Biphasic estradiol-induced AKT phosphorylation is modulated by PTEN via MAP kinase in HepG2 cells

We reported previously in HepG2 cells that estradiol induces cell cycle progression throughout the G1-S transition by the parallel stimulation of both PKC-alpha and ERK signaling molecules. The analysis of the cyclin D1 gene expression showed that only the MAP kinase pathway was involved. Here, the presence of rapid/nongenomic, estradiol-regulated, PI3K/AKT signal transduction pathway, its modulation by the levels of the tumor suppressor PTEN, its cross-talk with the ERK pathway, and its involvement in DNA synthesis and cyclin D1 gene promoter activity have all been studied in HepG2 cells. 17beta-Estradiol induced the rapid and biphasic phosphorylation of AKT. These phosphorylations were independent of each other, being the first wave of activation independent of the estrogen receptor (ER), whereas the second was dependent on ER. Both activations were dependent on PI3K activity; furthermore, the ERK pathway modulated AKT phosphorylation by acting on the PTEN levels. The results showed that the PI3K pathway, as well as ER, were strongly involved in both G1-S progression and cyclin D1 promoter activity by acting on its proximal region (-254 base pairs). These data indicate that in HepG2 cells, different rapid/nongenomic estradiol-induced signal transduction pathways modulate the multiple steps of G1-S phase transition.     

Interleukin-3 (IL-3)-induced c-fos activation is modulated by Gab2-calcineurin interaction

Interleukin-3 (IL-3) regulates cell growth by affecting various processes such as cell death, survival, and proliferation. Cues from the external environment are sensed by surface receptors, and complex signaling mechanisms arise within the cells, leading to specific functional outcomes. In this study, we demonstrate that the cytokine IL-3 induces the activation of the Ca(2+)-dependent phosphatase, calcineurin (Cn). Furthermore Cn dephosphorylates Gab2, resulting in c-fos activation and cell proliferation. We also report that there is a direct interaction between Cn and Gab2 upon IL-3 stimulation, and Akt can regulate this interaction.     

Aspirin and salicylates inhibit the IL-4- and IL-13-induced activation of STAT6.

Allergic diseases, including asthma, represent a major threat to human health. Over the three last decades, their incidence has risen in western countries. Aspirin treatment has been shown to improve allergic diseases, especially asthma, and the decreased use of aspirin has been hypothesized to contribute to the increase in childhood asthma. Because salicylate compounds suppress a number of enzymatic activities, and signaling through IL-4R participates in the development of allergic responses, we tested the effect of salicylates on IL-4 signal transduction. We found that treatment of cell lines and primary cells with aspirin and salicylates, but not acetaminophen, inhibited the activation of STAT6 by IL-4 and IL-13. This effect correlated with the inhibition of IL-4-induced CD23 expression. Although salicylates inhibited the in vivo activation of Janus kinases, their kinase activity was not affected in vitro by salicylates, suggesting that other kinases were involved in IL-4-induced STAT6 activation. Furthermore, we found that an Src kinase was involved in STAT6 activation because 1) Src kinase activity was induced by IL-4, 2) Src kinase activity, but not Janus kinase, was inhibited by salicylates in vitro, 3) cells expressing viral Src had constitutive STAT6 phosphorylation, and 4) cells lacking Src showed low STAT6 phosphorylation in response to IL-4. Because STAT6 activation by IL-4 and IL-13 participates in the development of allergic diseases, our results provide a mechanism to explain the beneficial effects of aspirin and salicylate treatment of these diseases.     

Mechanisms of endothelin-1-induced MAP kinase activation in adrenal glomerulosa cells

G protein-coupled receptors (GPCRs) such as angiotensin II, bradykinin and endothelin-1 (ET-1) are critically involved in the regulation of adrenal function, including aldosterone production from zona glomerulosa cells. Whereas, substantial data are available on the signaling mechanisms of ET-1 in cardiovascular tissues, such information in adrenal glomerulosa cells is lacking. Bovine adrenal glomerulosa (BAG) cells express receptors for endothelin-1 (ET-1) and their stimulation caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2 at Tyr402), extracellularly regulated signal kinases (ERK1/2), and their dependent proteins, p90 ribosomal S6 kinase (RSK-1) and CREB. ET-1 elicited these responses predominantly through activation of a G_i-linked cascade with a minor contribution from the G_q/PKC pathway. Whereas, selective inhibition of EGF-R kinase with AG1478 caused complete inhibition of EGF-induced ERK/RSK-1/CREB activation, it caused only partial reduction (30–40%) of such ET-1-induced responses. Consistent with this, inhibition of matrix metalloproteinases (MMPs) with GM6001 reduced ERK1/2 activation by ET-1, consistent with partial involvement of the MMP-dependent EGF-R activation in this cascade. Activation of ERK/RSK-1/CREB by both ET-1 and EGF was abolished by inhibition of Src, indicating its central role in ET-1 signaling in BAG cells. Moreover, the signaling characteristics of ET-1 in cultured BAG cells closely resembled those observed in clonal adrenocortical H295R cells. The ET-1-induced proliferation of BAG and H295 R cells was much smaller than that induced by Ang II or FGF. These data demonstrate that ET-1 causes ERK/RSK-1/CREB phosphorylation predominantly through activation of G_i and Src, with a minor contribution from MMP-dependent EGF-R transactivation.     

Sprouty2-mediated inhibition of fibroblast growth factor signaling is modulated by the protein kinase DYRK1A

Raf-MEK-extracellular signal-regulated kinase (Erk) signaling initiated by growth factor-engaged receptor tyrosine kinases (RTKs) is modulated by an intricate network of positive and negative feedback loops which determine the specificity and spatiotemporal characteristics of the intracellular signal. Well-known antagonists of RTK signaling are the Sprouty proteins. The activity of Sprouty proteins is modulated by phosphorylation. However, little is known about the kinases responsible for these posttranslational modifications. We identify DYRK1A as one of the protein kinases of Sprouty2. We show that DYRK1A interacts with and regulates the phosphorylation status of Sprouty2. Moreover, we identify Thr75 on Sprouty2 as a DYRK1A phosphorylation site in vitro and in vivo. This site is functional, since its mutation enhanced the repressive function of Sprouty2 on fibroblast growth factor (FGF)-induced Erk signaling. Further supporting the idea of a functional interaction, DYRK1A and Sprouty2 are present in protein complexes in mouse brain, where their expression overlaps in several structures. Moreover, both proteins copurify with the synaptic plasma membrane fraction of a crude synaptosomal preparation and colocalize in growth cones, pointing to a role in nerve terminals. Our results suggest, therefore, that DYRK1A positively regulates FGF-mitogen-activated protein kinase signaling by phosphorylation-dependent impairment of the inhibitory activity of Sprouty2.     

IL-6-induced Bcl6 variant 2 supports IL-6-dependent myeloma cell proliferation and survival through STAT3

IL-6 is a growth and survival factor for myeloma cells, although the mechanism by which it induces myeloma cell proliferation through gene expression is largely unknown. Microarray analysis showed that some B-cell lymphoma-associated oncogenes such as Bcl6, which is absent in normal plasma cells, were upregulated by IL-6 in IL-6-dependent myeloma cell lines. We found that Bcl6 variant 2 was upregulated by STAT3. ChIP assay and EMSA showed that STAT3 bound to the upstream region of variant 2 DNA. Expression of p53, a direct target gene of Bcl6, was downregulated in the IL-6-stimulated cells, and this process was impaired by an HDAC inhibitor. Bcl6 was knocked down by introducing small hairpin RNA, resulting in decreased proliferation and increased sensitivity to a DNA damaging agent. Thus, STAT3-inducible Bcl6 variant 2 appears to generate an important IL-6 signal that supports proliferation and survival of IL-6-dependent myeloma cells.     

PAR1-mediated RhoA activation facilitates CCL2-induced chemotaxis in PC-3 cells

Patients with advanced prostate cancer often exhibit increased activation of the coagulation system. The key activator of the coagulation cascade is the serine protease thrombin which is capable of eliciting numerous cellular responses. We previously reported that the thrombin receptor PAR1 is overexpressed in prostate cancer. To investigate further the role of PAR1 in prostate cancer metastasis, we examined the effects of thrombin activation on cell adhesion and motility in PC-3 prostate cancer cells. Activation of PAR1-induced dynamic cytoskeletal reorganization and reduced PC-3 binding to collagen I, collagen IV, and laminin (P < 0.01) but not fibronectin. Expression of the cell surface integrin receptors did not change as assessed by flow cytometry. Immunofluorescence microscopy revealed that PAR1 stimulation caused reorganization of the focal adhesions, suggesting that PAR1 activation in PC-3 cells may be modulating cell adhesion through integrin function but not expression. Furthermore, RhoA was activated upon stimulation with thrombin with subsequent cell contraction, decreased cell adhesion, and induced migration towards monocyte chemoattractant protein 1 (MCP-1; CCL2). Thus, it appears that thrombin stimulation plays a role in prostate cancer metastasis by decreasing cell adhesion to the extracellular matrix and positioning the cell in a "ready state" for migration in response to a chemotactic signal. Further exploration is needed to determine whether PAR1 activation affects other signaling pathways involved in prostate cancer.     

LPS and TNFalpha induce SOCS3 mRNA and inhibit IL-6-induced activation of STAT3 in macrophages

The human N-acetylglucosaminyltransferase I gene was introduced in the genome of Trichoderma reesei strain VTT-D-80133. Expression was studied after induction from the cellobiohydrolase I promoter. Successful in vivo transfer of GlcNAc was demonstrated by analyzing the neutral N-glycans which were synthesized on cellobiohydrolase I. Final proof of the formation of GlcNAcMan5GlcNAc2 was obtained by NMR analysis.