CXC chemokine ligand 12-induced focal adhesion kinase activation and segregation into membrane domains is modulated by regulator of G protein signaling 1 in pro-B cells

CXCL12-induced chemotaxis and adhesion to VCAM-1 decrease as B cells differentiate in the bone marrow. However, the mechanisms that regulate CXCL12/CXCR4-mediated signaling are poorly understood. We report that after CXCL12 stimulation of progenitor B cells, focal adhesion kinase (FAK) and PI3K are inducibly recruited to raft-associated membrane domains. After CXCL12 stimulation, phosphorylated FAK is also localized in membrane domains. The CXCL12/CXCR4-FAK pathway is membrane cholesterol dependent and impaired by metabolic inhibitors of G(i), Src family, and the GTPase-activating protein, regulator of G protein signaling 1 (RGS1). In the bone marrow, RGS1 mRNA expression is low in progenitor B cells and high in mature B cells, implying developmental regulation of CXCL12/CXCR4 signaling by RGS1. CXCL12-induced chemotaxis and adhesion are impaired when FAK recruitment and phosphorylation are inhibited by either membrane cholesterol depletion or overexpression of RGS1 in progenitor B cells. We conclude that the recruitment of signaling molecules to specific membrane domains plays an important role in CXCL12/CXCR4-induced cellular responses.     

Controlling BMP growth factor bioavailability: The extracellular matrix as multi skilled platform

Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily of signaling ligands which comprise a family of pluripotent cytokines regulating a multitude of cellular events. Although BMPs were originally discovered as potent factors extractable from bone matrix that are capable to induce ectopic bone formation in soft tissues, their mode of action has been mostly studied as soluble ligands in absence of the physiologically relevant cellular microenvironment. This micro milieu is defined by supramolecular networks of extracellular matrix (ECM) proteins that specifically target BMP ligands, present them to their cellular receptors, and allow their controlled release. Here we focus on functional interactions and mechanisms that were described to control BMP bioavailability in a spatio-temporal manner within the respective tissue context. Structural disturbance of the ECM architecture due to mutations in ECM proteins leads to dysregulated BMP signaling as underlying cause for connective tissue disease pathways. We will provide an overview about current mechanistic concepts of how aberrant BMP signaling drives connective tissue destruction in inherited and chronic diseases.     

p38MAPK inhibitors attenuate cytokine production by bone marrow stromal cells and reduce stroma-mediated proliferation of acute lymphoblastic leukemia cells

Objective: The bone marrow microenvironment provides critical support for the growth and survival of acute lymphoblastic leukemia cells and protection against the effects of chemotherapeutic agents. Although the mechanisms are not fully understood, it is likely that they are mediated at least in part by stromal derived cytokines and chemokines. Methods: Cell proliferation was measured by 3H-thymidine assays, survival by Annexin V/PI staining, gene expression by microarray, cytokine protein levels by antibody microarrays and/or ELISA and cellular signaling by western blotting. Results: We have demonstrated that inhibition of p38MAPK in bone marrow stromal cells reduced the production of IL-6, VEGF, PDGF and CXCL12. In addition to the known role of CXCL12 in ALL cell stromal-dependent proliferation, we have shown that VEGF and PDGF also provide important proliferative cues for ALL cells, both as exogenous single agents and as bone marrow stromal culture-derived factors. In contrast we could not detect a significant role for IL-6 in ALL stromal-dependent proliferation. Consistent with these findings inhibition of p38MAPK significantly reduced stromal-dependent proliferation of ALL cells. Conclusion: These findings suggest that inhibition of p38MAPK may provide a useful adjunct to current treatment strategies by retarding ALL cell growth.     

CXCL12 (SDF-1) and CXCL13 (BCA-1) chemokines significantly induce proliferation and collagen type I expression in osteoblasts from osteoarthritis patients

To evaluate the role of CXC chemokines CXCL8 (IL8), CXCL10 (IP-10), CXCL12 (SDF-1), and CXCL13 (BCA-1) in bone remodeling, we analyzed their effects on osteoblasts (OBs) obtained from subchondral trabecular bone tissue of osteoarthritis (OA) and post-traumatic (PT) patients. The expression of CXC receptors/ligands (CXCR1/CXCL8, CXCR2/CXCL8, CXCR3/CXCL10, CXCR4/CXCL12, and CXCR5/CXCL13) was analyzed in cultured OBs by flow cytometry and immunocytochemistry. Functional assays on CXC chemokine-treated-OBs in the presence or absence of their specific inhibitors were performed to analyze cellular proliferation and the enzymatic response to chemokine activation. The expression of chemokine ligands/receptors was also confirmed in bone tissue samples by immunohistochemical analysis. Collagen type I and alkaline phosphatase mRNA expression were analyzed on CXCL12- and CXCL13-treated OBs by real-time PCR. OBs from both OA and PT patients expressed high levels of CXCR3 and CXCR5 and lower amounts of CXCR1 and CXCR4. CXCL12 and CXCL13, only in OBs from OA patients, induced a significant proliferation that was also confirmed by specific blocking experiments. Moreover, OBs from OA patients released a higher amount of CXCL13 than those of PT patients while no differences were found for CXCL12. In the remodeling area of bone tissue samples, immunohistochemical analysis confirmed that OBs expressed CXCL12/CXCR4 and CXCL13/CXCR5 both in OA and PT samples. CXCL12 and CXCL13 upregulated collagen type I mRNA expression in OBs from OA patients. These data suggest that CXCL12 and CXCL13 may directly modulate cellular proliferation and collagen type I in OA patients, so contributing to the remodeling process that occurs in the evolution of this disease.     

Migration potential and gene expression profile of human mesenchymal stem cells induced by CCL25

Recruitment of mesenchymal stem cells (MSC) to tissue damages is a promising approach for in situ tissue regeneration. The physiological mechanisms and regulatory processes of MSC trafficking to injured tissue remain poorly understood. However, the pivotal role of chemokines in MSC recruitment has already been shown.The aim of this study was to determine the migratory potential and the gene expression profile of MSC stimulated with the CC chemokine CCL25 (TECK). Bone marrow derived human MSC were exposed to different doses of CCL25 in a standardized chemotaxis assay. Microarray gene expression profiling and pathway analysis were performed for CCL25 stimulated MSC.Maximum migration of MSC towards CCL25 was observed at 10³ nM. Microarray analysis revealed an induction of molecules directly involved in chemotaxis and homing of bone marrow cells (CXCL1-3, CXCL8, PDE4B), cytoskeletal and membrane reorganisation (CXCL8, PLD1, IGFBP1), cellular polarity (PLD1), and cell movement (CXCL1-3, CXCL6, CXCL8, PTGS2, PDE4B, TGM2). Respective chemokine secretion was confirmed by protein membrane-array analysis. The activation of CXCR2 ligands (CXCL1-3, CXCL5-6, CXCL8) and a LIF-receptor/gp130 ligand (LIF) indicated an involvement of the respective signaling pathways during initiation of chemotaxis and migration.These results suggest CCL25 as a new potential candidate for further in situ regeneration approaches.     

Noncanonical Wnt signaling in stromal cells regulates B-lymphogenesis through interleukin-7 expression

The regulation of early B cell development and the interaction of hematopoietic precursors with stromal cells in the bone marrow (BM) are controlled by various secreted signaling molecules. Several recent studies showed Wnt signaling involved in B-lymphogenesis through stromal cells. However, the molecules modulated by Wnt signaling in stromal cells regulating B-lymphogenesis have not been identified yet. Interleukin (IL)-7 and CXC chemokine ligand (CXCL) 12 are known to be express in stromal cells, and both molecules are essential for B-lymphogenesis. In the present study, we examined the role of Wnt signaling in regulating IL-7 and CXCL12 expression and in affecting B-lymphogenesis. In mouse stromal ST2 cells, expression of IL-7 and CXCL12 mRNA was augmented by noncanonical Wnt5a. When mouse BM-derived cells were cultured on Wnt5a-overexpressing ST2 cells, an increased number of B220+/IgM- B-lymphoid precursor cells was observed. These results show that Wnt5a regulates IL-7 gene expression in stromal cells and suggest the possibility that noncanonical Wnt regulates B-lymphogenesis via IL-7 expression in stromal cells.     

Quantitative phosphoproteomics of CXCL12 (SDF-1) signaling

CXCL12 (SDF-1) is a chemokine that binds to and signals through the seven transmembrane receptor CXCR4. The CXCL12/CXCR4 signaling axis has been implicated in both cancer metastases and human immunodeficiency virus type 1 (HIV-1) infection and a more complete understanding of CXCL12/CXCR4 signaling pathways may support efforts to develop therapeutics for these diseases. Mass spectrometry-based phosphoproteomics has emerged as an important tool in studying signaling networks in an unbiased fashion. We employed stable isotope labeling with amino acids in cell culture (SILAC) quantitative phosphoproteomics to examine the CXCL12/CXCR4 signaling axis in the human lymphoblastic CEM cell line. We quantified 4,074 unique SILAC pairs from 1,673 proteins and 89 phosphopeptides were deemed CXCL12-responsive in biological replicates. Several well established CXCL12-responsive phosphosites such as AKT (pS473) and ERK2 (pY204) were confirmed in our study. We also validated two novel CXCL12-responsive phosphosites, stathmin (pS16) and AKT1S1 (pT246) by Western blot. Pathway analysis and comparisons with other phosphoproteomic datasets revealed that genes from CXCL12-responsive phosphosites are enriched for cellular pathways such as T cell activation, epidermal growth factor and mammalian target of rapamycin (mTOR) signaling, pathways which have previously been linked to CXCL12/CXCR4 signaling. Several of the novel CXCL12-responsive phosphoproteins from our study have also been implicated with cellular migration and HIV-1 infection, thus providing an attractive list of potential targets for the development of cancer metastasis and HIV-1 therapeutics and for furthering our understanding of chemokine signaling regulation by reversible phosphorylation.     

MiR-886-3p down regulates CXCL12 (SDF1) expression in human marrow stromal cells

Stromal Derived Factor 1 (SDF1 or CXCL12), is a chemokine known to be critical for the migration of cells in several tissue systems including the homing of the hematopoietic stem cell (HSC) to its niche in the bone marrow. A comparative analysis of miRNA expression profiles of two stromal cell lines, distinguishable by function and by CXCL12 expression (CXCL12+ and CXCL12-), revealed that the CXCL12- cells expressed>40 fold more miR-886-3p than the CXCL12+ cells. Screening studies showed that when miR-886-3p was transfected into the CXCL12+ stromal cells, the expression of CXCL12 was down-regulated by as much as 85% when compared to appropriate controls, and results in the loss of CXCL12-directed chemotaxis. Similar reductions in CXCL12 were obtained with the transfection of miR-886-3p into primary stromal cell cultures. Additional studies showed that miR-886-3p specifically targeted the 3' untranslated region (UTR) of CXCL12 mRNA. These data suggest a role for miRNA in modulating the expression of CXCL12, a gene product with a critical role in hematopoietic regulation.     

The IKKα-dependent NF-κB p52/RelB noncanonical pathway is essential to sustain a CXCL12 autocrine loop in cells migrating in response to HMGB1

HMGB1 is a chromatin architectural protein that is released by dead or damaged cells at sites of tissue injury. Extracellular HMGB1 functions as a proinflammatory cytokine and chemoattractant for immune effector and progenitor cells. Previously, we have shown that the inhibitor of NF-κB kinase (IKK)β- and IKKα-dependent NF-κB signaling pathways are simultaneously required for cell migration to HMGB1. The IKKβ-dependent canonical pathway is needed to maintain expression of receptor for advanced glycation end products, the ubiquitously expressed receptor for HMGB1, but the target of the IKKα non-canonical pathway was not known. In this study, we show that the IKKα-dependent p52/RelB noncanonical pathway is critical to sustain CXCL12/SDF1 production in order for cells to migrate toward HMGB1. Using both mouse bone marrow-derived macrophages and mouse embryo fibroblasts (MEFs), it was observed that neutralization of CXCL12 by a CXCL12 mAb completely eliminated chemotaxis to HMGB1. In addition, the HMGB1 migration defect of IKKα KO and p52 KO cells could be rescued by adding recombinant CXCL12 to cells. Moreover, p52 KO MEFs stably transduced with a GFP retroviral vector that enforces physiologic expression of CXCL12 also showed near normal migration toward HMGB1. Finally, both AMD3100, a specific antagonist of CXCL12's G protein-coupled receptor CXCR4, and an anti-CXCR4 Ab blocked HMGB1 chemotactic responses. These results indicate that HMGB1-CXCL12 interplay drives cell migration toward HMGB1 by engaging receptors of both chemoattractants. This novel requirement for a second receptor-ligand pair enhances our understanding of the molecular mechanisms regulating HMGB1-dependent cell recruitment to sites of tissue injury.     

CXCL12 chemokine up-regulates bone resorption and MMP-9 release by human osteoclasts: CXCL12 levels are increased in synovial and bone tissue of rheumatoid arthritis patients

Chemokines are involved in a number of inflammatory pathologies and some of them show a pivotal role in the modulation of osteoclast development. Therefore, we evaluated the role of CXCL12 chemokine on osteoclast differentiation and function and we analyzed its expression on synovial and bone tissue biopsies from rheumatoid arthritis (RA) patients. Osteoclasts were obtained by 7 days in vitro differentiation with RANKL and M-CSF of CD11b positive cells in the presence or absence of CXCL12. The total number of osteoclast was analyzed by Tartrate-resistant acid phosphatase (TRAP)-staining and bone-resorbing activity was assessed by pit assay. MMP-9 and TIMP-1 release was evaluated by ELISA assay. CXCL12 expression on biopsies from RA patients was analyzed by immunohistochemistry. Osteoclasts obtained in the presence of CXCL12 at 10 nM concentration displayed a highly significant increase in bone-resorbing activity as measured by pit resorption assay, while the total number of mature osteoclasts was not affected. The increased resorption is associated with overexpression of MMP-9. Immunostaining for CXCL12 on synovial and bone tissue biopsies from both rheumatoid arthritis (RA) and osteoarthritis (OA) samples revealed a strong increase in the expression levels under inflammatory conditions. CXCL12 chemokine showed a clear activating role on mature osteoclast by inducing bone-resorbing activity and specific MMP-9 enzymatic release. Moreover, since bone and synovial biopsies from RA patients showed an elevated CXCL12 expression, these findings may provide useful tools for achieving a full elucidation of the complex network that regulates osteoclast function in course of inflammatory diseases.     

Involvement of mTOR in CXCL12 mediated T cell signaling and migration

Background

CXCL12 is a pleiotropic chemokine involved in multiple different processes such as immune regulation, inflammatory responses, and cancer development. CXCL12 is also a potent chemokine involved in chemoattraction of T cells to the site of infection or inflammation. Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that modulates different cellular processes, such as metabolism, nutrient sensing, protein translation, and cell growth. The role of mTOR in CXCL12-mediated resting T cell migration has yet to be elucidated.

Methodology/Principal Findings

Rapamycin, an inhibitor of mTOR, significantly inhibits CXCL12 mediated migration of both primary human resting T cells and human T cell leukemia cell line CEM. p70^S6K1, an effector molecule of mTOR signaling pathway, was knocked down by shRNA in CEM cells using a lentiviral gene transfer system. Using p70^S6K1 knock down cells, we demonstrate the role of mTOR signaling in T cell migration both in vitro and in vivo.

Conclusions

Our data demonstrate a new role for mTOR in CXCL12-induced T cell migration, and enrich the current knowledge regarding the clinical use of rapamycin.     

Downregulation of CXCL12 signaling and altered hematopoietic stem and progenitor cell trafficking in a murine model of acute Anaplasma phagocytophilum infection

Infection with a variety of bacterial pathogens results in hematopoietic stem and progenitor cell (HSPC) mobilization. The mechanism and kinetics of HSPC mobilization during infection are largely unknown. Previously, we found altered HSPC activity in bone marrow, spleen and blood during infection with Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis. We hypothesized that altered CXCL12/CXCR4 signaling, a central pathway for HSPC homing to, and retention within, the bone marrow, plays a role in infection-induced alterations in HSPC number and trafficking. Mice were infected with A. phagocytophilum. Lineage-cKit+ HSPCs were enumerated and proliferation determined. CXCL12 and CXCR4 mRNA were quantified along with CXCL12 protein, and CXCR4 surface, intracellular and total protein expression in HSPCs was determined. Increased bone marrow proliferation of HSPCs began at 2 d post-infection followed by HSPC mobilization and splenic homing. Proliferation of resident HSPCs contributed to increased splenic HSPC numbers. Bone marrow CXCL12 mRNA and protein levels were decreased at 4-8 d post-infection concurrent with HSPC mobilization. CXCR4 protein parameters were decreased in bone marrow HSPCs throughout 2-6 d post-infection. Reduction of CXCL12/CXCR4 signaling simultaneously occurs with HSPC mobilization from bone marrow. Findings suggest that deranged CXCL12/CXCR4 signaling plays a causal role in HSPC mobilization during acute A. phagocytophilum infection.     

CXCL12-Mediated Murine Neural Progenitor Cell Movement Requires PI3Kβ Activation

The migratory route of neural progenitor/precursor cells (NPC) has a central role in central nervous system development. Although the role of the chemokine CXCL12 in NPC migration has been described, the intracellular signaling cascade involved remains largely unclear. Here we studied the molecular mechanisms that promote murine NPC migration in response to CXCL12, in vitro and ex vivo. Migration was highly dependent on signaling by the CXCL12 receptor, CXCR4. Although the JAK/STAT pathway was activated following CXCL12 stimulation of NPC, JAK activity was not necessary for NPC migration in vitro. Whereas CXCL12 activated the PI3K catalytic subunits p110α and p110β in NPC, only p110β participated in CXCL12-mediated NPC migration. Ex vivo experiments using organotypic slice cultures showed that p110β blockade impaired NPC exit from the medial ganglionic eminence. In vivo experiments using in utero electroporation nonetheless showed that p110β is dispensable for radial migration of pyramidal neurons. We conclude that PI3K p110β is activated in NPC in response to CXCL12, and its activity is necessary for immature interneuron migration to the cerebral cortex.     

CXCR7/CXCR4 heterodimer constitutively recruits beta-arrestin to enhance cell migration

G protein-coupled receptor hetero-oligomerization is emerging as an important regulator of ligand-dependent transmembrane signaling, but precisely how receptor heteromers affect receptor pharmacology remains largely unknown. In this study, we have attempted to identify the functional significance of the heteromeric complex between CXCR4 and CXCR7 chemokine receptors. We demonstrate that co-expression of CXCR7 with CXCR4 results in constitutive recruitment of β-arrestin to the CXCR4·CXCR7 complex and simultaneous impairment of G(i)-mediated signaling. CXCR7/CXCR4 co-expression also results in potentiation of CXCL12 (SDF-1)-mediated downstream β-arrestin-dependent cell signaling pathways, including ERK1/2, p38 MAPK, and SAPK as judged from the results of experiments using siRNA knockdown to deplete β-arrestin. Interestingly, CXCR7/CXCR4 co-expression enhances cell migration in response to CXCL12 stimulation. Again, inhibition of β-arrestin using either siRNA knockdown or a dominant negative mutant abrogates the enhanced CXCL12-dependent migration of CXCR4/CXCR7-expressing cells. These results show how CXCR7, which cannot signal directly through G protein-linked pathways, can nevertheless affect cellular signaling networks by forming a heteromeric complex with CXCR4. The CXCR4·CXCR7 heterodimer complex recruits β-arrestin, resulting in preferential activation of β-arrestin-linked signaling pathways over canonical G protein pathways. CXCL12-dependent signaling of CXCR4 and its role in cellular physiology, including cancer metastasis, should be evaluated in the context of potential functional hetero-oligomerization with CXCR7.     

The relevance of the chemokine receptor ACKR3/CXCR7 on CXCL12-mediated effects in cancers with a focus on virus-related cancers

Recent studies have highlighted the importance of understanding the molecular determinants of CXCL12-mediated effects in cancers. Once previously thought to interact exclusively with CXCR4, CXCL12 also binds with high affinity to CXCR7 (recently renamed ACKR3), which belongs to an atypical chemokine receptor family whose members fail to activate G_αi proteins but interact with β-arrestins. In addition to its capacity to control CXCL12 bioavailability, ACKR3 can either enhance or dampen CXCR4-mediated signaling and activity. In light of the most recent findings, we have examined the role of ACKR3 in cancer, including a subset of virus-related cancers.     

Neutrophil mobilization from the bone marrow during polymicrobial sepsis is dependent on CXCL12 signaling

Neutrophils are essential for successful host eradication of bacterial pathogens and for survival to polymicrobial sepsis. During inflammation, the bone marrow provides a large reserve of neutrophils that are released into the peripheral circulation where they traverse to sites of infection. Although neutrophils are essential for survival, few studies have investigated the mechanisms responsible for neutrophil mobilization from the bone marrow during polymicrobial sepsis. Using a cecal ligation and puncture model of polymicrobial sepsis, we demonstrated that neutrophil mobilization from the bone marrow is not dependent on TLR4, MyD88, TRIF, IFNARα/β, or CXCR2 pathway signaling during sepsis. In contrast, we observed that bone marrow CXCL12 mRNA abundance and specific CXCL12 levels are sharply reduced, whereas splenic CXCR4 mRNA and cell surface expression are increased during sepsis. Blocking CXCL12 activity significantly reduced blood neutrophilia by inhibiting bone marrow release of granulocytes during sepsis. However, CXCL12 inhibition had no impact on the expansion of bone marrow neutrophil precursors and hematopoietic progenitors. Bone marrow neutrophil retention by CXCL12 blockade prevented blood neutrophilia, inhibited peritoneal neutrophil accumulation, allowed significant peritoneal bacterial invasion, and increased polymicrobial sepsis mortality. We concluded that changes in the pattern of CXCL12 signaling during sepsis are essential for neutrophil bone marrow mobilization and host survival but have little impact on bone marrow granulopoiesis.