Homology-2 Domain-containing Research Articles

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation

Autophagy is regulated for IFN-gamma-mediated antimicrobial efficacy; however, its molecular effects for IFN-gamma signaling are largely unknown. Here, we show that autophagy facilitates IFN-gamma-activated Jak2-STAT1. IFN-gamma induces autophagy in wild-type but not in autophagy protein 5 (Atg5(-/-))-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-gamma induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5(-/-) or Atg7(-/-) MEFs are, independent of changes in IFN-gamma receptor expression, resistant to IFN-gamma-activated Jak2-STAT1, which suggests that autophagy is important for IFN-gamma signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-gamma-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-gamma-induced activation of STAT1 in Atg5(-/-) MEFs. Our study provides evidence that there is a link between autophagy and both IFN-gamma signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation.

Arecoline induces HA22T/VGH hepatoma cells to undergo anoikis - involvement of STAT3 and RhoA activation

BackgroundOur previous study showed that, in basal cell carcinoma cells, arecoline reduces levels of the tumor cell survival factor interleukin-6 (IL-6), increases levels of tumor suppressor factor p53, and elicits cell cycle arrest, followed by apoptosis. In preliminarily studies, we observed that arecoline induces detachment of the human-derived hepatoma cell line HA22T/VGH from the extracellular matrix. In the present study, we explored the fate of the detached HA22T/VGH cells and investigated the underlying mechanism.MethodsHA22T/VGH cells or primary cultured rat hepatocytes were treated with arecoline, then changes in morphology, viability, apoptosis, and the expression of surface β1-integrin, apoptosis-related proteins, and IL-6 were examined. Furthermore, activation of the signal transducer and activator of transcription 3 (STAT3) pathway and the RhoA/Rock signaling pathway, including p190RhoGAP and Src homology-2 domain-containing phosphatase SHP2, was examined.ResultsA low concentration of arecoline (≤ 100 μg/ml) caused cytoskeletal changes in HA22T/VGH cells, but not hepatocytes, and this was accompanied by decreased β1-integrin expression and followed by apoptosis, indicating that HA22T/VGH cells undergo anoikis after arecoline treatment. IL-6 expression and phosphorylation of STAT3, which provides protection against anoikis, were inhibited and levels of downstream signaling proteins, including Bcl-XL and Bcl-2, were decreased, while Bax expression, mitochondrial cytochrome c release, and caspase-3 activity were increased. In addition, phosphorylation/activation of p190RhoGAP, a RhoA inhibitor, and of its upstream regulator, SHP2, was inhibited by arecoline treatment, while Rho/Rock activation was increased. Addition of the RhoA inhibitor attenuated the effects of arecoline.ConclusionsThis study demonstrated that arecoline induces anoikis of HA22T/VGH cells involving inhibition of STAT3 and increased RhoA/Rock activation and that the STAT3 and RhoA/Rock signaling pathways are connected.

Glycogen Synthase Kinase-3β Facilitates IFN-γ-Induced STAT1 Activation by Regulating Src Homology-2 Domain-Containing Phosphatase 2

Glycogen synthase kinase-3beta (GSK-3beta)-modulated IFN-gamma-induced inflammation has been reported; however, the mechanism that activates GSK-3beta and the effects of activation remain unclear. Inhibiting GSK-3beta decreased IFN-gamma-induced inflammation. IFN-gamma treatment rapidly activated GSK-3beta via neutral sphingomyelinase- and okadaic acid-sensitive phosphatase-regulated dephosphorylation at Ser(9), and proline-rich tyrosine kinase 2 (Pyk2)-regulated phosphorylation at Tyr(216). Pyk2 was activated through phosphatidylcholine-specific phospholipase C (PC-PLC)-, protein kinase C (PKC)-, and Src-regulated pathways. The activation of PC-PLC, Pyk2, and GSK-3beta was potentially regulated by IFN-gamma receptor 2-associated Jak2, but it was independent of IFN-gamma receptor 1. Furthermore, Jak2/PC-PLC/PKC/cytosolic phospholipase A(2) positively regulated neutral sphingomyelinase. Inhibiting GSK-3beta activated Src homology-2 domain-containing phosphatase 2 (SHP2), thereby preventing STAT1 activation in the late stage of IFN-gamma stimulation. All these results showed that activated GSK-3beta synergistically affected IFN-gamma-induced STAT1 activation by inhibiting SHP2.

Ginkgo biloba extract EGb® 761 exerts anti‐angiogenic effects via activation of tyrosine phosphatases

The standardised Ginkgo biloba extract EGb 761 (Dr. Willmar Schwabe Pharmaceuticals, Karlsruhe, Germany) is one of the most widely used herbal remedies. Indications for this extract range from dementia to peripheral vascular disease, based on well-documented vascular effects. Surprisingly, the actions of EGb 761 on angiogenesis as a function of vascular cells have not been investigated to date. The anti-cancer activity of EGb 761 in vitro and epidemiological data showing reduced risk for ovarian cancer in regular users have prompted us to investigate this issue. We show an anti-angiogenic profile of EGb 761 in vitro (inhibited proliferation, migration and tube formation of endothelial cells) and in vivo in the chicken chorio-allantoic membrane (CAM) assay. An analysis of the underlying mechanisms indicates inhibition of growth factor-induced extracellular signal-regulated kinase (ERK) phosphorylation by EGb 761. Inhibitory effects of EGb 761 on ERK as well as of the upstream kinases map-erk-kinase (MEK) and rapidly growing fibrosarcoma (Raf)-1 could be completely reversed by pre-treatment with sodium vanadate (inhibitor of tyrosine phosphatases). Sodium vanadate also reversed the EGb 761-induced inhibition of endothelial cell migration. Focusing on tyrosine phosphatases upstream of the Raf-MEK-ERK cascade, we identified the tyrosine phosphatase Src homology-2 domain-containing phosphatase 1 (SHP-1) as one target of EGb 761. SHP-1 was rapidly activated by EGb 761, and silencing SHP-1 (siRNA) abrogated reduction of endothelial proliferation by EGb 761. In summary, we identify EGb 761 as a potent anti-angiogenic drug. The underlying mechanism is the activation of protein tyrosine phosphatases, leading to inhibition of the Raf-MEK-ERK pathway. These findings provide a rational basis for using EGb 761 for an additional therapeutic indication: anti-angiogenesis-based tumour prevention and adjuvant therapy.

Inhibitors of Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2 (Shp2) Based on Oxindole Scaffolds

Screening of the NCI diversity set of compounds has led to the identification of 5 (NSC-117199), which inhibits the protein tyrosine phosphatase (PTP) Shp2 with an IC50 of 47 microM. A focused library incorporating an isatin scaffold was designed and evaluated for inhibition of Shp2 and Shp1 PTP activities. Several compounds were identified that selectively inhibit Shp2 over Shp1 and PTP1B with low to submicromolar activity. A model for the binding of the active compounds is proposed.

The tyrosine phosphatase Shp2 (PTPN11) in cancer

Diverse cellular processes are regulated by tyrosyl phosphorylation, which is controlled by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). De-regulated tyrosyl phosphorylation, evoked by gain-of-function mutations and/or over-expression of PTKs, contributes to the pathogenesis of many cancers and other human diseases. PTPs, because they oppose the action of PTKs, had been considered to be prime suspects for potential tumor suppressor genes. Surprisingly, few, if any, tumor suppressor PTPs have been identified. However, the Src homology-2 domain-containing phosphatase Shp2 (encoded by PTPN11) is a bona fide proto-oncogene. Germline mutations in PTPN11 cause Noonan and LEOPARD syndromes, whereas somatic PTPN11 mutations occur in several types of hematologic malignancies, most notably juvenile myelomonocytic leukemia and, more rarely, in solid tumors. Shp2 also is an essential component in several other oncogene signaling pathways. Elucidation of the events underlying Shp2-evoked transformation may provide new insights into oncogenic mechanisms and novel targets for anti-cancer therapy.

Integrin-Mediated Cell Adhesion and Spreading Engage Different Sources of Reactive Oxygen Species

The tightly regulated production of intracellular reactive oxygen species (ROS) participates in several biologic processes such as cellular growth, programmed cell death, senescence, and adhesion. It is increasingly evident that the same enzymatic processes that were originally linked to ROS generation during host defence or apoptosis execution are also involved in redox-mediated signal transduction. We investigated in murine NIH3T3 fibroblasts the contribution of a variety of redox-dependent events during signal transduction initiated by integrin engagement due to fibronectin stimulation and report that a mitochondrial ROS release occurs, strictly confined to the early phase of extracellular matrix (ECM) contact (10 min). Besides, 5-lipoxygenase (5-LOX) is engaged by integrin receptor ligation as another ROS source, contributing to the more-intense, second ROS burst (45 min), possibly orchestrating the spreading of cells in response to ECM contact. To define a potential mechanism for ROS signaling, we demonstrate that on integrin recruitment, the Src homology-2 domain-containing phosphatase 2 (SHP-2) undergoes a reversible oxidization/inactivation to which mitochondrial and 5-lipoxygenase ROS contribute differentially. In keeping with a key role of oxidants during integrin signaling, the inactivation of SHP-2 prevents the dephosphorylation and inactivation of SHP-2 substrates (p125FAK and SHPS-1), thus enabling the continued propagation of the signal arising by integrin engagement.