The role of PKC and PKD in CXCL12 and CXCL13 directed malignant melanoma and acute monocytic leukemic cancer cell migration.

Cancer metastasis is the leading cause of cancer related mortality. Chemokine receptors and proteins in their downstream signalling axis represent desirable therapeutic targets for the prevention of metastasis. Despite this, current therapeutics have experienced limited success in clinical trials due to a lack of insight into the downstream signalling pathway of specific chemokine receptor cascades in different tumours. In this study, we investigated the role of protein kinase C (PKC) and protein kinase D (PKD) in CXCL12 and CXCL13 stimulated SK-MEL-28 (malignant melanoma) and THP-1 (acute monocytic leukaemia) cell migration. While PKC and PKD had no active role in CXCL12 or CXCL13 stimulated THP-1 cell migration, PKC and PKD inhibition reduced CXCL12 stimulated migration and caused profound effects upon the cytoskeleton of SK-MEL-28 cells. Furthermore, only PKC and not PKD inhibition reduced CXCL13 stimulated migration in SK-MEL-28 cells however PKC inhibition failed to stimulate any changes to the actin cytoskeleton. These findings indicate that PKC inhibitors would be a useful therapeutic for the prevention of both CXCL12 and CXCL13 stimulated migration and PKD inhibitors for CXCL12 stimulated migration in malignant melanoma.

M1 Macrophages Increase Endothelial Permeability and Enhance p38 Phosphorylation via PPAR-γ/CXCL13-CXCR5 in Sepsis.

PURPOSE: Vascular endothelial hyperpermeability and barrier disruption are involved in the initiation and development of sepsis. M1 macrophages promote inflammation in sepsis by releasing pro-inflammatory cytokines and chemokines. This study was designed to investigate the functional relationships between M1 macrophages and human umbilical vein endothelial cells (HUVECs), as well as the underlying molecular mechanisms. METHODS: HUVECs were co-cultured with THP-1-derived M1 macrophages pretreated with or without rosiglitazone (RSG), a peroxisome proliferator-activated receptor (PPAR)-γ agonist. C-X-C chemokine receptor type (CXCR)5 was knocked down by short hairpin RNA lentivirus. Cecal ligation and puncture were used to induce sepsis in a mouse model. Endothelial permeability was evaluated using transendothelial electrical resistance and fluorescein isothiocyanate (FITC)-dextran assays. RESULTS: Chemokine ligand (CXCL)13 was upregulated in M1 macrophages than M0 macrophages, as well as in the culture medium. In HUVECs co-cultured with M1 macrophages, transendothelial electrical resistance decreased, FITC-dextran flux increased, p38 phosphorylation was strengthened, and the expression of tight junction proteins (zonula occludens protein-1, occludin, and claudin-4) decreased. CXCR5 RNA interference or RSG pretreatment partially reversed these effects. A luciferase reporter assay revealed that CXCL13 was a direct target of PPAR-γ. RSG treatment decreased serum levels of creatinine, blood urea nitrogen, CXCL13, tumor necrosis factor-α, and interleukin-6, downregulated CXCL13 in peritoneal macrophages, and enhanced the survival rate of sepsis mice. CONCLUSION: M1 macrophages induced endothelial hyperpermeability and promoted p38 phosphorylation in sepsis by inhibiting PPAR-γ to increase CXCL13 production. PPAR-γ/CXCL13-CXCR5 signaling could be a promising novel therapeutic target for sepsis.

Study of bone repair mediated by recombination BMP-2/ recombination CXC chemokine Ligand-13-loaded hollow hydroxyapatite microspheres/chitosan composite.

AIMS: The present study aimed to investigate the mechanism of bone repair mediated by recombination BMP-2 (rhBMP-2)/recombination CXC chemokine ligand-13 (rhCXCL13)-loaded hollow hydroxyapatite (HA) microspheres/chitosan (CS) composite. MATERIALS AND METHODS: Firstly, the biological activity of rhBMP-2 and rhCXCL13 released from the complex was investigated. Secondly, the effect of rhBMP-2 sustained release solution on ALP activity and rhCXCL13 sustained release solution on cell migration of rat bone marrow mesenchyme stem cells was tested. Thirdly, osteoblasts differentiation test, X-ray scoring and three-point bending test were performed. Finally, the mRNAs expression of osteogenic marker genes and the protein expression of Runx2 was tested by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting (WB), respectively. KEY FINDINGS: RhBMP-2 could significantly promote the proliferation and differentiation, and RhCXCL13 could promote the migration of rat bone marrow MSCs. Detection of ALP activity and calcium salt deposition showed that rhBMP-2 and rhCXCL13 could significantly improve the biological activity and promote cell differentiation ability. X-ray scoring of radius and flexural strength test showed that rhBMP-2 and rhCXCL13 could promote bone healing and improve the bending resistance of bone tissue. The in vitro molecular experiments including RT-PCR and WB further demonstrated the roles of rhBMP-2 and rhCXCL13 in bone formation and bone repair. SIGNIFICANCE: Our results indicated that the hollow HA microspheres/CS composite could be effective as a delivery vehicle for rhBMP-2 and rhCXCL13 in bone regeneration and bone repair. In this process, rhBMP-2 may promote bone regeneration by regulating bone marrow MSCs cells recruited by rhCXCL13.

Induction of Follicular-Like CXCR5+ CD8+ T Cells by TGF-ß1/IL-23 Is Limited During HIV Infection.

Follicular CD4+ T cells are the main HIV reservoirs due to, among other factors, the low frequency of CD8+ T cells in lymphoid follicles. Follicular CXCR5+ CD8+ T cells are associated with HIV control, but their differentiation conditions are yet undefined. In this study, we explored the in vitro effect of transforming growth factor (TGF)-ß1, interleukin (IL)-12, and IL-23 on the induction of CXCR5, the follicle homing receptor, in human circulating CD8+ T cells from seronegative, and treated HIV-infected individuals. The combination of TGF-ß1 plus IL-23 induced the highest expression of CXCR5 in purified CD8+ T cells. These CXCR5+ CD8+ T cells also expressed a transcriptional and phenotypic profile similar to that of follicular CD4+ T cells, such as the upregulation of BCL6, inducible costimulator and CD40L, and downregulation of PRDM1. These cells responded in vitro to CXCL13 and had low expression of CCR7. In addition, after polyclonal stimulation, they produced IL-21, interferon-γ, and de novo perforin. However, in comparison with seronegative individuals, CD8+ T cells from HIV-infected patients had a lower response to TGF-ß1/IL-23, a defect that was restored with the blockade of the programmed cell death 1 inhibitory receptor. Thus, TGF-ß1 plus IL-23 induce follicular-like CXCR5+ CD8+ T cells in seronegative individuals, but in HIV-infected patients there is a limited response which could impair the generation of this cell population.

MicroRNA-155 Suppresses Mesangial Cell Proliferation and TGF-ß1 Production via Inhibiting CXCR5-ERK Signaling Pathway in Lupus Nephritis.

Increasing evidence shows miR-155 plays an important role in regulating inflammatory processes in systemic lupus erythematosus (SLE), especially in lupus nephritis (LN). Because the chemokine CXCL13 is implicated in the pathogenesis of LN, here we examined whether miR-155 can modulate the activity of CXCL13 or its receptor CXCR5. We determined the expression of CXCL13 in normal and MRL/lpr mice and found elevated levels of CXCL13 in the kidneys of MRL/lpr mice compared with normal kidneys. Besides, CXCL13 expression was mainly detected in the glomerulus, specifically to mesangial areas. We then transfected a miR-155 mimic in human renal mesangial cells (HRMCs) to overexpress miR-155 and detected decreased protein levels of CXCR5 by western blot analysis. Transfection of the miR-155 mimic into CXCL13-treated HRMCs resulted in a significantly reduced proliferation rate of HRMCs as measured by the cell-counting assay and flow cytometry. Moreover, increased intracellular miR-155 also led to decreased phosphorylation of ERK and TGF-ß1 production. Together, these results revealed that miR-155 may play a role in the pathogenesis of LN.

Ageing adversely affects the migration and function of marginal zone B cells.

Marginal zone (MZ) B cells are positioned within the spleen to capture blood-borne antigen and immune complexes and deliver them to follicular dendritic cells in the B-cell follicles. We show that within the spleens of aged mice antigen capture by MZ B cells, and their ability to shuttle between the follicle and MZ, were impaired. The ability of aged MZ B cells to migrate towards the MZ chemoattractant sphingosine-1-phosphate was increased, suggesting that aged MZ B cells had a greater propensity to be retained within the MZ. An extrinsic impairment in aged B-cell migration towards the MZ was demonstrated using bone marrow chimeras. The follicular shuttling of MZ B cells derived from either young or aged bone marrow was similarly reduced in aged recipient spleens, showing that ageing effects on splenic stromal cells were responsible for the impaired follicular shuttling of MZ B cells. MZ B cells rapidly mount T-cell-independent (TI) antibody-responses to microbial polysaccharide antigen. In aged mice the ability to produce immunoglobulins in response to the TI type 1 antigen TNP-LPS was impaired. These ageing-related changes to the MZ and MZ B cells have implications for the clearance of blood-borne pathogens. Indeed elderly people have increased susceptibility to Streptococcus pneumoniae, a TI antigen, and decreased responses to vaccination. A thorough analysis of the mechanisms that underpin the ageing-related decline in the status of the MZ and MZ B cells will help the design of novel treatments to improve immunity in the elderly.

Long non-coding RNA MEG3 inhibits microRNA-125a-5p expression and induces immune imbalance of Treg/Th17 in immune thrombocytopenic purpura.

BACKGROUND: The imbalance of Treg/Th17 cells is an important pathogenic factor for immune thrombocytopenic purpura (ITP). We previously reported miR-125a-5p targeted CXCL13 and participated in the process of ITP. In the present study, the role of miR-125a-5p in regulating Treg/Th17 ratio and its potential molecular mechanism were investigated. METHOD: A total of 30 adults with ITP and 30 healthy subjects were included. MEG3 expression in peripheral blood derived CD4+ T cells from ITP patients and healthy subjects were detected by real-time PCR. In vitro experiments, the effects of inhibiting or overexpressing MEG3 on the expression of miR-125a-5p, Foxp3 and ROTγt in CD4+ T cells were investigated. RESULTS: MEG3 expression was increased in CD4+ T cells of patients with ITP. Dexamethasone decreased MEG3 expression level of CD4+ T cells in vitro. MEG3 directly interacted with miR-125a-5p and MEG3 overexpression inhibited miR-125a-5p expression in CD4+ T cells exposed to dexamethasone. MEG3 down-regulation or miR-125a-5p overexpression promoted Foxp3 expression and inhibited RORγt expression. CONCLUSION: MEG3 interacted with miR-125a-5p and inhibited its expression, and MEG3/miR-125a-5p contributed to induce immune imbalance of Treg/Th17 in ITP.

CXCL13 inhibits microRNA-23a through PI3K/AKT signaling pathway in adipose tissue derived-mesenchymal stem cells.

OBJECTIVES: Adipose tissue derived-mesenchymal stem cells (AMSCs) are one of the most widely used MSCs in the cell therapy for regenerative medicine. In the current study, the role of CXCL13 in AMSCs and its potential signaling pathway were investigated. METHODS: AMSCs were isolated from adipose tissue of healthy subjects. After administrating the cells with CXCL13, the expression levels of miR-23a and runt-related transcription factor 2 (Runx2) were assessed by real-time PCR and western blot. The alterations of phosphoinositide-3 kinase (PI3K)/Akt, stress-activated protein kinase (SAPK)/c-jun kinase (JNK), and extracellular-signal-regulated kinase (ERK1/2) pathways were also evaluated. RESULTS: CXCL13 down-regulated miR-23a and up-regulated Runx2 expression in AMSCs. The inhibitor specific for PI3K/AKT, but not SAPK/JNK and ERK ERK1/2, reversed the effects of CXCL13 on miR-23a and Runx2 expression. CONCLUSION: CXCL13 inhibits miR-23a expression through modulating PI3K/AKT pathway in AMSCs.

Inflammation without neuronal death triggers striatal neurogenesis comparable to stroke.

Ischemic stroke triggers neurogenesis from neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ) and migration of newly formed neuroblasts toward the damaged striatum where they differentiate to mature neurons. Whether it is the injury per se or the associated inflammation that gives rise to this endogenous neurogenic response is unknown. Here we showed that inflammation without corresponding neuronal loss caused by intrastriatal lipopolysaccharide (LPS) injection leads to striatal neurogenesis in rats comparable to that after a 30 min middle cerebral artery occlusion, as characterized by striatal DCX+ neuroblast recruitment and mature NeuN+/BrdU+ neuron formation. Using global gene expression analysis, changes in several factors that could potentially regulate striatal neurogenesis were identified in microglia sorted from SVZ and striatum of LPS-injected and stroke-subjected rats. Among the upregulated factors, one chemokine, CXCL13, was found to promote neuroblast migration from neonatal mouse SVZ explants in vitro. However, neuroblast migration to the striatum was not affected in constitutive CXCL13 receptor CXCR5(-/-) mice subjected to stroke. Infarct volume and pro-inflammatory M1 microglia/macrophage density were increased in CXCR5(-/-) mice, suggesting that microglia-derived CXCL13, acting through CXCR5, might be involved in neuroprotection following stroke. Our findings raise the possibility that the inflammation accompanying an ischemic insult is the major inducer of striatal neurogenesis after stroke.

The Effect of C-X-C Motif Chemokine 13 on Hepatocellular Carcinoma Associates with Wnt Signaling.

OBJECTS: To investigate the effect of CXCL13 (C-X-C motif chemokine 13) on hepatocellular carcinoma and clarify the potential mechanisms. METHODS: 32 patients with hepatocellular carcinoma and 12 healthy controls were recruited for analyzing the expression of CXCL13 by RT-PCR (reverse transcription-polymerase chain reaction). ELISA (enzyme-linked immune-sorbent assay) was used to test the concentration of serum CXCL13. The interaction between CXCL13 and Wnt signaling was analyzed by western blot. In vitro PBMCs cultured with HepG2 supernatant, the levels of IL-12, IL4, IL-6, and IL-17, and four IgG subclasses were detected by ELISA. RESULTS: The rate of high expression CXCL13 was 63.4% in advanced HCC patients, and the serum CXCL13 was also at a high level in stage IV HCC patients. Meanwhile CXCL13 level was positively correlated with serum ALT (Alanine Transaminase) and AST (Aspartate Aminotransferase). CXCL13 and Wnt/ß-catenin signaling shared a positive feedback loop. Furthermore, CXCL13 could obviously promote the expressions of IL-12 and IL-17, and induce IgG4 secreted by B cells. CONCLUSIONS: The effect of CXCL13 on promoting liver cancer is related to the activation of Wnt/ß-catenin pathway and the facilitation of IL-12, IL-17 and IgG4. CXCL13 plays an important role in the progression of HCC, and it may act as a potential target for the diagnosis and treatment of HCC.

CXCL13 promotes the effect of bone marrow mesenchymal stem cells (MSCs) on tendon-bone healing in rats and in C3HIOT1/2 cells.

OBJECTIVES: Mesenchymal stem cells (MSCs) are potential effective therapy for tissue repair and bone regeneration. In present study, the effects of CXC chemokine ligand-13 (CXCL13) were evaluated on tendon-bone healing of rats. METHODS: Tendon bone healing of the rat model was established and biomechanical testing was performed at 2, 4, 8 weeks after surgery. Murine mesenchymal cell line (C3HIOT1/2 cells) was cultured. The expression of miRNA-23a was detected by real-time PCR. The protein expression of ERK1/2, JNK and p38 was detected by western blotting. MiR-23a mimic and inhibitor were used to overexpress or silence the expression of miR-23a. RESULTS: MSCs significantly elevated the levels of ultimate load to failure, stiffness and stress in specimens of rats, the effects of which were enhanced by CXCL13. The expression of miR-23a was down-regulated and the protein of ERK1/2 level was up-regulated by CXCL13 treatment in both in vivo and in vitro experiments. ERK1/2 expression was elevated by overexpression of miR-23a and reduced by miR-23a inhibitor. CONCLUSIONS: These findings revealed that CXCL13 promoted the tendon-bone healing in rats with MSCs treatment, and implied that the activation of ERK1/2 via miR-23a was involved in the process of MSCs treated bone regeneration.

WIP regulates persistence of cell migration and ruffle formation in both mesenchymal and amoeboid modes of motility.

The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCR) regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein) family. WIP (WASP Interacting Protein) is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor) but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP) and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast) and amoeboid (B lymphocytes) motility.

Differential G protein subunit expression by prostate cancer cells and their interaction with CXCR5.

BACKGROUND: Prostate cancer (PCa) cell lines and tissues differentially express CXCR5, which positively correlate with PCa progression, and mediate PCa cell migration and invasion following interaction with CXCL13. However, the differential expression of G protein α, ß, and γ subunits by PCa cell lines and the precise combination of these proteins with CXCR5 has not been elucidated. METHODS: We examined differences in G protein expression of normal prostate (RWPE-1) and PCa cell lines (LNCaP, C4-2B, and PC3) by western blot analysis. Further, we immunoprecipitated CXCR5 with different G protein subunits, and CXCR4, following CXCL13 stimulation. To investigate constitutive coupling of CXCR5 with CXCR4 and PAR-1 we performed invasion assay in PCa cells transfected with Gαq/i2 or Gα13 siRNA, following CXCL13 treatment. We also investigated Rac and RhoA activity by G-LISA activation assay in PCa cells following CXCL13/thrombin stimulation. RESULT: Of the 22 G proteins studied, Gαi1-3, Gß1-4, Gγ5, Gγ7, and Gγ10 were expressed by both normal and PCa cell lines. Gαs was moderately expressed in C4-2B and PC3 cell lines, Gαq/11 was only present in RWPE-1 and LNCaP cell lines, while Gα12 and Gα13 were expressed in C4-2B and PC3 cell lines. Gγ9 was expressed only in PCa cell lines. Gα16, Gß5, Gγ1-4, and Gγ13 were not detected in any of the cell lines studied. Surprisingly, CXCR4 co-immunoprecipitated with CXCR5 in PCa cell lines irrespective of CXCL13 treatment. We also identified specific G protein isoforms coupled to CXCR5 in its resting and active states. Gαq/11/Gß3/Gγ9 in LNCaP and Gαi2/Gß3/Gγ9 in C4-2B and PC3 cell lines, were coupled to CXCR5 and disassociated following CXCL13 stimulation. Interestingly, Gα13 co-immunoprecipitated with CXCR5 in CXCL13-treated, but not in untreated PCa cell lines. Inhibition of Gαq/i2 significantly decreased the ability of cells to invade, whereas silencing Gα13 did not affect CXCL13-dependent cell invasion. Finally, CXCL13 treatment significantly increased Rac activity in Gαq/i2 dependent manner, but not RhoA activity, in PCa cell lines. CONCLUSIONS: These findings offer insight into molecular mechanisms of PCa progression and can help to design some therapeutic strategies involving CXCR5 and/or CXCL13 blockade and specific G protein inhibition to abrogate PCa metastasis.

RhoH is critical for cell-microenvironment interactions in chronic lymphocytic leukemia in mice and humans.

Trafficking of B-cell chronic lymphocytic leukemia (CLL) cells to the bone marrow and interaction with supporting stromal cells mediates important survival and proliferation signals. Previous studies have demonstrated that deletion of Rhoh led to a delayed disease onset in a murine model of CLL. Here we assessed the impact of RhoH on homing, migration, and cell-contact dependent interactions of CLL cells. Rhoh(-/-) CLL cells exhibited reduced marrow homing and subsequent engraftment. In vitro migration toward the chemokines CXCL12 and CXCL13 and cell-cell interactions between Rhoh(-/-) CLL cells and the supporting microenvironment was reduced. In the absence of RhoH the distribution of phosphorylated focal adhesion kinase, a protein known to coordinate activation of the Rho GTPases RhoA and Rac, appeared less polarized in chemokine-stimulated Rhoh(-/-) CLL cells, and activation and localization of RhoA and Rac was dysregulated leading to defective integrin function. These findings in the Rhoh(-/-) CLL cells were subsequently demonstrated to closely resemble changes in GTPase activation observed in human CLL samples after in vitro and in vivo treatment with lenalidomide, an agent with known influence on microenvironment protection, and suggest that RhoH plays a critical role in prosurvival CLL cell-cell and cell-microenvironment interactions with this agent.

Small molecule inhibitors of the Pyk2 and FAK kinases modulate chemoattractant-induced migration, adhesion and Akt activation in follicular and marginal zone B cells.

B-lymphocytes produce protective antibodies but also contribute to autoimmunity. In particular, marginal zone (MZ) B cells recognize both microbial components and self-antigens. B cell trafficking is critical for B cell activation and is controlled by chemoattactants such as CXCL13 and sphingosine 1-phosphate (S1P). The related tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2) regulate cell migration and adhesion but their roles in B cells are not fully understood. Using a novel Pyk2-selective inhibitor described herein (PF-719), as well as a FAK-selective inhibitor, we show that both Pyk2 and FAK are important for CXCL13- and S1P-induced migration of B-2 cells and MZ B cells. In contrast, LFA-1-mediated adhesion required only Pyk2 whereas activation of the Akt pro-survival kinase required FAK but not Pyk2. Thus Pyk2 and FAK mediate critical processes in B cells and these inhibitors can be used to further elucidate their functions in B cells.

LPS-induced migration of peritoneal B-1 cells is associated with upregulation of CXCR4 and increased migratory sensitivity to CXCL12.

B-1 cells, which constitute a predominant lymphocyte subset in serosal cavities and produce most of natural antibodies, are subdivided into the CD5(+) B-1a and CD5(-) B-1b cell subpopulations, but the differential roles of B-1a and B-1b cells are not well understood. We report that B-1a cells preferentially migrate out of the peritoneal cavity and upregulate the expression of CXCR4 with heightened sensitivity to CXCL12 and CXCL13 upon LPS treatment compared to B-1b and B-2 cells. Whereas B-1a cells were slightly more abundant than B-1b and B-2 cells in the homeostatic condition, the number of B-1a cells preferentially decreased 48 hr after LPS treatment. The decrease in the peritoneal B-1a cell number was accompanied with increased migration of B-1a cells toward CXCL-12 and CXCL-13 in in vitro transmigration assay using peritoneal B cells from LPS treated mice. The expression level of CXCR4, but not of CXCR5, was also more prominently increased in B-1a cells upon LPS stimulation. LPS-stimulated B-1a cells did not accumulate in omental milky spots in contrast to B-2 cells. These results suggest that B-1a cells actively migrate out of the peritoneal cavity through the regulation of the migratory responsiveness to chemokines and actively participate in systemic immune responses.

Mice deficient in MIM expression are predisposed to lymphomagenesis.

Missing in metastasis (MIM) is a member of newly emerged inverse Bin-Amphiphysin-Rvs (BAR) domain protein family and a putative metastasis suppressor. Although reduced MIM expression has been associated with bladder, breast and gastric cancers, evidence for the role of MIM in tumor progression remains scarce and controversial. Herein we characterized a MIM knockout mouse strain and observed that MIM-deficient mice often developed enlarged spleens. Autopsy and histological analysis revealed that nearly 78% of MIM(-/-) mice developed tumors with features similar to diffuse large B lymphoma during a period from 1 to 2 years. MIM(-/-) mice also exhibited abnormal distribution of B cells in lymphoid organs with decrease in the spleen but increase in the bone marrow and the peripheral blood. Furthermore, the bone marrow of MIM(-/-) mice contained a higher percentage of pre-B2 cells but fewer immature B-cells than wild-type mice. In response to CXCL13, a B-cell chemokine released from splenic stromal cells, MIM-deficient B-cells did not undergo chemotaxis or morphological changes in response to the chemokine and also did not internalize CXCR5, the receptor of CXCL13. Microarray analyses demonstrated that MIM is the only member of the I-BAR domain family that was highly expressed in human B cells. However, low or absent MIM expression was common in either primary B-cell malignancies or established B-cell acute lymphocytic leukemia or lymphomas. Thus, our data demonstrate for the first time an important role for MIM in B-cell development and suggest that predisposition of MIM-null mice to lymphomagenesis may involve aberrant interactions between B lineage cells and the lymphoid microenvironment.

On sample size of the kruskal-wallis test with application to a mouse peritoneal cavity study.

As the nonparametric generalization of the one-way analysis of variance model, the Kruskal-Wallis test applies when the goal is to test the difference between multiple samples and the underlying population distributions are nonnormal or unknown. Although the Kruskal-Wallis test has been widely used for data analysis, power and sample size methods for this test have been investigated to a much lesser extent. This article proposes new power and sample size calculation methods for the Kruskal-Wallis test based on the pilot study in either a completely nonparametric model or a semiparametric location model. No assumption is made on the shape of the underlying population distributions. Simulation results show that, in terms of sample size calculation for the Kruskal-Wallis test, the proposed methods are more reliable and preferable to some more traditional methods. A mouse peritoneal cavity study is used to demonstrate the application of the methods.

PI3Kp110-, Src-, FAK-dependent and DOCK2-independent migration and invasion of CXCL13-stimulated prostate cancer cells.

BACKGROUND: Most prostate cancer (PCa)-related deaths are due to metastasis, which is mediated in part by chemokine receptor and corresponding ligand interaction. We have previously shown that PCa tissue and cell lines express high levels of the chemokine receptor CXCR5, than compared to their normal counterparts, and interaction of CXCR5 with its specific ligand (CXCL13) promoted PCa cell invasion, migration, and differential matrix metalloproteinase (MMP) expression. This study dissects some of the molecular mechanisms following CXCL13-CXCR5 interaction that mediate PCa cell migration and invasion. RESULTS: Using Western blot analysis, kinase-specific cell-based ELISAs, and migration and invasion assays, we show that PCa cell lines differentially express phosphoinositide-3 kinase (PI3K) catalytic subunit isoforms and dedicator of cytokinesis 2 (DOCK2). Specifically, we show that PC3 and normal prostatic epithelial (RWPE-1), but not LNCaP cell lines expressed DOCK2, while RWPE, PC3, and LNCaP cell lines expressed PI3K-p110alpha and -p110beta. Moreover, PC3 selectively expressed PI3K-p110gamma, but LNCaP and RWPE cell lines expressed PI3Kp110delta. CXCL13 caused CXCR5-dependent activation of the PI3Kp85alpha in LNCaP cells, and p85alpha as well as -p101 in PC3 cells. CXCL13-CXCR5 interaction regulated LNCaP and PC3 cell migration and invasion through extracellular signal-regulated kinase 1/2 (ERK1/2) activation that was primarily dependent on the PI3Kp110 isoform(s), Src, and focal adhesion kinase (FAK), but not DOCK2. CONCLUSIONS: While additional studies will be needed to determine the PI3K-independent (i.e., DOCK2-mediated) and -dependent events that dictate PCa cell responsiveness to CXCL13, these data provide evidence of the existence of cell type- and stimulus-specific signaling events that support migration and invasion of PCa cells.

A human CXCL13-induced actin polymerization assay measured by fluorescence plate reader.

The chemokine receptor CXCR5 is predominantly expressed on mature B cells and follicular T-helper cells. CXCR5 and its ligand CXCL13 participate in ectopic germinal center formation at the inflammatory sites of multiple immune diseases such as rheumatoid arthritis, multiple sclerosis, and Sjogren's syndrome. Therefore, disrupting CXCL13-induced chemotaxis may be a fruitful approach for developing therapeutics in treating these diseases. Cells undergo cytoskeletal rearrangement prior to chemotaxis, and therefore actin polymerization can be used as a surrogate readout more proximal to chemokine receptor activation than chemotaxis. Conventionally, actin polymerization is measured by fluorescence microscopy or flow cytometry, which are either of low throughput or in need of special instruments. We developed a 96-well actin polymerization assay that can process 1,000 to 1,500 samples a day. This assay uses a standard laboratory fluorescence microplate reader as the detection instrument and was optimized for various experimental conditions such as cell density, actin filament staining reagent, staining buffer, and cell culture conditions. We demonstrate that this actin polymerization assay in 96-well format exhibits the expected pharmacology for human CXCR5 and is suitable as a primary functional assay to screen neutralizing scFv in crude bacterial peri-preps and a secondary assay for small compound collections.