Pyk2 is required for neutrophil degranulation and host defense responses to bacterial infection

The appropriate regulation of neutrophil activation is critical for maintaining host defense and limiting inflammation. Polymorphonuclear neutrophils (PMNs) express a number of cytoplasmic tyrosine kinases that regulate signaling pathways leading to activation. One of the most highly expressed, but least studied, kinases in PMNs is proline rich kinase 2 (Pyk2). By analogy to the related focal adhesion kinase, Pyk2 has been implicated in regulating PMN adhesion and migration; however, its physiologic function has yet to be described. Using pyk2(-/-) mice, we found that this kinase was required for integrin-mediated degranulation responses, but was not involved in adhesion-induced cell spreading or activation of superoxide production. Pyk2-deficient PMNs also manifested reduced migration on fibrinogen-coated surfaces. The absence of Pyk2 resulted in a severe reduction in paxillin and Vav phosphorylation following integrin ligation, which likely accounts for the poor degranulation and cell migration. Pyk2(-/-) mice were unable to efficiently clear infection with Staphylococcus aureus in a skin abscess model, owing in part to the poor release of granule contents at the site of infection. However, Pyk2-deficient PMNs responded normally to soluble agonists, demonstrating that this kinase functions mainly in the integrin pathway. These data demonstrate the unrealized physiologic role of this kinase in regulating the adhesion-mediated release of PMN granule contents.     

The juxtamembrane region of the epidermal growth factor receptor is required for phosphorylation of Galpha(s)

We have previously demonstrated that Galpha(s) associates with the juxtamembrane region of the epidermal growth factor (EGF) receptor (EGFR) and that the EGFR can phosphorylate and activate this G protein (H. Poppleton et al., 1996, J. Biol. Chem. 271, 6947-6951; H. Sun et al., 1995, Proc. Natl. Acad. Sci. USA 92, 2229-2233). In this report, we have employed peptides EGFR-13 and EGFR-14 (corresponding to amino acids 645-657 and 679-692 in the EGFR, respectively) which disrupt the association of Galpha(s) with the EGFR to investigate whether or not this region of the EGFR is required for phosphorylation of the G protein. EGFR-13 increased the tyrosine phosphorylation of G(alpha)s by two-fold whereas EGFR-14 decreased the phosphorylation of the G protein. Phosphorylation of EGFR-13 on the threonine residue corresponding to Thr654 of the EGFR obliterated the ability of the peptide to increase Galpha(s) phosphorylation. EGFR-13 and EGFR-14, but not phospho-EGFR-13, competed for the association of the EGFR with Galpha(s). A peptide betaIII-2 corresponding to amino acids Arg259-Lys273 in the beta2-adrenergic receptor which competes for association of Galpha(s) with the EGFR and increases protein tyrosine kinase activity of the EGFR could mimic the effects of EGFR-13. Among the three peptides (EGFR-13, EGFR-14, and betaIII-2) that interfere with association of Galpha(s) to the EGFR, only EGFR-13 and betaIII-2 have been shown to activate the G protein. Polylysine which increases EGFR tyrosine kinase activity but does not interfere with association of Galpha(s) and EGFR also augmented phosphorylation of Galpha(s) by the EGFR. Phosphopeptide mapping demonstrated that EGFR-13 and polylysine increased phosphorylation of Galpha(s) by the EGFR on the same additional sites. Collectively, these data suggest that the interaction of Galpha(s) with residues 645-657 of the EGFR, or a peptide corresponding to this sequence alters the conformation of the G protein and/or the EGFR such that Galpha(s) is readily phosphorylated by the EGFR. The peptide EGFR-14, which does not activate Galpha(s), does not allow for the efficient phosphorylation of the G protein even though it does elevate the intrinsic tyrosine kinase activity of the EGFR. The hyperphosphorylation of Galpha(s) by EGFR is likely to require the contact of the G protein with EGFR-13 region (aa 645-657 in the EGFR) as well as augmentation of EGFR kinase activity.     

Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing

Keratinocyte proliferation and migration are essential to cutaneous wound healing and are, in part, mediated in an autocrine fashion by epidermal growth factor receptor (EGFR)-ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR. OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and caused suppression of keratinocyte migration in vitro. Soluble EGFR-immunoglobulin G-Fcgamma fusion protein, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.     

The ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.

To examine the role of the ligand binding domain of epidermal growth factor receptor in its dimerization, we studied the dimerization of a truncated form of the receptor that resembles v-erbB in that it lacks a ligand binding domain. Receptor dimerization was determined by sedimentation analysis on sucrose density gradients at different concentrations of Triton X-100. At high concentrations of Triton X-100 (0.2%), the truncated receptor occurred as a monomer and displayed low basal autophosphorylation. By contrast, at low concentrations of Triton X-100 (0.01%), it existed as a dimer and exhibited high basal autophosphorylation. The ability of the truncated receptor to dimerize indicates that the ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.     

TRPM7 is required for ovarian cancer cell growth,migration and invasion

Our previous study demonstrated that the melastatin-related transient receptor potential channel 7 (TRPM7) was highly expressed in ovarian carcinomas and its overexpression was significantly associated with poor prognosis in ovarian cancer patients. However, the function of TRPM7 in ovarian cancer is mostly unknown. In this study, we examined the roles of TRPM7 in ovarian cancer cell proliferation, migration and invasion. We found that short hairpin RNA interference-mediated silence of TRPM7 significantly inhibited cell proliferation, colony formation, migration and invasion in multiple ovarian cancer cell lines. Mechanistic investigation revealed that silence of TRPM7 decreased phosphorylation levels of Akt, Src and p38 and increased filamentous actin and focal adhesion number in ovarian cancer cells. Thus, our results suggest that TRPM7 is required for proliferation, migration and invasion of ovarian cancer cells through regulating multiple signaling transduction pathways and the formation of focal adhesions.     

Human EMR2, a novel EGF-TM7 molecule on chromosome 19p13.1, is closely related to CD97

The epidermal growth factor (EGF)-TM7 proteins [EMR1, (EGF-like molecule containing mucin-like hormone receptor 1) F4/80, and CD97] constitute a recently defined class B GPCR subfamily and are predominantly expressed on leukocytes. These molecules possess N-terminal EGF-like domains coupled to a seven-span transmembrane (7TM) moiety via a mucin-like spacer domain. Genomic mapping analysis has suggested a possible EGF-TM7 gene family on the human chromosome 19p13 region. In this study, a new member of the EGF-TM7 family, EMR2, which shares strikingly similar molecular characteristics with CD97, is described. In addition to mapping closely to CD97 on human chromosome 19p13.1, EMR2 contains a total of five tandem EGF-like domains and expresses similar protein isoforms consisting of various numbers of EGF-like domains as a result of alternative RNA splicing. Furthermore, EMR2 and CD97 exhibit highly homologous EGF-like domains and share identical gene organization, indicating that both genes are the products of a recent gene duplication event. The homologous EGF-like domains enable the identification of both EMR2 and CD97 by monoclonal antibodies (mAbs) raised against the first EGF-like domain of CD97, whereas mAbs directed against the extracellular spacer domain of CD97 are able to differentiate these two proteins. Both EMR2 and CD97 are highly expressed in immune tissues; however, unlike CD97, which is ubiquitously expressed in most cell types, EMR2 expression is restricted to monocytes/Mφ and granulocytes. EMR2 fails to interact with CD55, the cellular ligand for CD97, suggesting the possibility of a different cellular ligand(s). EMR2 may therefore have a unique function in cells of monocyte/Mφ and granulocyte lineages.     

The G protein G alpha(13) is required for growth factor-induced cell migration

Heterotrimeric G proteins are critical cellular signal transducers. They are known to directly relay signals from seven-transmembrane G protein-coupled receptors (GPCRs) to downstream effectors. On the other hand, receptor tyrosine kinases (RTKs), a different family of membrane receptors, signal through docking sites in their carboxy-terminal tails created by autophosphorylated tyrosine residues. Here we show that a heterotrimeric G protein, G alpha(13), is essential for RTK-induced migration of mouse fibroblast and endothelial cells. G alpha(13) activity in cell migration is retained in a C-terminal mutant that is defective in GPCR coupling, suggesting that the migration function is independent of GPCR signaling. Thus, G alpha(13) appears to be a critical signal transducer for RTKs as well as GPCRs. This broader role of G alpha(13) in cell migration initiated by two types of receptors could provide a molecular basis for the vascular system defects exhibited by G alpha(13) knockout mice.     

Targeting of AMSH to endosomes is required for epidermal growth factor receptor degradation

To reach the lysosomes, down-regulated receptors such as the epidermal growth factor receptor must first be sorted into internal vesicles of late endosomes (multivesicular bodies), a ubiquitin-dependent event that requires the coordinated function of the endosome sorting complex required for transport (ESCRT) proteins. Here we report that CHMP3, an ESCRT-III complex component, and associated molecule of SH3 domain of STAM (AMSH), a deubiquitinating enzyme, interact with each other in cells. A dominant-negative version of CHMP3, which specifically prevents targeting of AMSH to endosomes, inhibits degradation but not internalization of EGFR, suggesting that endosomal AMSH is a functional component of the multivesicular body pathway.     

Human epidermal growth factor (EGF) module-containing mucin-like hormone receptor 3 is a new member of the EGF-TM7 family that recognizes a ligand on human macrophages and activated neutrophils

The epidermal growth factor (EGF)-TM7 subgroup of G-protein-coupled receptors is composed predominantly of leukocyte-restricted glycoproteins defined by their unique hybrid structure, in which extracellular EGF-like domains are coupled to a seven-span transmembrane moiety via a mucin-like stalk. The EGF-TM7 group comprises mouse F4/80, human EGF module-containing mucin-like hormone receptor (EMR) 1, human EMR2, and human and mouse CD97, the genes for which map to human chromosome 19p13 and the syntenic regions of the mouse genome. In this study we describe the cloning and characterization of EMR3, a novel human EGF-TM7 molecule, and show the existence of its cellular ligand. The EMR3 gene maps closely to the existing members of the EGF-TM7 family on human chromosome 19p13.1 and, in common with other EGF-TM7 genes, is capable of generating different protein isoforms through alternative splicing. Two alternative splice forms have been isolated: one encoding a 652-amino acid cell surface protein consisting of two EGF-like domains, a mucin stalk, and a putative G-protein-coupled receptor domain and the other encoding a truncated soluble form containing only two EGF-like domains. As with other members of the EGF-TM7 family, EMR3 mRNA displays a predominantly leukocyte-restricted expression pattern, with highest levels in neutrophils, monocytes, and macrophages. Through the use of soluble EMR3 multivalent probes we have shown the presence of a ligand at the surface of monocyte-derived macrophages and activated human neutrophils. These interactions suggest a potential role for EMR3 in myeloid-myeloid interactions during immune and inflammatory responses.     

Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.     

CD47 mediates post-adhesive events required for neutrophil migration across polarized intestinal epithelia

Transepithelial migration of neutrophils (PMN) is a defining characteristic of active inflammatory states of mucosal surfaces. The process of PMN transepithelial migration, while dependent on the neutrophil beta 2 integrin CD11b/CD18, remains poorly understood. In these studies, we define a monoclonal antibody, C5/D5, raised against epithelial membrane preparations, which markedly inhibits PMN migration across polarized monolayers of the human intestinal epithelial cell line T84 in a bidirectional fashion. In T84 cells, the antigen defined by C5/D5 is upregulated by epithelial exposure to IFN-gamma, and represents a membrane glycoprotein of approximately 60 kD that is expressed on the basolateral membrane. While transepithelial migration of PMN was markedly inhibited by either C5/D5 IgG or C5/D5 Fab fragments, the antibody failed to inhibit both adhesion of PMN to T84 monolayers and adhesion of isolated T84 cells to the purified PMN integrin, CD11b/CD18. Thus, epithelial-PMN interactions blocked by C5/D5 appear to be downstream from initial CD11b/CD18-mediated adhesion of PMN to epithelial cells. Purification, microsequence analysis, and cross-blotting experiments indicate that the C5/D5 antigen represents CD47, a previously cloned integral membrane glycoprotein with homology to the immunoglobulin superfamily. Expression of the CD47 epitope was confirmed on PMN and was also localized to the basolateral membrane of normal human colonic epithelial cells. While C5/D5 IgG inhibited PMN migration even in the absence of epithelial, preincubation of T84 monolayers with C5/D5 IgG followed by antibody washout also resulted in inhibition of transmigration. These results suggest the presence of both neutrophil and epithelial components to CD47-mediated transepithelial migration. Thus, CD47 represents a potential new therapeutic target for downregulating active inflammatory disease of mucosal surfaces.     

T cell-derived interferon-γ is required for host defense to Toxoplasma gondii

Interferon-γ (IFN-γ) is important for host defense against various intracellular organisms including a protozoan pathogen Toxoplasma gondii. Various immune cells are recently shown to produce IFN-γ in T. gondii infection, however, it remains elusive which cell types are important for anti-T. gondii host defense so far. Here we generate a new IFN-γ reporter "GREVEN" mouse line in which a fusion protein of Venus and NanoLuc to analyze IFN-γ producing cells during T. gondii infection and find that CD4⁺, CD8⁺, γδ T cells and natural killer cells express Venus in a time dependent manner. Furthermore, Lck-Cre/Ifng^fl/fl mice are highly susceptible to T. gondii infection. Taken together, our results demonstrate that T cell-derived IFN-γ plays an important role in anti-T. gondii host defense.     

Dectin-1 is not required for the host defense to Cryptococcus neoformans

Dectin-1 is known as a sole receptor for beta-glucan, a major cell wall component of fungal microorganisms. In the current study, we examined the role of this molecule in the host defense to Cryptococcus neoformans, an opportunistic fungal pathogen in AIDS patients. There was no significant difference in the clinical course and cytokine production between dectin-1 gene-deficient and control mice. These results indicate that dectin-1 is not likely essential for the development of host protective responses to C. neoformans.     

Toll-like receptor 2 is required for innate, but not acquired, host defense to Borrelia burgdorferi.

Borrelia burgdorferi lipoproteins activate inflammatory cells through Toll-like receptor 2 (TLR2), suggesting that TLR2 could play a pivotal role in the host response to B. burgdorferi. TLR2 does play a critical role in host defense, as infected TLR2(-/-) mice harbored up to 100-fold more spirochetes in tissues than did TLR2(+/+) littermates. Spirochetes persisted at extremely elevated levels in TLR2-deficient mice for at least 8 wk following infection. Infected TLR2(-/-) mice developed normal Borrelia-specific Ab responses, as measured by quantity of Borrelia-specific Ig isotypes, the kinetics of class switching to IgG, and the complexity of the Ags recognized. These findings indicate that the failure to control spirochete levels in tissues is not due to an impaired acquired immune response. While macrophages from TLR2(-/-) mice were not responsive to lipoproteins, they did respond to nonlipoprotein components of sonicated spirochetes. These TLR2-independent responses could play a role during the inflammatory response to B. burgdorferi, as infected TLR2(-/-) mice developed greater ankle swelling than wild-type littermates. Thus, while TLR2-dependent signaling pathways play a major role in the innate host defense to B. burgdorferi, both inflammatory responses and the development of the acquired humoral response can occur in the absence of TLR2.     

The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy

AMP-activated protein kinase (AMPK) is a crucial energy sensor and plays a key role in integration of cellular functions to maintain homeostasis. Despite this, it is largely unknown whether targeting the AMPK pathway can be used as a therapeutic strategy for infectious diseases. Herein, we show that AMPK activation robustly induces antibacterial autophagy, which contributes to antimicrobial defense against Mycobacterium tuberculosis (Mtb). AMPK activation led to inhibition of Mtb-induced phosphorylation of the mechanistic target of rapamycin (MTOR) in macrophages. In addition, AMPK activation increased the genes involved in oxidative phosphorylation, mitochondrial ATP production, and biogenesis in Mtb-infected macrophages. Notably, peroxisome proliferator-activated receptor-gamma, coactivator 1α (PPARGC1A) was required for AMPK-mediated antimicrobial activity, as well as enhancement of mitochondrial function and biogenesis, in macrophages. Further, the AMPK-PPARGC1A pathway was involved in the upregulation of multiple autophagy-related genes via CCAAT/enhancer binding protein (C/EBP), β (CEBPB). PPARGC1A knockdown inhibited the AMPK-mediated induction of autophagy and impaired the fusion of phagosomes with MAP1LC3B (LC3B) autophagosomes in Mtb-infected macrophages. The link between autophagy, mitochondrial function, and antimicrobial activity was further demonstrated by studying LysMCre-mediated knockout of atg7, demonstrating mitochondrial ultrastructural defects and dysfunction, as well as blockade of antimicrobial activity against mycobacteria. Collectively, our results identify the AMPK-PPARGC1A axis as contributing to autophagy activation leading to an antimicrobial response, as a novel host defense mechanism.     

The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.     

Insulin-like growth factor receptor 1b is required for zebrafish primordial germ cell migration and survival

Insulin-like growth factor (IGF) signaling is a critical regulator of somatic growth during fetal and adult development, primarily through its stimulatory effects on cell proliferation and survival. IGF signaling is also required for development of the reproductive system, although its precise role in this regard remains unclear. We have hypothesized that IGF signaling is required for embryonic germline development, which requires the specification and proliferation of primordial germ cells (PGCs) in an extragonadal location, followed by directed migration to the genital ridges. We tested this hypothesis using loss-of-function studies in the zebrafish embryo, which possesses two functional copies of the Type-1 IGF receptor gene (igf1ra, igf1rb). Knockdown of IGF1Rb by morpholino oligonucleotides (MO) results in mismigration and elimination of primordial germ cells (PGCs), resulting in fewer PGCs colonizing the genital ridges. In contrast, knockdown of IGF1Ra has no effect on PGC migration or number despite inducing widespread somatic cell apoptosis. Ablation of both receptors, using combined MO injections or overexpression of a dominant-negative IGF1R, yields embryos with a PGC-deficient phenotype similar to IGF1Rb knockdown. TUNEL analyses revealed that mismigrated PGCs in IGF1Rb-deficient embryos are eliminated by apoptosis; overexpression of an antiapoptotic gene (Bcl2l) rescues ectopic PGCs from apoptosis but fails to rescue migration defects. Lastly, we show that suppression of IGF signaling leads to quantitative changes in the expression of genes encoding CXCL-family chemokine ligands and receptors involved in PGC migration. Collectively, these data suggest a novel role for IGF signaling in early germline development, potentially via cross-talk with chemokine signaling pathways.