Maya Kozlowski

SHP-1 Binds and Negatively Modulates the c-Kit Receptor by Interaction with Tyrosine 569 in the c-Kit Juxtamembrane Domain

ABSTRACT The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathioneS-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit–SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3–c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation.

SHP-1 negatively regulates neuronal survival by functioning as a TrkA phosphatase

Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.

Tumor Necrosis Factor-α Induces Functionally Active Hyaluronan-adhesive CD44 by Activating Sialidase through p38 Mitogen-activated Protein Kinase in Lipopolysaccharide-stimulated Human Monocytic Cells

Interaction of CD44, an adhesion molecule, with its ligand, hyaluronan (HA), in monocytic cells plays a critical role in cell migration, inflammation, and immune responses. Most cell types express CD44 but do not bind HA. The biological functions of CD44 have been attributed to the generation of the functionally active, HA-adhesive form of this molecule. Although lipopolysaccharide (LPS) and cytokines induce HA-adhesive CD44, the molecular mechanism underlying this process remains unknown. In this study, we show that LPS-induced CD44-mediated HA (CD44-HA) binding in monocytes is regulated by endogenously produced tumor necrosis factor (TNF)-α and IL-10. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation was required for LPS- and TNF-α-induced, but not IL-10-induced, CD44-HA-binding in normal monocytes. To dissect the signaling pathways regulating CD44-HA binding independently of cross-regulatory IL-10-mediated effects, IL-10-refractory promonocytic THP-1 cells were employed. LPS-induced CD44-HA binding in THP-1 cells was regulated by endogenously produced TNF-α. Our results also suggest that lysosomal sialidase activation may be required for the acquisition of the HA-binding form of CD44 in LPS- and TNF-α-stimulated monocytic cells. Studies conducted to understand the role of MAPKs in the induction of sialidase activity revealed that LPS-induced sialidase activity was dependent on p42/44 MAPK-mediated TNF-α production. Blocking TNF-α production by PD98059, a p42/44 inhibitor, significantly reduced the LPS-induced sialidase activity and CD44-HA binding. Subsequently, TNF-α-mediated p38 MAPK activation induced sialidase activity and CD44-HA binding. Taken together, our results suggest that TNF-α-induced p38 MAPK activation may regulate the induction of functionally active HA-binding form of CD44 by activating sialidase in LPS-stimulated human monocytic cells.

IL-6 Production Is Positively Regulated by Two Distinct Src Homology Domain 2-Containing Tyrosine Phosphatase-1 (SHP-1)–Dependent CCAAT/Enhancer-Binding Protein β and NF-κB Pathways and an SHP-1–Independent NF-κB Pathway in Lipopolysaccharide-Stimulated Bone Marrow-Derived Macrophages

Comparison of the inflammatory cytokine profile in bone marrow-derived macrophages (BMDMs) from normal and Src homology domain 2-containing tyrosine phosphatase-1 (SHP-1)–deficient Motheaten (me/me) mice revealed a dramatic suppression of IL-6 transcript and protein in me/me BMDMs after LPS stimulation. Interfering with SHP-1 expression using antisense SHP-1 oligonucleotides led to a significant downregulation of IL-6 in normal BMDMs. Conversely, reconstitution of me/me BMDMs with the SHP-1 gene using adenoviral vectors restored IL-6 production. Expression of only SHP-1 Src homology region 2 domains in normal BMDMs inhibited IL-6 production, confirming that IL-6 regulation depends on SHP-1 phosphatase activity. We further demonstrated that loss of SHP-1 function affects proper phosphorylation of Erk1/2 MAPKs and, to a lesser degree, of NF-κB downstream of TLR4 in BMDMs. Inefficient phosphorylation of Erk1/2 MAPKs abrogated the activation of C/EBPβ transcription factor, which was reversed on restoration of SHP-1 function and led to a concomitant enhancement of IL-6 production. We demonstrate that IL-6 production is regulated by a complex network of signaling pathways that include SHP-1–dependent activation of Erk1/2–C/EBPβ and NF-κB, in addition to SHP-1–independent IκB pathway through the activation of protein tyrosine kinases downstream of TLR4. Taken together, these results revealed for the first time, to our knowledge, a positive and critical role of SHP-1 in IL-6 regulation and dependence of Erk1/2–C/EBPβ pathway in addition to that of IκB on SHP-1 activity required for IL-6 induction after LPS stimulation.

Breast Cancer Cells Proliferation Is Regulated by Tyrosine Phosphatase SHP1 through c-jun N-Terminal Kinase and Cooperative Induction of RFX-1 and AP-4 Transcription Factors

In this study, we show that proliferation of breast cancer cells is suppressed by IGF-1–activated JNK MAPK pathway. The molecular mechanism by which c-jun-NH,-kinase (JNK) activation induces antiproliferative signals in IGF-1–stimulated breast cancer cells remains unknown. Tyrosine phosphatase SHP1 is known to negatively regulate signal transduction pathways activated by cell surface receptors including IGF-1. Moreover, SHP1 transcript and protein levels are increased in epithelial tumors. Therefore, we hypothesized that IGF-activated JNK induces expression of SHP1 in breast cancer cells. To further clarify the role of SHP1 in tumor growth, we correlated the proliferation rates of breast adenocarcinoma cells with SHP1 expression and JNK activation. We show that proliferation of serum- or IGF-1–stimulated breast adenocarcinoma cells is negatively regulated by SHP1 and show for the first time that IGF-1–activated JNK induces SHP1 expression in MCF-7 cells used as experimental model. In an attempt to understand the mechanism by which serum- or IGF-1–activated JNK induces SHP1 expression resulting in suppression of cell proliferation, we reveal for the first time that in serum- or IGF-1–stimulated breast cancer MCF-7 cells, JNK induces SHP1 expression through the binding of AP-4 and RFX-1 transcription factors to the epithelial tissue–specific SHP1 promoter. Overall, we show for the first time that IGF-1–stimulated proliferation of breast adenocarcinoma cells is negatively regulated by SHP1 through activation of JNK. Mol Cancer Res; 9(8); 1112–25. ©2011 AACR.

Polarization of Monocytic Myeloid-Derived Suppressor Cells by Hepatitis B Surface Antigen Is Mediated via ERK/IL-6/STAT3 Signaling Feedback and Restrains the Activation of T Cells in Chronic Hepatitis B Virus Infection.

Chronic hepatitis B virus (HBV) infection is characterized by T cell tolerance to virus. Although inhibition of T cell responses by myeloid-derived suppressor cells (MDSCs) has been observed in patients with chronic hepatitis B (CHB), the mechanism for expansion of MDSCs remains ambiguous. In this study, a significant increased frequency of monocytic MDSCs (mMDSCs) was shown positively correlated to level of HBsAg in the patients with CHB. We further found hepatitis B surface Ag (HBsAg) efficiently promoted differentiation of mMDSCs in vitro, and monocytes in PBMCs performed as the progenitors. This required the activation of ERK/IL-6/STAT3 signaling feedback. Importantly, the mMDSCs polarized by HBsAg in vitro acquired the ability to suppress T cell activation. Additionally, treatment of all-trans retinoic acid, an MDSC-targeted drug, restored the proliferation and IFN-γ production by HBV-specific CD4+ and CD8+ T cells in PBMCs from patients with CHB and prevented increase of viral load in mouse model. In summary, HBsAg maintains HBV persistence and suppresses T cell responses by promoting differentiation of monocytes into mMDSCs. A therapy aimed at the abrogation of MDSCs may help to disrupt immune suppression in patients with CHB.

IFN-γ-induced IL-27 and IL-27p28 expression are differentially regulated through JNK MAPK and PI3K pathways independent of Jak/STAT in human monocytic cells.

IL-27, a member of the IL-12 cytokine family, is a key immunoregulatory cytokine produced predominantly by monocytic cells and mediates innate and adaptive immune responses. IL-27 has been shown to be produced by human monocytic cells primed with IFN-γ and in response to a second stimulus such as LPS. In this study, we show for the first time that IFN-γ alone without any second stimulus can induce IL-27p28 gene expression and IL-27 protein production by human monocytic cells. The signaling pathways that govern IL-27 production in general, and particularly following stimulation of monocytic cells with IFN-γ are not known. We investigated the signaling pathways governing the regulation of IL-27 protein and its subunit IL-27p28 following stimulation with IFN-γ in primary human monocytic cells. Our results suggest that IFN-γ-mediated IL-27 protein but not IL-27p28 gene expression is positively regulated by the C-Jun N-terminal kinases (JNK), mitogen-activated protein kinases (MAPKs) and the phosphoinositide 3-kinase (PI3K), independent of the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) signaling in primary human monocytes.

SHP-1–Pyk2–Src Protein Complex and p38 MAPK Pathways Independently Regulate IL-10 Production in Lipopolysaccharide-Stimulated Macrophages

The role of tyrosine phosphatase Src homology region 2 domain-containing phosphatase (SHP)-1 in LPS-activated cytokine production and inflammation was investigated by determining TNF-α and IL-10 production in splenic macrophages employing SHP-1–null (me/me) mouse model. LPS-stimulated me/me splenic macrophages secreted significantly less IL-10 with concomitantly elevated levels of TNF-α compared with wild-type (WT) macrophages irrespective of LPS dose and duration of stimulation. IL-10 significantly inhibited LPS-induced TNF-α production in both me/me and WT macrophages. The critical requirement for SHP-1 in regulating LPS-induced IL-10 and TNF-α production was confirmed by interfering with SHP-1 expression in WT macrophages and by reconstituting me/me macrophages with the SHP-1 gene. To delineate the role of SHP-1 in positive regulation of LPS-induced IL-10 production, signaling proteins representing SHP-1 targets were examined. The results reveal that tyrosine kinases Src and proline-rich tyrosine kinase 2 (Pyk2) regulate SHP-1–dependent LPS-induced IL-10 production and infer that optimal LPS-induced IL-10 production requires an assembly of a protein complex consisting of SHP-1–Pyk2–Src proteins. Moreover, LPS-induced IL-10 production also requires activation of the p38 MAPK independent of SHP-1 function. Overall, to our knowledge our results show for the first time that SHP-1 acts as a positive regulator of LPS-induced IL-10 production in splenic macrophages through two distinct and independent SHP-1–Pyk2–Src and p38 MAPK pathways.

Innate detection of hepatitis B and C virus and viral inhibition of the response

Viral hepatitis caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) infections poses a significant burden to the public health system. Although HBV and HCV differ in structure and life cycles, they share unique characteristics, such as tropism to infect hepatocytes and association with hepatic and extrahepatic disorders that are of innate immunity nature. In response to HBV and HCV infection, the liver innate immune cells eradicate pathogens by recognizing specific molecules expressed by pathogens via distinct cellular pattern recognition receptors whose triggering activates intracellular signalling pathways inducing cytokines, interferons and anti‐viral response genes that collectively function to clear infections. However, HBV and HCV evolve strategies to inactivate innate signalling factors and as such establish persistent infections without being recognized by the innate immunity. We review recent insights into how HBV and HCV are sensed and how they evade innate immunity to establish chronicity. Understanding the mechanisms of viral hepatitis is mandatory to develop effective and safe therapies for eradication of viral hepatitis.

Oral administered particulate yeast-derived glucan promotes hepatitis B virus clearance in a hydrodynamic injection mouse model.

Hepatitis B virus (HBV) persistent infection is associated with ineffective immune response for the clearance of virus. Immunomodulators represent an important class of therapeutics, which potentially could be beneficial for the treatment of HBV infection. The particulate yeast-derived glucan (PYDG) has been shown to enhance the innate and adaptive immune responses. We therefore, assessed the efficacy of PYDG in enhancing HBV specific immune responses by employing the hydrodynamic injection-based (HDI) HBV transfection mouse model. Mice were intragatric administered PYDG daily for 9 weeks post pAAV/HBV1.2 hydrodynamic injection. PYDG treatment significantly promoted HBV DNA clearance and production of HBsAb compared to control mice. PYDG treatment resulted in recruitment of macrophages, dendritic cells (DCs) and effector T cells to the liver microenvironment, accompanied by a significantly augmented DCs maturation and HBV-specific IFN-γ and TNF-α production by T cell. In addition, enhanced production of Th1 cytokines in liver tissue interstitial fluid (TIF) was associated with PYDG administration. Live imaging showed the accumulation of PYDG in the mouse liver. Our results demonstrate that PYDG treatment significantly enhances HBV-specific Th1 immune responses, accompanied by clearance of HBV DNA, and therefore holds promise for further development of therapeutics against chronic hepatitis B.