Granulocyte chemotaxis and disease expression are differentially regulated by GRK subtype in an acute inflammatory arthritis model (K/BxN)

Duke University, Department of Medicine, Division of Cardiology, Durham, NC 27110, USA
Clinical Immunology (Impact Factor: 3.67). 10/2008; 129(1):115-122. DOI: 10.1016/j.clim.2008.06.008


Chemokine receptors are G-protein coupled receptors (GPCRs) phosphorylated by G-protein receptor kinases (GRKs) after ligand-mediated activation. We hypothesized that GRK subtypes differentially regulate granulocyte chemotaxis and clinical disease expression in the K/BxN model.Methods
Clinical, histologic, and cytokine responses in GRK6−/−, GRK5−/−, GRK2+/−, and wildtype mice were evaluated using K/BxN serum transfer. Granulocyte chemotaxis was analyzed by transendothelial migration assays.ResultsBoth GRK6−/− and GRK2+/− mice had increased arthritis disease severity (p < 0.001); whereas GRK5−/− was not different from controls. Acute weight loss was enhanced in GRK6−/− and GRK2+/− mice (p < 0.001, days 3–10). However, GRK6−/− mice uniquely had more weight loss (> 10%), elevated serum IL-6, and enhanced migration toward LTB4 and C5a in vitro.ConclusionsGRK6 and -2, but not GRK5, are involved in the pathogenesis of acute arthritis in the K/BxN model. In particular, GRK6 may dampen inflammatory responses by regulating granulocyte trafficking toward chemoattractants.

Download full-text


Available from: Teresa K Tarrant, Sep 22, 2014
  • Source
    • "GRK6 typically has a negative regulatory role in CXCR4 activation and CXCL12-induced cell migration [24,26]. For example, GRK6 deficiency is associated with impaired desensitization and enhanced CXCR4-mediated neutrophil migration and has been implicated in the pro-inflammatory response seen in rheumatoid arthritis [26,36]. However, in HeLa cells, siRNA-based functional screening identified GRK6 as a critical positive regulator of integrin-mediated cell adhesion and migration [37]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Metastasis in medulloblastoma (MB) is associated with poor survival. Recent genetic studies revealed MB to comprise distinct molecular subgroups, including the sonic hedgehog (SHH) subgroup that exhibits a relatively high rate of progression. To identify targeted therapeutics against metastasis, a better understanding of the regulation of MB cell migration is needed. G protein-coupled receptor kinases (GRKs) have been implicated in cancer metastasis through their regulation of G-protein coupled receptors (GPCRs) involved in growth factor (GF)-mediated cell migration. However, the specific roles and regulation of GRKs in MB have not been investigated. Microarray mRNA analysis was performed for GRKs, GPCRs, and GFs in 29 human MB, and real time RT-PCR was used to detect GRK6 expression in MB cells. Lenti- or retro-virus infection, and siRNA or shRNA transfection, of MB cells was used to overexpress and knockdown target genes, respectively. Western blot was used to confirm altered expression of proteins. The effect of altered target protein on cell migration was determined by Boyden chamber assay and xCELLigence migration assays. We observed co-overexpression of PDGFRA, CXCR4, and CXCL12 in the SHH MB subtype compared to non-SHH MB (5, 7, and 5-fold higher, respectively). GRK6, which typically acts as a negative regulator of CXCR4 signaling, is downregulated in MB, relative to other GRKs, while the percentage of GRK6 expression is lower in MB tumors with metastasis (22%), compared to those without metastasis (43%). In SHH-responsive MB cells, functional blockade of PDGFR abolished CXCR4-mediated signaling. shPDGFR transfected MB cells demonstrated increased GRK6 expression, while PDGF or 10% FBS treatment of native MB cells reduced the stability of GRK6 by inducing its proteosomal degradation. Overexpression or downregulation of Src, a key mediator of GF receptor/PDGFR signaling, similarly inhibited or induced GRK6 expression, respectively. siRNA downregulation of GRK6 enhanced CXCR4 signaling and promoted MB migration, while lentiviral-GRK6 overexpression suppressed CXCR4 signaling, potentiated the effect of AMD3100, a CXCR4 antagonist, and impaired migration. Our findings demonstrate a novel mechanism of GF receptor/PDGFR-Src-mediated dysregulation of CXCR4 signaling that promotes MB cell migration, which could potentially be exploited for therapeutic targeting in SHH MB.
    Molecular Cancer 03/2013; 12(1):18. DOI:10.1186/1476-4598-12-18 · 4.26 Impact Factor
  • Source
    • "Chemotaxis experiments were performed as described (Tarrant et al., 2008) on Ly6C-enriched splenocytes. Briefly, mouse spleens were manually disrupted and filtered through 70 ␮m mesh, RBCs lysed, and enriched for Ly6C+ cells by magnetic separation using anti-Ly6C-biotin beads (Miltenyi Biotec, MA). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Polymorphism at the GPSM3 gene locus is inversely associated with four systemic autoimmune diseases, including rheumatoid arthritis and ankylosing spondylitis. G-protein signaling modulator-3 (GPSM3) expression is most pronounced in myeloid cells, in which it targets heterotrimeric G-protein Gαi subunits of chemokine receptors, critical to immune function. To begin to explore the regulatory role of GPSM3 in monocytes, human THP-1 and primary mouse myeloid cells were cultured under stimulus conditions; GPSM3 was found by immunoblotting to be expressed at highest levels in the mature monocyte. To evaluate the effects of GPSM3 deficiency on a myeloid-dependent autoimmune disease, collagen antibody-induced arthritis (CAIA) was induced in Gpsm3-/- and control mice, which were then analyzed for clinical score, paw swelling, intra-articular proinflammatory markers, and histopathology. Mice lacking GPSM3 were protected from CAIA, and expression of monocyte-representative pro-inflammatory chemokine receptors and cytokines in paws of Gpsm3-/- mice were decreased. Flow cytometry, apoptosis, and transwell chemotaxis experiments were conducted to further characterize the effect of GPSM3 deficiency on survival and chemokine responsiveness of monocytes. GPSM3-deficient myeloid cells had reduced migration ex vivo to CCL2, CX3CL1, and chemerin and enhanced apoptosis in vitro. Our results suggest that GPSM3 is an important regulator of monocyte function involving mechanisms of differentiation, survival, and chemotaxis, and deficiency in GPSM3 expression is protective in acute inflammatory arthritis.
    Molecular Immunology 12/2012; 54(2):193-198. DOI:10.1016/j.molimm.2012.12.001 · 2.97 Impact Factor
  • Source
    • "It has been well documented that the expression levels of GRK2 are altered in specific cell types in human diseases including active relapsing-remitting multiple sclerosis (MS) or with secondary progressive MS (Vroon et al., 2005), Alzheimer's disease (Leosco et al., 2007), and rheumatoid arthiritis (Lombardi et al., 1999). Related to these changes in GRK2 levels in humans, rodent models with decreased GRK2 levels exhibit altered disease pathogenesis ((Tarrant et al., 2008) (Vroon et al., 2005) (Nijboer et al., 2008). In addition to the human diseases outlined above, GRK2 levels are increased in immune cells from patients with sepsis. "
    [Show abstract] [Hide abstract]
    ABSTRACT: G-protein-coupled receptor kinase 2 (GRK2) is a member of a kinase family originally discovered for its role in the phosphorylation and desensitization of G-protein-coupled receptors. It is expressed in high levels in myeloid cells and its levels are altered in many inflammatory disorders including sepsis. To address the physiological role of myeloid cell-specific GRK2 in inflammation, we generated mice bearing GRK2 deletion in myeloid cells (GRK2▵mye). GRK2▵mye mice exhibited exaggerated inflammatory cytokine/chemokine production, and organ injury in response to lipopolysaccharide (LPS, a TLR4 ligand) when compared to wild-type littermates (GRK2fl/fl). Consistent with this, peritoneal macrophages from GRK2▵mye mice showed enhanced inflammatory cytokine levels when stimulated with LPS. Our results further identify TLR4-induced NF-κB1p105-ERK pathway to be selectively regulated by GRK2. LPS-induced activation of NF-κB1p105-MEK-ERK pathway is significantly enhanced in the GRK2▵mye macrophages compared to GRK2fl/fl cells and importantly, inhibition of the p105 and ERK pathways in the GRK2▵mye macrophages, limits the enhanced production of LPS-induced cytokines/chemokines. Taken together, our studies reveal previously undescribed negative regulatory role for GRK2 in TLR4-induced p105-ERK pathway as well as in the consequent inflammatory cytokine/chemokine production and endotoxemia in mice.
    Journal of Cellular Physiology 03/2011; 226(3):627-37. DOI:10.1002/jcp.22384 · 3.84 Impact Factor
Show more