Syndecan-4 is a signaling molecule for stromal cell-derived factor-1 (SDF-1)/ CXCL12

Stromal cell-derived factor-1 (SDF-1)/CXCL12, the ligand for CXCR4, induces signal transduction. We previously showed that CXCL12 binds to high- and low-affinity sites expressed by primary cells and cell lines, and forms complexes with CXCR4 as expected and also with a proteoglycan, syndecan-4, but does not form complexes with syndecan-1, syndecan-2, CD44 or beta-glycan. We also demonstrated the occurrence of a CXCL12-independent heteromeric complex between CXCR4 and syndecan-4. However, our data ruled out the glycosaminoglycan-dependent binding of CXCL12 to HeLa cells facilitating the binding of this chemokine to CXCR4. Here, we demonstrate that CXCL12 directly binds to syndecan-4 in a glycosaminoglycan-dependent manner. We show that upon stimulation of HeLa cells by CXCL12, CXCR4 becomes tyrosine phosphorylated as expected, while syndecan-4 (but not syndecan-1, syndecan-2 or beta-glycan) also undergoes such tyrosine phosphorylation. Moreover, tyrosine-phosphorylated syndecan-4 from CXCL12-stimulated HeLa cells physically coassociates with tyrosine phosphorylated CXCR4. Pretreatment of the cells with heparitinases I and III prevented the tyrosine phosphorylation of syndecan-4, which suggests that the heparan sulfate-dependent binding of SDF-1 to this proteoglycan is involved. Finally, by reducing syndecan-4 expression using RNA interference or by pretreating the cells with heparitinase I and III mixture, we suggest the involvement of syndecan-4 and heparan sulfate in p44/p42 mitogen-activated protein kinase and Jun N-terminal/stress-activated protein kinase activation by action of CXCL12 on HeLa cells. However, these treatments did not modify the calcium mobilization induced by CXCL12 in these cells. Therefore, syndecan-4 behaves as a CXCL12 receptor, selectively involved in some transduction pathways induced by SDF-1, and heparan sulfate plays a role in these events.     

The shedding of syndecan-4 and syndecan-1 from HeLa cells and human primary macrophages is accelerated by SDF-1/CXCL12 and mediated by the matrix metalloproteinase-9

We recently demonstrated that stromal cell-derived factor-1(SDF-1/CXCL12) forms complexes with CXCR4, but also with syndecan-4expressed by human primary lymphocytes and macrophages, andHeLa cells. We also suggested that syndecan-4 behaves as a SDF-1-signalingmolecule. Here, we demonstrate that SDF-1 strongly acceleratesthe shedding of syndecan-4 ectodomains and to a lesser extentthat of syndecan-1 from HeLa cells. The fact that this accelerationwas not inhibited by the CXCR4 antagonist AMD3100, anti-CXCR4mAb 12G5, and CXCR4 gene silencing suggests its CXCR4-independence.Pre-treating the cells with heparitinases I, III, or with theprotein kinase C (PKC) inhibitor, bisindolylmaleimide, significantlyinhibited this accelerated shedding, which suggests the involvementof both cell-surface heparan sulfate and PKC transduction pathway.In contrast, Map Kinase or NF-B pathway inhibitors had no effect.Moreover, SDF-1 increases the matrix metalloproteinase-9 (MMP-9)mRNA level as well as MMP-9 activity in HeLa cells, and MMP-9silencing by RNA interference strongly decreases the syndecan-1and -4 ectodomain shedding accelerated by SDF-1. Finally, SDF-1also accelerates in a CXCR4-independent manner, the sheddingof syndecan-1 and -4 from human primary macrophages, which issignificantly inhibited by anti-MMP-9 antibodies. This stronglyindicates the role of MMP-9 in these events occurring in botha tumoral cell line and in human primary macrophages. BecauseMMP-9 plays a crucial role in extracellular matrix degradationduring cancer cell metastasis and invasion, and shed ectodomainsof syndecans may likely be involved in tumor cell proliferation,these data further indicate the multiplicity of the roles playedby SDF-1 on tumor cell biology.     

Syndecan-1 and syndecan-4 are involved in RANTES/CCL5-induced migration and invasion of human hepatoma cells

Background

We previously demonstrated that the CC-chemokine Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES)/CCL5 exerts pro-tumoral effects on human hepatoma Huh7 cells through its G protein-coupled receptor, CCR1. Glycosaminoglycans play major roles in these biological events.

Methods

In the present study, we explored 1/ the signalling pathways underlying RANTES/CCL5-mediated hepatoma cell migration or invasion by the use of specific pharmacological inhibitors, 2/ the role of RANTES/CCL5 oligomerization in these effects by using a dimeric RANTES/CCL5, 3/ the possible involvement of two membrane heparan sulfate proteoglycans, syndecan-1 (SDC-1) and syndecan-4 (SDC-4) in RANTES/CCL5-induced cell chemotaxis and spreading by pre-incubating cells with specific antibodies or by reducing SDC-1 or -4 expression by RNA interference.

Results and conclusion

The present data suggest that focal adhesion kinase phosphorylation, phosphoinositide 3-kinase-, mitogen-activated protein kinase- and Rho kinase activations are involved in RANTES/CCL5 pro-tumoral effects on Huh7 cells. Interference with oligomerization of the chemokine reduced RANTES/CCL5-mediated cell chemotaxis. This study also indicates that SDC-1 and -4 may be required for HepG2, Hep3B and Huh7 human hepatoma cell migration, invasion or spreading induced by the chemokine. These results also further demonstrate the involvement of glycosaminoglycans as the glycosaminoglycan-binding deficient RANTES/CCL5 variant, in which arginine 47 was replaced by lysine, was devoid of effect.

General significance

The modulation of RANTES/CCL5-mediated cellular effects by targeting the chemokine-syndecan interaction could represent a new therapeutic approach for hepatocellular carcinoma.     

A syndecan-4/CXCR4 complex expressed on human primary lymphocytes and macrophages and HeLa cell line binds the CXC chemokine stromal cell-derived factor-1 (SDF-1)

The stromal cell-derived factor-1 (SDF-1) is a CXC chemokine, which plays critical roles in migration, proliferation, and differentiation of leukocytes. SDF-1 is the only known ligand of CXCR4, the coreceptor of X4 HIV strains. We show that SDF-1 binds to high- and low-affinity sites on HeLa cells. Coimmunoprecipitation studies demonstrate that glycanated and oligomerized syndecan-4 but neither syndecan-1, syndecan-2, betaglycan, nor CD44 forms complexes with SDF-1 and CXCR4 on these cells as well as on primary lymphocytes or macrophages. Moreover, biotinylated SDF-1 directly binds in a glycosaminoglycans (GAGs)-dependent manner to electroblotted syndecan-4, and colocalization of SDF-1 with syndecan-4 was visualized by confocal microscopy. Glycosaminidases pretreatment of the HeLa cells or the macrophages decreases the binding of syndecan-4 to the complex formed by it and SDF-1. In addition, this treatment also decreases the binding of the chemokine to CXCR4 on the primary macrophages but not on the HeLa cells. Therefore GAGs-dependent binding of SDF-1 to the cells facilitates SDF-1 binding to CXCR4 on primary macrophages but not on HeLa cell line. Finally, an SDF-1-independent heteromeric complex between syndecan-4 and CXCR4 was visualized on HeLa cells by confocal microscopy as well as by electron microscopy. Moreover, syndecan-4 from lymphocytes, monocyte derived-macrophages, and HeLa cells coimmunoprecipitated with CXCR4. This syndecan-4/CXCR4 complex is likely a functional unit involved in SDF-1 binding. The role of these interactions in the pathophysiology of SDF-1 deserves further study.     

Stromal cell-derived factor-1/chemokine (C-X-C motif) ligand 12 stimulates human hepatoma cell growth, migration, and invasion

In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The aim of this study was to determine whether the chemokine stromal cell-derived factor 1 (SDF-1) induces the growth, migration, and invasion of human hepatoma cells. We show that SDF-1 G protein-coupled receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), and SDF-1 mRNA are expressed in human hepatoma Huh7 cells, which secrete and bind SDF-1. This binding depends on CXCR4 and glycosaminoglycans. SDF-1 associates with CXCR4, and syndecan-4 (SDC-4), a heparan sulfate proteoglycan at the plasma membrane of Huh7 cells, induces the growth of Huh7 cells by promoting their entry into the cell cycle, and inhibits the tumor necrosis factor-alpha-mediated apoptosis of the cells. SDF-1 also reorganizes Huh7 cytoskeleton and induces tyrosine phosphorylation of focal adhesion kinase. Finally, SDF-1 activates matrix metalloproteinase-9, resulting in increased migration and invasion of Huh7 cells. These biological effects of SDF-1 were strongly inhibited by the CXCR4 antagonist AMD3100, by a glycosaminoglycan, heparin, as well as by beta-D-xyloside treatment of the cells, or by c-jun NH(2)-terminal kinase/stress-activated protein kinase inhibitor. Therefore, the CXCR4, glycosaminoglycans, and the mitogen-activated protein kinase signaling pathways are involved in these events. The fact that reducing SDC-4 expression by RNA interference decreased SDF-1-induced Huh7 hepatoma cell migration and invasion strongly indicates that SDC-4 may be an auxiliary receptor for SDF-1. Finally, the fact that CXCR4 is expressed in hepatocellular carcinoma cells from liver biopsies indicates that the in vitro results reported here could be extended to in vivo conditions.     

CXCL12/CXCR4 Axis Triggers the Activation of EGF Receptor and ERK Signaling Pathway in CsA-Induced Proliferation of Human Trophoblast Cells

Introduction

Our previous study has demonstrated Cyclosporin A (CsA) promotes the proliferation of human trophoblast cells. Therefore, we further investigate the intracellular signaling pathway involved in the CsA-induced proliferation of human trophoblast cells.

Methods

Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the regulation of CsA on CXCL12 secretion in human trophoblast cells. Immunofluorescence analysis and western blotting analysis were used to investigate the role of CXCL12/CXCR4 axis in the CsA-induced epidermal growth factor receptor (EGFR) phosphorylation in human trophoblast cells. 5-bromo-2′-deoxyuridine (BrdU) cell proliferation assay was performed to analyze the involvement of EGFR and its downstream extracellular signal-regulated protein kinase (ERK) signaling pathway in the CsA-induced proliferation of human trophoblast cells.

Results

Low concentration of CsA promoted the secretion of CXCL12, and recombinant human CXCL12 promoted the phosphorylation of EGFR in primary human trophoblast cells and choriocarcinoma cell line JEG-3. The inhibition of CXCL12 or CXCR4 by either neutralizing antibodies or small interfering RNA (siRNA) could completely block the CsA-induced EGFR phosphorylation. The CsA-induced proliferation of human trophoblast cells was effectively abrogated by the EGFR inhibitor AG1478 as well as the ERK inhibitor U0126, but not by the PI3K/PKB inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. CsA promoted the activation of ERK in JEG-3 cells, which was markedly abrogated in the presence of CXCL12 siRNA, or CXCR4 siRNA, or AG1478.

Conclusions

CsA may promote EGFR activation via CXCL12/CXCR4 axis, and EGFR downstream ERK signaling pathway may be involved in the CsA-induced proliferation of human trophoblast cells.     

T Cell Specific Adapter Protein (TSAd) Interacts with Tec Kinase ITK to Promote CXCL12 Induced Migration of Human and Murine T Cells

Background

The chemokine CXCL12/SDF-1α interacts with its G-protein coupled receptor CXCR4 to induce migration of lymphoid and endothelial cells. T cell specific adapter protein (TSAd) has been found to promote migration of Jurkat T cells through interaction with the G protein β subunit. However, the molecular mechanisms for how TSAd influences cellular migration have not been characterized in detail.

Principal Findings

We show that TSAd is required for tyrosine phosphorylation of the Lck substrate IL2-inducible T cell kinase (Itk). Presence of Itk Y511 was necessary to boost TSAd''s effect on CXCL12 induced migration of Jurkat T cells. In addition, TSAd''s ability to promote CXCL12-induced actin polymerization and migration of Jurkat T lymphocytes was dependent on the Itk-interaction site in the proline-rich region of TSAd. Furthermore, TSAd-deficient murine thymocytes failed to respond to CXCL12 with increased Itk phosphorylation, and displayed reduced actin polymerization and cell migration responses.

Conclusion

We propose that TSAd, through its interaction with both Itk and Lck, primes Itk for Lck mediated phosphorylation and thereby regulates CXCL12 induced T cell migration and actin cytoskeleton rearrangements.     

CXC Chemokine Receptor 7 (CXCR7) Regulates CXCR4 Protein Expression and Capillary Tuft Development in Mouse Kidney

Background

The CXCL12/CXCR4 axis is involved in kidney development by regulating formation of the glomerular tuft. Recently, a second CXCL12 receptor was identified and designated CXCR7. Although it is established that CXCR7 regulates heart and brain development in conjunction with CXCL12 and CXCR4, little is known about the influence of CXCR7 on CXCL12 dependent kidney development.

Methodology/Principal Findings

We provided analysis of CXCR7 expression and function in the developing mouse kidney. Using in situ hybridization, we identified CXCR7 mRNA in epithelial cells including podocytes at all nephron stages up to the mature glomerulus. CXCL12 mRNA showed a striking overlap with CXCR7 mRNA in epithelial structures. In addition, CXCL12 was detected in stromal cells and the glomerular tuft. Expression of CXCR4 was complementary to that of CXCR7 as it occurred in mesenchymal cells, outgrowing ureteric buds and glomerular endothelial cells but not in podocytes. Kidney examination in CXCR7 null mice revealed ballooning of glomerular capillaries as described earlier for CXCR4 null mice. Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme. Malformation of the glomerular tuft in CXCR7 null mice was associated with mesangial cell clumping.

Conclusions/Significance

We established that there is a similar glomerular pathology in CXCR7 and CXCR4 null embryos. Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries.     

The Potent Anti-HIV Activity of CXCL12γ Correlates with Efficient CXCR4 Binding and Internalization

We previously demonstrated that the naturally occurring splice variant stromal cell-derived factor 1γ/CXCL12γ is the most potent CXCL12 isoform in blocking X4 HIV-1, with weak chemotactic activity. A conserved BBXB domain (B for basic and X for any residue) located in the N terminus (²⁴KHLK²⁷) is found in all six isoforms of CXCL12. To determine whether the potent antiviral activity of CXCL12γ is due to the presence of the extra C-terminal BBXB domains, we mutated each domain individually as well as in combination. Although binding of CXCL12γ to heparan sulfate proteoglycan (HSPG) was 10-fold higher than that observed with CXCL12α, the results did not demonstrate a direct correlation between HSPG binding and the potent antiviral activity. CXCL12γ mutants lacking the conserved BBXB domain (designated γB1) showed increased binding to HSPG but reduced anti-HIV activity. In contrast, the mutants lacking the C-terminal second and/or third BBXB domain but retaining the conserved domain (designated B2, B3, and B23) showed decreased binding to HSPG but increased anti-HIV activity. The B2, B3, and B23 mutants were associated with enhanced CXCR4 binding, receptor internalization, and restored chemotaxis. Internalization of CXCR4 was more potent with CXCL12γ than with CXCL12α and was significantly reduced when the conserved BBXB domain was mutated. We concluded that the observed potent anti-HIV-1 activity of CXCL12γ is due to increased affinity for CXCR4 and to efficient receptor internalization.Chemokines are small, structurally related chemoattractant cytokines characterized by conserved cysteine residues. Based on the positions of the first N-terminal cysteines, chemokines fall into four subfamilies. The CC and CXC subfamilies have been well characterized. The CC subfamily includes the following: regulated on activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory peptides 1 (MIP-1). The prototype of the CXC subfamily is interleukin-8 (IL-8)/CXCL8. The C chemokine (lymphotactine) and CX3C chemokine (fractalkine) subfamilies were recently identified (reviewed in reference 30). The physiological activities of chemokines are mediated by the selective recognition and activation of chemokine receptors belonging to the seven-membrane-domain G-protein-coupled receptor superfamily (GPCRs). In addition, chemokines also bind to glycosaminoglycans (GAGs) through distinct binding sites. Chemokine binding to GAGs on cells, particularly endothelial cells, results in chemotactic chemokine gradients that allow correct presentation of chemokines to leukocytes, therefore enabling target cells to cross the endothelial barrier and migrate into tissues (reviewed in reference 10).Stromal cell-derived factor 1 (SDF-1)/CXCL12 is a member of the CXC chemokine family and is a key regulator of B-cell lymphopoiesis, hematopoietic stem cell mobilization, and leukocyte migration (reviewed in reference 10). CXCL12 was originally thought to mediate these processes through the single receptor CXCR4 (9). However, later studies demonstrated that RDC-1/CXCR7 is also a receptor for CXCL12 (6, 11). CXCL12 has also been shown to block HIV-1 infection (5). There are two known human splice variants of CXCL12, referred to as CXCL12α and CXCL12β (27). The genomic structure of the CXCL12 gene revealed that human CXCL12α and CXCL12β are encoded by a single gene and arise by alternative splicing. The cDNAs corresponding to CXCL12α and CXCL12β encode proteins of 89 and 93 amino acids, respectively. A third splice variant, classified as CXCL12γ, has been identified in rats (14). The human equivalent of CXCL12γ was recently identified among other splice variants of CXCL12 (33). The novel human splice variants CXCL12γ, CXCL12ɛ, CXCL12δ, and CXCL12θ (also reported as CXCL12ϕ [33]) are expressed through alternative splicing events that result in different exons being added to the same first three exons. Therefore, all six splice variants of CXCL12 are identical in the first 88 amino acid residues from the amino terminus.It has been demonstrated that CXCL12α and -β are expressed in numerous tissues, with the highest expression levels in the liver, pancreas, and spleen (33). The mRNA encoding CXCL12γ was detectable in the adult human heart but hardly detectable in several other tissues. On the other hand, CXCL12δ, -ɛ, and -θ could be detected in several human adult and fetal tissues, with the pancreas expressing the highest levels (33). Recent studies have demonstrated the tissue expression of CXCL12γ in the adult heart (24). We previously demonstrated that CXCL12γ is the most potent anti-HIV-1 inhibitor, with the weakest chemotactic activity and no detectable enhancing activity for hematopoietic progenitor cell survival or replating capacity (2). The first three exons present in the CXCL12γ splice variant are identical to those found in CXCL12α and CXCL12β. The fourth exon, however, contains a large number of basic residues that result in at least four additional BBXB domains in addition to the conserved ²⁴KHLK²⁷ domain (33). It is not known whether the additional BBXB domains in the C terminus of CXCL12γ result in higher affinity for heparan sulfate proteoglycan (HSPG) and whether differences in HSPG binding could explain the observed anti-HIV-1 potency or the low chemotactic activity.The BBXB motif on RANTES has been suggested as the principal site for high-affinity binding to heparan sulfate. This binding controls receptor selectivity (22). It was previously demonstrated that a mutation of CXCL12α in the ²⁴KHLK²⁷ domain reduces the antiviral activity at least 50 percent without affecting the chemotactic activity (4, 29). It was proposed that chemokine binding to HSPG might concentrate the chemokine near the CXCR4 receptor or form a haptotactic chemokine gradient.In this study, we analyzed the mechanism of the potent antiviral activity of CXCL12γ. We examined the role of the additional BBXB domains of CXCL12γ in the observed biological activities of CXCL12γ. Mutations in CXCL12γ were introduced to knock out the BBXB domains either individually or in combination. We analyzed receptor internalization and binding affinities of the mutant chemokines for CXCR4 and HSPG. The results demonstrate that the potent anti-HIV activity of CXCL12γ is due to its efficient binding and internalization of CXCR4. The results provide important insight into the structure-function relationship of CXCL12γ and suggest that determinants other than the BBXB domains are involved in the observed biological activities of CXCL12γ.     

HIV glycoprotein gp120 enhances mesenchymal stem cell migration by upregulating CXCR4 expression

Background

HIV infection and/or the direct pathogenic effects of circulating HIV proteins impairs the physiological function of mesenchymal stem cells (MSCs), and contribute to the pathogenesis of age-related clinical comorbidities in people living with HIV. The SDF-1/CXCR4 pathway is vital for modulating MSC proliferation, migration and differentiation. HIV glycoprotein gp120 inhibits SDF-1 induced chemotaxis by downregulating the expression and function of CXCR4 in monocytes, B and T cells. The influence of gp120 on CXCR4 expression and migration in MSCs is unknown.

Small molecule inhibitors of protein interaction with glycosaminoglycans (SMIGs), a novel class of bioactive agents with anti-inflammatory properties

Background

Small molecule inhibitors of biologically important protein–glycosaminoglycan (GAG) interactions have yet to be identified.

Methods

Compound libraries were screened in an assay of L-selectin–IgG binding to heparin (a species of heparan sulfate [HS-GAG]). Hits were validated, IC-50s established and direct binding of hits to HS-GAGs was investigated by incubating compounds alone with heparin. Selectivity of inhibitors was assessed in 11 different protein-GAG binding assays. Anti-inflammatory activity of selected compounds was evaluated in animal models.

Results

Screening identified a number of structurally-diverse planar aromatic cationic amines. Scaffolds similar to known GAG binders, chloroquine and tilorone, were also identified. Inhibitors displayed activity also against bovine kidney heparan sulfate. Direct binding of compounds to GAGs was verified by incubating compounds with heparin alone. Selectivity of inhibitors was demonstrated in a panel of 11 heparin binding proteins, including selectins, chemokines (IL-8, IP-10), Beta Amyloid and cytokines (VEGF, IL-6). A number of selected lead compounds showed dose-dependent efficacy in peritonitis, paw edema and delayed type hypersensitivity.

Conclusions

A new class of compounds, SMIGs, inhibits protein–GAG interaction by direct binding to GAGs. Although their IC-50s were in the low micro-molar range, SMIGs binding to HS-GAGs appeared to be stable in physiological conditions, indicating high avidity binding. SMIGs may interfere with major checkpoints for inflammatory and autoimmune events.

General significance

SMIGs are a class of structurally-diverse planar aromatic cationic amines that have an unusual mode of action — inhibiting protein–GAG interactions via direct and stable binding to GAGs. SMIGs may have therapeutic potential in inflammatory and autoimmune disorders.     

Osteopontin enhances the expression and activity of MMP-2 via the SDF-1/CXCR4 axis in hepatocellular carcinoma cell lines

Background and Aims

Osteopontin, SDF-1α, and MMP-2 are important secreted molecules involved in the pathophysiology of human hepatocellular carcinoma (HCC). This study investigates the effect of the SDF-1α/CXCR4 axis on expression and activity of MMP-2 induced by osteopontin.

Methods

The expression of CXCR4, SDF-1α, MMP-2 and their associated cellular signaling cascades, involving Akt and MAP Kinases, were determined by Western blotting. The activities of MMP-2 and MMP-9 were assayed by gel zymography. The role of the osteopontin receptors integrin α_vβ₃ and CD44v6 was evaluated using neutralizing antibodies. We also established CXCR4-deficient SMMC7721 cell lines by transfection with miRNA-CXCR4 plasmids and determined cell invasion activity in a transwell assay.

Results

In comparison with untreated cells, recombinant human osteopontin (rhOPN) up-regulated CXCR4, SDF-1α, and MMP-2 expression about 5-, 4-, and 6-fold on the protein levels through binding to integrin α_vβ₃ and CD44v6 in hepatocellular carcinoma cells (SMMC7721 and HepG2). Inhibition of the SDF-1α/CXCR4 axis down-regulated the rhOPN-induced MMP-2 expression and activity. rhOPN also activated Akt, p38 and JNK. Down-regulation of CXCR4 decreased the rhOPN-induced invasion in SMMC7721 cells.

Conclusion

These results indicate that rhOPN up-regulates MMP-2 through the SDF-1α/CXCR4 axis, mediated by binding to integrin α_vβ₃ and CD44v6 and activating the PI-3K/Akt and JNK pathways in HepG2 and SMMC7721 cells. Therefore, the osteopontin-SDF-1α/CXCR4-MMP-2 system may be a new therapeutic target for treating HCC progression.     

Inhibition of the CXCL12/CXCR4-Axis as Preventive Therapy for Radiation-Induced Pulmonary Fibrosis

Background

A devastating late injury caused by radiation is pulmonary fibrosis. This risk may limit the volume of irradiation and compromise potentially curative therapy. Therefore, development of a therapy to prevent this toxicity can be of great benefit for this patient population. Activation of the chemokine receptor CXCR4 by its ligand stromal cell-derived factor 1 (SDF-1/CXCL12) may be important in the development of radiation-induced pulmonary fibrosis. Here, we tested whether MSX-122, a novel small molecule and partial CXCR4 antagonist, can block development of this fibrotic process.

Methodology/Principal Findings

The radiation-induced lung fibrosis model used was C57BL/6 mice irradiated to the entire thorax or right hemithorax to 20 Gy. Our parabiotic model involved joining a transgenic C57BL/6 mouse expressing GFP with a wild-type mouse that was subsequently irradiated to assess for migration of GFP+ bone marrow-derived progenitor cells to the irradiated lung. CXCL12 levels in the bronchoalveolar lavage fluid (BALF) and serum after irradiation were determined by ELISA. CXCR4 and CXCL12 mRNA in the irradiated lung was determined by RNase protection assay. Irradiated mice were treated daily with AMD3100, an established CXCR4 antagonist; MSX-122; and their corresponding vehicles to determine impact of drug treatment on fibrosis development. Fibrosis was assessed by serial CTs and histology. After irradiation, CXCL12 levels increased in BALF and serum with a corresponding rise in CXCR4 mRNA within irradiated lungs consistent with recruitment of a CXCR4+ cell population. Using our parabiotic model, we demonstrated recruitment of CXCR4+ bone marrow-derived mesenchymal stem cells, identified based on marker expression, to irradiated lungs. Finally, irradiated mice that received MSX-122 had significant reductions in development of pulmonary fibrosis while AMD3100 did not significantly suppress this fibrotic process.

Conclusions/Significance

CXCR4 inhibition by drugs such as MSX-122 may alleviate potential radiation-induced lung injury, presenting future therapeutic opportunities for patients requiring chest irradiation.     

Syndecan-3 is selectively pro-inflammatory in the joint and contributes to antigen-induced arthritis in mice

Introduction

Syndecans are heparan sulphate proteoglycans expressed by endothelial cells. Syndecan-3 is expressed by synovial endothelial cells of rheumatoid arthritis (RA) patients where it binds chemokines, suggesting a role in leukocyte trafficking. The objective of the current study was to examine the function of syndecan-3 in joint inflammation by genetic deletion in mice and compare with other tissues.

Methods

Chemokine C-X-C ligand 1 (CXCL1) was injected in the joints of syndecan-3−/−and wild-type mice and antigen-induced arthritis performed. For comparison chemokine was administered in the skin and cremaster muscle. Intravital microscopy was performed in the cremaster muscle.

Results

Administration of CXCL1 in knee joints of syndecan-3−/−mice resulted in reduced neutrophil accumulation compared to wild type. This was associated with diminished presence of CXCL1 at the luminal surface of synovial endothelial cells where this chemokine clustered and bound to heparan sulphate. Furthermore, in the arthritis model syndecan-3 deletion led to reduced joint swelling, leukocyte accumulation, cartilage degradation and overall disease severity. Conversely, CXCL1 administration in the skin of syndecan-3 null mice provoked increased neutrophil recruitment and was associated with elevated luminal expression of E-selectin by dermal endothelial cells. Similarly in the cremaster, intravital microscopy showed increased numbers of leukocytes adhering and rolling in venules in syndecan-3−/−mice in response to CXCL1 or tumour necrosis factor alpha.

Conclusions

This study shows a novel role for syndecan-3 in inflammation. In the joint it is selectively pro-inflammatory, functioning in endothelial chemokine presentation and leukocyte recruitment and cartilage damage in an RA model. Conversely, in skin and cremaster it is anti-inflammatory.     

Platelet-derived CXCL12 regulates monocyte function,survival, differentiation into macrophages and foam cells through differential involvement of CXCR4–CXCR7

Platelets store and release CXCL12 (SDF-1), which governs differentiation of hematopoietic progenitors into either endothelial or macrophage-foam cells. CXCL12 ligates CXCR4 and CXCR7 and regulates monocyte/macrophage functions. This study deciphers the relative contribution of CXCR4–CXCR7 in mediating the effects of platelet-derived CXCL12 on monocyte function, survival, and differentiation. CXCL12 and macrophage migration inhibitory factor (MIF) that ligate CXCR4–CXCR7 induced a dynamic bidirectional trafficking of the receptors, causing CXCR4 internalization and CXCR7 externalization during chemotaxis, thereby influencing relative receptor availability, unlike MCP-1. In vivo we found enhanced accumulation of platelets and platelet-macrophage co-aggregates in peritoneal fluid following induction of peritonitis in mice. The relative surface expression of CXCL12, CXCR4, and CXCR7 among infiltrated monocytes was also enhanced as compared with peripheral blood. Platelet-derived CXCL12 from collagen-adherent platelets and recombinant CXCL12 induced monocyte chemotaxis specifically through CXCR4 engagement. Adhesion of monocytes to immobilized CXCL12 and CXCL12-enriched activated platelet surface under static and dynamic arterial flow conditions were mediated primarily through CXCR7 and were counter-regulated by neutralizing platelet-derived CXCL12. Monocytes and culture-derived-M1–M2 macrophages phagocytosed platelets, with the phagocytic potential of culture-derived-M1 macrophages higher than M2 involving CXCR4–CXCR7 participation. CXCR7 was the primary receptor in promoting monocyte survival as exerted by platelet-derived CXCL12 against BH3-mimetic induced apoptosis (phosphatidylserine exposure, caspase-3 activation, loss of mitochondrial transmembrane potential). In co-culture experiments with platelets, monocytes predominantly differentiated into CD163⁺ macrophages, which was attenuated upon CXCL12 neutralization and CXCR4/CXCR7 blocking antibodies. Moreover, OxLDL uptake by platelets induced platelet apoptosis, like other platelet agonists TRAP and collagen-related peptide (CRP). CXCL12 facilitated phagocytosis of apoptotic platelets by monocytes and M1–M2 macrophages, also promoted their differentiation into foam cells via CXCR4 and CXCR7. Thus, platelet-derived CXCL12 could regulate monocyte-macrophage functions through differential engagement of CXCR4 and CXCR7, indicating an important role in inflammation at site of platelet accumulation.Platelets are central players in regulation of inflammation at the site of thrombosis.^{1, 2, 3} When platelets are activated they release a variety of pro-inflammatory mediators including the chemokine CXCL12 (SDF-1).^{4, 5, 6, 7} CXCL12 binds to its chemokine receptors CXCR4 and CXCR7 and regulates cell migration, adhesion and survival.^{8, 9, 10, 11}Recently, platelets have been recognized to store substantial amounts of CXCL12 in their alpha-granules and release the chemokine upon activation.^{5, 6} Platelet-derived CXCL12 propagates migration and subsequent differentiation of CD34⁺ progenitor cells^{5, 12} into either an endothelial or a macrophage/foam cell phenotype depending on the culture conditions.^{12, 13} Release of CXCL12 from platelets is enhanced in acute coronary syndromes and correlates with the number of circulating CD34⁺ progenitor cells and platelet/CD34⁺ co-aggregates.^{14, 15} Enhanced levels of platelet–CXCL12 are associated with preservation of left ventricular function following myocardial infarction in humans.¹⁶ Administration of recombinant CXCL12 preserves myocardial function following transient ischemia in mice.¹⁷Monocytes/macrophages have a critical role in vascular inflammation and disease progression of atherosclerosis.¹⁸ Monocytes express both CXCR4 and CXCR7 although their role in monocyte function is still incompletely understood.^{9, 19}In the present study, we explored the effect of platelet-derived CXCL12 on monocyte function and the differential role of CXCR4 and CXCR7 for monocyte function and differentiation. We found that both chemokine receptors have a decisive but differential role for platelet-dependent monocyte function.     

Attenuation of osteoarthritis via blockade of the SDF-1/CXCR4 signaling pathway

Introduction

This study was performed to evaluate the attenuation of osteoarthritic (OA) pathogenesis via disruption of the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signaling with AMD3100 in a guinea pig OA model.

Methods

OA chondrocytes and cartilage explants were incubated with SDF-1, siRNA CXCR4, or anti-CXCR4 antibody before treatment with SDF-1. Matrix metalloproteases (MMPs) mRNA and protein levels were measured with real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The 35 9-month-old male Hartley guinea pigs (0.88 kg ± 0.21 kg) were divided into three groups: AMD-treated group (n = 13); OA group (n = 11); and sham group (n = 11). At 3 months after treatment, knee joints, synovial fluid, and serum were collected for histologic and biochemical analysis. The severity of cartilage damage was assessed by using the modified Mankin score. The levels of SDF-1, glycosaminoglycans (GAGs), MMP-1, MMP-13, and interleukin-1 (IL-1β) were quantified with ELISA.

Results

SDF-1 infiltrated cartilage and decreased proteoglycan staining. Increased glycosaminoglycans and MMP-13 activity were found in the culture media in response to SDF-1 treatment. Disrupting the interaction between SDF-1 and CXCR4 with siRNA CXCR4 or CXCR4 antibody attenuated the effect of SDF-1. Safranin-O staining revealed less cartilage damage in the AMD3100-treated animals with the lowest Mankin score compared with the control animals. The levels of SDF-1, GAG, MMP1, MMP-13, and IL-1β were much lower in the synovial fluid of the AMD3100 group than in that of control group.

Conclusions

The binding of SDF-1 to CXCR4 induces OA cartilage degeneration. The catabolic processes can be disrupted by pharmacologic blockade of SDF-1/CXCR4 signaling. Together, these findings raise the possibility that disruption of the SDF-1/CXCR4 signaling can be used as a therapeutic approach to attenuate cartilage degeneration.     

Deubiquitination of CXCR4 by USP14 Is Critical for Both CXCL12-induced CXCR4 Degradation and Chemotaxis but Not ERK Activation

The chemokine receptor CXCR4 plays important roles in the immune and nervous systems. Abnormal expression of CXCR4 contributes to cancer and inflammatory and neurodegenerative disorders. Although ligand-dependent CXCR4 ubiquitination is known to accelerate CXCR4 degradation, little is known about counter mechanisms for receptor deubiquitination. CXCL12, a CXCR4 agonist, induces a time-dependent association of USP14 with CXCR4, or its C terminus, that is not mimicked by USP2A, USP4, or USP7, other members of the deubiquitination catalytic family. Co-localization of CXCR4 and USP14 also is time-dependent following CXCL12 stimulation. The physical interaction of CXCR4 and USP14 is paralleled by USP14-catalyzed deubiquitination of the receptor; knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4 deubiquitination, whereas overexpression of USP14 promotes CXCR4 deubiquitination. We also observed that ubiquitination of CXCR4 facilitated receptor degradation, whereas overexpression of USP14 or RNAi-induced knockdown of USP14 blocked CXCL12-mediated CXCR4 degradation. Most interestingly, CXCR4-mediated chemotactic cell migration was blocked by either overexpression or RNAi-mediated knockdown of USP14, implying that a CXCR4-ubiquitin cycle on the receptor, rather than a particular ubiquitinated state of the receptor, is critical for the ligand gradient sensing and directed motility required for chemokine-mediated chemotaxis. Our observation that a mutant of CXCR4, HA-3K/R CXCR4, which cannot be ubiquitinated and does not mediate a chemotactic response to CXCL12, indicates the importance of this covalent modification not only in marking receptors for degradation but also for permitting CXCR4-mediated signaling. Finally, the indistinguishable activation of ERK by wild typeor 3K/R-CXCR4 suggests that chemotaxis in response to CXCL12 may be independent of the ERK cascade.The CXCR4 (CXC chemokine receptor 4) is a member of the chemokine receptor family, which belongs to the superfamily of G protein-coupled receptors (GPCRs)² (1). Its ligand, CXCL12, also known as SDF-1α, also binds to RDC1, another chemokine receptor that is being proposed to be renamed as CXCR7 (2). CXCR4 mediates CXCL12-induced migration of peripheral blood lymphocytes (3), CD34⁺ progenitor cells (4), and pre- and pro-B cell lines (5). CXCR4 also plays an important role in the development of the immune system, because mouse embryos lacking either expression of the CXCR4 receptor or of its CXCL12 ligand are embryonic lethal and also manifest abnormalities in B cell lymphopoiesis and bone marrow myelopoiesis (3, 6, 7). The altered cerebellar neuron migration in mice null for the CXCR4 receptor also suggests a role for this receptor in central nervous system development. Abnormal expression and/or function of CXCR4 have been implicated in a number of diseases, including human immunodeficiency virus infection (8), cardiovascular disease (9), allergic inflammatory disease (10), neuroinflammation (11), neurodegenerative diseases (12, 13), and cancers (14-24).Stimulation of CXCR4 triggers various intracellular signaling cascades (1, 14, 25-27), such as extracellular signal-regulated kinase (ERK), which likely contribute to CXCR4-induced cell proliferation, differentiation, and/or migration. Ligand stimulation of CXCR4 also induces endocytosis of these receptors, which are targeted to lysosomes for degradation through a pathway involving ubiquitination of the C-terminal lysine residues (28). CXCR4 ubiquitination can be catalyzed by a member of the HECT family of E3 ligases, AIP4 (atrophin-interacting protein 4) (29, 30). The ubiquitinated CXCR4 is delivered to the endosomal compartments via a regulated pathway involving several adaptor proteins (31).It has been noted that deubiquitination also regulates the fate and function of ubiquitin-conjugated proteins. Deubiquitinating enzymes, which catalyze the removal of ubiquitin from ubiquitin-conjugated proteins, represent the largest family of enzymes in the ubiquitin system, implying the possibility that substrate selectivity is even greater for these enzymes than for those that catalyze ubiquitin ligation. Little is known about the mechanisms of CXCR4 deubiquitination and their regulation by receptor ligands. A proteomics study revealed that the steady state level of USP14 was increased upon CXCL12 stimulation of target cells (32), and preliminary studies revealed that ligand stimulation led to enhanced association of USP14 with the CXCR4. The present studies were undertaken to ascertain the functional consequences of this interaction, the selectivity of CXCR4 for USP14, when compared with three other deubiquitinating enzymes, USP2a, USP4, and USP7, and the impact of modifying the ubiquitinated state of the receptor on CXCR4 turnover, CXCL12-evoked chemotaxis, and CXCL12-induced activation of ERK.     

Cooperation of binding sites at the hydrophilic domain of cell-surface sulfatase Sulf1 allows for dynamic interaction of the enzyme with its substrate heparan sulfate

Background

Sulf1 is a cell-surface sulfatase removing internal 6-O-sulfate groups from heparan sulfate (HS) chains. Thereby it modulates the activity of HS-dependent growth factors. For HS interaction Sulf1 employs a unique hydrophilic domain (HD).

Methods

Affinity-chromatography, AFM-single-molecule force spectroscopy (SMFS) and immunofluorescence on living cells were used to analyze specificity, kinetics and structural basis of this interaction.

Results

Full-length Sulf1 interacts broadly with sulfated glycosaminoglycans (GAGs) showing, however, higher affinity toward HS and heparin than toward chondroitin sulfate or dermatan sulfate. Strong interaction depends on the presence of Sulf1-substrate groups, as Sulf1 bound significantly weaker to HS after enzymatic 6-O-desulfation by Sulf1 pretreatment, hence suggesting autoregulation of Sulf1/substrate association. In contrast, HD alone exhibited outstanding specificity toward HS and did not interact with chondroitin sulfate, dermatan sulfate or 6-O-desulfated HS. Dynamic SMFS revealed an off-rate of 0.04/s, i.e., ~ 500-fold higher than determined by surface plasmon resonance. SMFS allowed resolving the dynamics of single dissociation events in each force–distance curve. HD subdomain constructs revealed heparin interaction sites in the inner and C-terminal regions of HD.

Conclusions

Specific substrate binding of Sulf1 is mediated by HD and involves at least two separate HS-binding sites. Surface plasmon resonance K_D-values reflect a high avidity resulting from multivalent HD/heparin interaction. While this ensures stable cell–surface HS association, the dynamic cooperation of binding sites at HD and also the catalytic domain enables processive action of Sulf1 along or across HS chains.

General significance

HD confers a novel and highly dynamic mode of protein interaction with HS.