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Aggregation-independent modulation of proteoglycan binding by neutralization of C-terminal acidic residues in the chemokine macrophage inflammatory protein 1alpha.

ABSTRACT: Members of the chemokine family of proteins mediate their biological effects through interaction with a family of seven-transmembrane G-protein-coupled receptors. This interaction is complicated by the biochemical properties of chemokines, such as their ability to form self aggregates and their ability to bind to proteoglycans. With some chemokines there is a clear interrelationship between these interactions; the chemokine platelet factor 4 binds preferentially to proteoglycans in its aggregated form. Little is known about the role of aggregation in the proteoglycan binding of other chemokines. Here we demonstrate that the aggregation status of the chemokine macrophage inflammatory protein 1alpha (MIP-1alpha) has no detectable effect on its affinity for proteoglycans. Furthermore, we demonstrate that the alteration of acidic amino acid residues in MIP-1alpha influences the affinity of its interactions with heparin as these residues are progressively neutralized, leading to an enhanced binding affinity for heparin. Thus, with MIP-1alpha, aggregation is not a determinant of proteoglycan binding; however, overall charge does seem to have a major role in the interaction. These results therefore add to our understanding of the nature of the interaction between MIP-1alpha and proteoglycans and suggests that the basic amino acids might not be the sole regulators of proteoglycan binding.

SUBMITTER: Ottersbach K

PROVIDER: S-EPMC1221674 | biostudies-other | 2001 Mar

REPOSITORIES: biostudies-other

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Project description:BackgroundSynovial macrophages perform a multitude of functions that include clearance of cell debris and foreign bodies, tissue immune surveillance, and resolution of inflammation. The functional diversity of macrophages is enabled by distinct subpopulations that express unique surface markers. Proteoglycan-4 (PRG4) is an important regulator of synovial hyperplasia and fibrotic remodeling, and the involvement of macrophages in PRG4's synovial role is yet to be defined. Our objectives were to study the PRG4's importance to macrophage homeostatic regulation in the synovium and infiltration of pro-inflammatory macrophages in acute synovitis and investigate whether macrophages mediated synovial fibrosis in Prg4 gene-trap (Prg4GT/GT) murine knee joints.MethodsMacrophage phenotyping in Prg4GT/GT and Prg4+/+ joints was performed by flow cytometry using pan-macrophage markers, e.g., CD11b, F4/80, and surface markers of M1 macrophages (CD86) and M2 macrophages (CD206). Characterizations of the various macrophage subpopulations were performed in 2- and 6-month-old animals. The expression of inflammatory markers, IL-6, and iNOS in macrophages that are CD86+ and/or CD206+ was studied. The impact of Prg4 recombination on synovial macrophage populations of 2- and 6-month-old animals and infiltration of pro-inflammatory macrophages in response to a TLR2 agonist challenge was determined. Macrophages were depleted using liposomal clodronate and synovial membrane thickness, and the expression of fibrotic markers α-SMA, PLOD2, and collagen type I (COL-I) was assessed using immunohistochemistry.ResultsTotal macrophages in Prg4GT/GT joints were higher than Prg4+/+ joints (p<0.0001) at 2 and 6 months, and the percentages of CD86+/CD206- and CD86+/CD206+ macrophages increased in Prg4GT/GT joints at 6 months (p<0.0001), whereas the percentage of CD86-/CD206+ macrophages decreased (p<0.001). CD86+/CD206- and CD86+/CD206+ macrophages expressed iNOS and IL-6 compared to CD86-/CD206+ macrophages (p<0.0001). Prg4 re-expression limited the accumulation of CD86+ macrophages (p<0.05) and increased CD86-/CD206+ macrophages (p<0.001) at 6 months. Prg4 recombination attenuated synovial recruitment of pro-inflammatory macrophages in 2-month-old animals (p<0.001). Clodronate-mediated macrophage depletion reduced synovial hyperplasia, α-SMA, PLOD2, and COL-I expressions in the synovium (p<0.0001).ConclusionsPRG4 regulates the accumulation and homeostatic balance of macrophages in the synovium. In its absence, the synovium becomes populated with M1 macrophages. Furthermore, macrophages exert an effector role in synovial fibrosis in Prg4GT/GT animals.

Project description:Sprouting of angiogenic perivascular cells is thought to be highly dependent upon autocrine and paracrine growth factor stimulation. Accordingly, we report that corneal angiogenesis induced by ectopic FGF implantation is strongly impaired in NG2/CSPG4 proteoglycan (PG) null mice known to harbour a putative deficit in pericyte proliferation/mobilization. Conversely, no significant differences were seen between wild type and knockout corneas when VEGF was used as an angiocrine factor. Perturbed responsiveness of NG2-deficient pericytes to paracrine and autocrine stimulation by several FGFs could be confirmed in cells isolated from NG2 null mice, while proliferation induced by other growth factors was equivalent in wild type and knockout cells. Identical results were obtained after siRNA-mediated knock-down of NG2 in human smooth muscle-like cell lines, as also demonstrated by the decreased levels of FGF receptor phosphorylation detected in these NG2 deprived cells. Binding assays with recombinant proteins and molecular interactions examined on live cells asserted that FGF-2 bound to NG2 in a glycosaminoglycan-independent, core protein-mediated manner and that the PG was alone capable of retaining FGF-2 on the cell membrane for subsequent receptor presentation. The use of dominant-negative mutant cells, engineered by combined transduction of NG2 deletion constructs and siRNA knock-down of the endogenous PG, allowed us to establish that the FGF co-receptor activity of NG2 is entirely mediated by its extracellular portion. In fact, forced overexpression of the NG2 ectodomain in human smooth muscle-like cells increased their FGF-2-induced mitosis and compensated for low levels of FGF receptor surface expression, in a manner equivalent to that produced by overexpression of the full-length NG2. Upon FGF binding, the cytoplasmic domain of NG2 is phosphorylated, but there is no evidence that this event elicits signal transductions that could bypass the FGFR-mediated ones. Pull-down experiments, protein-protein binding assays and flow cytometry FRET coherently revealed an elective ligand-independent association of NG2 with FGFR1 and FGFR3. The NG2 cooperation with these receptors was also corroborated functionally by the outcome of FGF-2 treatments of cells engineered to express diverse NG2/FGFR combinations. Comprehensively, the findings suggest that perivascular NG2 may serve as a dual modulator of the availability/accessibility of FGF at the cell membrane, as well as the resulting FGFR transducing activity.

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Aggregation-independent modulation of proteoglycan binding by neutralization of C-terminal acidic residues in the chemokine macrophage inflammatory protein 1alpha.

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