an activated BMP signaling pathway. The Hex BRE binds Smad4 and Smad1-Smad4 complexes in vitro, and in transfection assays, it is responsive to Smad1 and Smad4 but not to Smad2 and Smad4 or Smad3 and Smad4. The BRE contains two copies of a GCCGnCGC-like motif that in Drosophila is the binding site for Mad and Madea followed by two CAGAG boxes that are similar to sequences required for transforming growth factor-beta/activin responsiveness of several vertebrate genes. Mutation of the GC elements, but not the two CAGAG boxes, abolishes Smads responsiveness in the intact Hex promoter, whereas mutations in both the GC elements and CAGAG boxes show that they act cooperatively to confer Smads responsiveness to the Hex promoter. The Hex BRE can confer Smads responsiveness to a heterologous promoter, and in this context, both the GC-rich elements and the CAGAG boxes are required for Smads-dependent promoter activity. An element almost identical to the Hex BRE is present within the BMP-responsive Nkx2-5 gene promoter, suggesting that the Hex BRE represents a common response element for genes regulated by BMP signaling in the foregut region of the embryo.     

A role for protein kinase cepsilon in the inhibitory effect of epidermal growth factor on calcium-stimulated chloride secretion in human colonic epithelial cells

Epidermal growth factor (EGF) inhibits carbachol-induced chloride secretion in T(84) colonic epithelial cells and has been shown to activate phosphatidylinositol (PI) 3-kinase, leading to inhibition of a basolateral potassium conductance. We asked whether the inhibitory effect of EGF on secretion is due to activation of specific isoforms of protein kinase C (PKC) by PI 3-kinase. Western analysis revealed that PKCalpha, gamma, epsilon, eta, mu, lambda/iota, and zeta were expressed in T(84) cells. Ro318220 (an inhibitor active against PKCepsilon, 10 micrometer) but not G?6983 (an inhibitor active against PKCzeta, 10 micrometer) reversed the inhibitory effect of EGF (100 ng/ml) on carbachol-stimulated chloride secretion. EGF induced the rapid translocation of PKCepsilon from the cytoplasm to the membrane. Wortmannin (50 micrometer) and LY294002 (20 nm), which are PI 3-kinase inhibitors that by themselves had no effect on PKCepsilon activity, significantly suppressed PKCepsilon translocation activated by EGF. LY294002 also reversed the inhibitory action of EGF on chloride secretion. PI (3,4)P(2) increased membrane-associated PKCepsilon and reduced carbachol-induced (86)Rb(+) efflux. Antisense oligonucleotides against PKCepsilon decreased PKCepsilon mass and prevented the inhibitory effect of EGF on carbachol-induced (86)Rb(+) efflux. Thus, the inhibitory effect of EGF on carbachol-induced chloride secretion involves the activation of PKCepsilon mediated by PI 3-kinase. Our findings contribute to the understanding of the cellular mechanisms that control chloride secretion.     

Regulation of tumor phenotypes by caveolin-1 and sphingolipid-controlled membrane signaling complexes

Aberrant (glyco)sphingolipid expression deeply affects several properties of tumor cells that are involved in tumor progression and metastasis formation: cell adhesion (to the extracellular matrix or to the endothelium of blood vessels), motility, recognition and invasion of host tissues. In particular, (glyco)sphingolipids might contribute to the modulation of integrin-dependent interactions of tumor cells (determining their adhesion, motility and invasiveness) with the extracellular matrix as well as with host cells present in the stromal compartment of the tumor. A model based on solid experimental evidence has been proposed: (glyco)sphingolipids at the cell surface interact with plasma membrane receptors (e.g., integrin receptors and growth factor receptors) and adapter molecules (including tetraspanins) forming signaling complexes that are able to influence the activity of signal transduction molecules oriented at the cytosolic surface of the plasma membrane (mainly the Src kinases pathway members). The function of these signaling complexes appears to be strictly dependent on their (glyco)sphingolipid composition, and likely on specific sphingolipid-protein interactions. From this point of view, particularly intriguing is the connection between (glyco)sphingolipids and caveolin-1, a membrane protein that plays multiple roles as a suppressor of tumor growth and metastasis in ovarian, breast and colon human carcinomas.     

Regulation of the TAK1 signaling pathway by protein phosphatase 2C

Protein phosphatase 2C (PP2C) is implicated in the negative regulation of stress-activated protein kinase cascades in yeast and mammalian cells. In this study, we determined the role of PP2Cbeta-1, a major isoform of mammalian PP2C, in the TAK1 signaling pathway, a stress-activated protein kinase cascade that is activated by interleukin-1, transforming growth factor-beta, or stress. Ectopic expression of PP2Cbeta-1 inhibited the TAK1-mediated mitogen-activated protein kinase kinase 4-c-Jun amino-terminal kinase and mitogen-activated protein kinase kinase 6-p38 signaling pathways. In vitro, PP2Cbeta-1 dephosphorylated and inactivated TAK1. Coimmunoprecipitation experiments indicated that PP2Cbeta-1 associates with the central region of TAK1. A phosphatase-negative mutant of PP2Cbeta-1, PP2Cbeta-1 (R/G), acted as a dominant negative mutant, inhibiting dephosphorylation of TAK1 by wild-type PP2Cbeta-1 in vitro. In addition, ectopic expression of PP2Cbeta-1(R/G) enhanced interleukin-1-induced activation of an AP-1 reporter gene. Collectively, these results indicate that PP2Cbeta negatively regulates the TAK1 signaling pathway by direct dephosphorylation of TAK1.     

Nuclear factor-kappaB p65 inhibits mitogen-activated protein kinase signaling pathway in radioresistant breast cancer cells

The molecular mechanism by which tumor cells increase their resistance to therapeutic radiation remains to be elucidated. We have previously reported that activation of nuclear factor-kappaB (NF-kappaB) is causally associated with the enhanced cell survival of MCF+FIR cells derived from breast cancer MCF-7 cells after chronic exposure to fractionated ionizing radiation. The aim of the present study was to reveal the context of NF-kappaB pathways in the adaptive radioresistance. Using cell lines isolated from MCF+FIR populations, we found that the elevated NF-kappaB activity was correlated with enhanced clonogenic survival, and increased NF-kappaB subunit p65 levels were associated with a decrease in phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK in all radioresistant MCF+FIR cell lines. Further irradiation with 30 fractions of radiation also inhibited MEK/ERK phosphorylation in paired cell lines of MCF+FIR and parental MCF-7 cells. Activation of ataxia-telangiectasia mutated (ATM) protein, a sensor to radiation-induced DNA damage, was elevated with increased interaction with NF-kappaB subunits p65 and p50. The interaction between p65 and MEK was also enhanced in the presence of activated ATM. In contrast, both interaction and nuclear translocation of p65/ERK were reduced. Inhibition of NF-kappaB by overexpression of mutant IkappaB increased ERK phosphorylation. In addition, MEK/ERK inhibitor (PD98059) reduced the interaction between p65 and ERK. Taken together, these results suggest that NF-kappaB inhibits ERK activation to enhance cell survival during the development of tumor adaptive radioresistance.