Functional CXCR4 Research Articles

CXCL12 enhances human neural progenitor cell survival through a CXCR7- and CXCR4-mediated endocytotic signaling pathway.

Chemokine CXCL12 is widely expressed in the central nervous system and essential for the proper functioning of human neural progenitor cells (hNPCs). Although CXCL12 is known to function through its receptor CXCR4, recent data have suggested that CXCL12 binds to chemokine receptor CXCR7 with higher affinity than to CXCR4. However, little is known about the function of CXCR7 in hNPCs. Using a primary hNPC culture system, we demonstrated that CXCL12 promotes hNPC survival in the events of camptothecin-induced apoptosis or growth factor deprivation, and that this effect requires both CXCR7 and CXCR4. Through fluorescence-activated cell sorting analysis and immunocytochemistry, we determined that CXCR7 is mainly localized in the early endosome, while CXCR4 is more broadly expressed at the cell surface and on both early and recycling endosomes. Furthermore, we found that endocytosis is required for the prosurvival function of CXCL12. Using dual-color total internal reflection fluorescence microscopy and immunoprecipitation, we demonstrated that CXCR7 quickly trafficks to plasma membrane in mediating CXCL12 endocytosis and colocalizes with CXCR4 after CXCL12 treatment. Investigating the molecular mechanisms, we found that ERK1/2 endocytotic signaling pathway is essential for hNPC survival upon apoptotic challenges. Consistent with these findings, a significantly higher number of apoptotic NPCs were found in the developing brain of CXCR7 knockout mice. In conclusion, CXCL12 protects hNPCs from apoptotic challenges through CXCR7- and CXCR4-mediated endocytotic signaling. Since survival of hNPCs is important for neurogenesis, CXCR7 may become a new therapeutic target to properly regulate critical processes of brain development.

Migration of cardiomyocytes is essential for heart regeneration in zebrafish

Adult zebrafish possess a significant ability to regenerate injured heart tissue through proliferation of pre-existing cardiomyocytes, which contrasts with the inability of mammals to do so after the immediate postnatal period. Zebrafish therefore provide a model system in which to study how an injured heart can be repaired. However, it remains unknown what important processes cardiomyocytes are involved in other than partial de-differentiation and proliferation. Here we show that migration of cardiomyocytes to the injury site is essential for heart regeneration. Ventricular amputation induced expression of cxcl12a and cxcr4b, genes encoding a chemokine ligand and its receptor. We found that cxcl12a was expressed in the epicardial tissue and that Cxcr4 was expressed in cardiomyocytes. We show that pharmacological blocking of Cxcr4 function as well as genetic loss of cxcr4b function causes failure to regenerate the heart after ventricular resection. Cardiomyocyte proliferation was not affected but a large portion of proliferating cardiomyocytes remained localized outside the injury site. A photoconvertible fluorescent reporter-based cardiomyocyte-tracing assay demonstrates that cardiomyocytes migrated into the injury site in control hearts but that migration was inhibited in the Cxcr4-blocked hearts. By contrast, the epicardial cells and vascular endothelial cells were not affected by blocking Cxcr4 function. Our data show that the migration of cardiomyocytes into the injury site is regulated independently of proliferation, and that coordination of both processes is necessary for heart regeneration.

Grk2 is an essential regulator of CXCR7 signalling in astrocytes.

We previously demonstrated that in astrocytes, SDF-1/CXCL12 exclusively signals through CXCR7 despite the additional presence of the alternate SDF-1/CXCL12 receptor, CXCR4. In addition, we provided evidence that astrocytic CXCR7-signalling involves a G protein-dependent mechanism. This is insofar remarkable as in all other cell types studied to date, CXCR7 either acts as a scavenger chemokine receptor, a modulator of CXCR4, or a non-classical chemokine receptor, signalling through ß-arrestin. To begin to unravel the molecular framework impinging the selective function of CXCR7 on a given cell type, we have now analysed the role of G protein-coupled receptor kinases (Grks) in astrocytic CXCR7 signalling. We demonstrate that Grk2 mediates signalling of SDF-1/CXCL12-bound CXCR7 as suggested by the finding that SDF-1/CXCL12-induced activation of Erk1/2 and Akt is abrogated following RNAi-mediated inhibition of Grk2, but not of Grk3, Grk5, or Grk6. We further unravel that Grk2 additionally controls signalling of SDF-1/CXCL12-bound CXCR7 in astrocytes by mediating internalization and subsequent silencing of CXCR7. Finally, we demonstrate that Grk2 is likewise expressed by microglial cells and Schwann cells, cell types in which CXCR7 does not act as a classical chemokine receptor. In conclusion, our findings establish that Grk2 tightly controls CXCR7 signalling in astrocytes, but does not imprint the cell type-specific function of this chemokine receptor.

The role of the CXCR4/CXCL12 axis and its clinical implications in gastric cancer.

Gastric cancer is the second leading cause of cancer deaths worldwide. Despite the extensive body of research on gastric cancer, the prognosis of patients with advanced gastric cancer remains poor, and therapy for advanced gastric cancer relies largely on cytotoxic chemotherapy. Therefore, identifying the distinct molecular pathways underlying disease progression and treatment resistance may lead to novel therapeutic approaches, as well as improve the quality of life and survival of patients. The chemokine CXCL12 and its receptor CXCR4 are now known to play an important role in cancer development and progression. Here, we review the expression and function of CXCR4 and CXCL12, as well as their clinical relevance in gastric cancer. We also cover the current molecular mechanism, specifically the cell-signaling pathway, by which gastric cancer progresses through the CXCR4/CXCL12 axis, and discuss the potential of that axis as a therapeutic target in the treatment of gastric cancer.

Transcriptional Network Analysis Identifies BACH1 as a Master Regulator of Breast Cancer Bone Metastasis

The application of functional genomic analysis of breast cancer metastasis has led to the identification of a growing number of organ-specific metastasis genes, which often function in concert to facilitate different steps of the metastatic cascade. However, the gene regulatory network that controls the expression of these metastasis genes remains largely unknown. Here, we demonstrate a computational approach for the deconvolution of transcriptional networks to discover master regulators of breast cancer bone metastasis. Several known regulators of breast cancer bone metastasis such as Smad4 and HIF1 were identified in our analysis. Experimental validation of the networks revealed BACH1, a basic leucine zipper transcription factor, as the common regulator of several functional metastasis genes, including MMP1 and CXCR4. Ectopic expression of BACH1 enhanced the malignance of breast cancer cells, and conversely, BACH1 knockdown significantly reduced bone metastasis. The expression of BACH1 and its target genes was linked to the higher risk of breast cancer recurrence in patients. This study established BACH1 as the master regulator of breast cancer bone metastasis and provided a paradigm to identify molecular determinants in complex pathological processes.

Chemokine CXCL12 and its receptors in the developing central nervous system: Emerging themes and future perspectives

Homeostatic chemokine CXCL12 (also known as SDF-1) and its receptor CXCR4 are indispensable for the normal development of the nervous system. This chemokine system plays a plethora of functions in numerous neural developmental processes, from which the underlying molecular and cellular mechanisms are beginning to be unravelled. Recent identification of CXCR7 as a second receptor for CXCL12 provides opportunities to gain deeper insights into how CXCL12 operates in the nervous system. Here, we review the diverse roles of CXCL12 in the developing central nervous system, summarize the recent progress in uncovering CXCR7 functions, and discuss the emerging common themes from these works and future perspectives.

Erratum to “Ferritin in Adult-Onset Still’s Disease: Just a Useful Innocent Bystander?”

There is an error in the citations of our previous paper as reference [51] G. Zandman-Goddard, M. Blank, P. Langevitz et al., “Antiserum amyloid component P antibodies in patients with systemic lupus erythematosus correlate with disease activity,” Annals of the Rheumatic Diseases, vol. 64, no. 12, pp. 1698–1702, 2005 should be replaced by the following references: [1] Li, X., et al., “Epidermal Growth Factor-Ferritin H-Chain Protein Nanoparticles for Tumor Active Targeting,” Small, 2012; [2] Jezequel, P., et al., “Validation of tumor-associated macrophage ferritin light chain as a prognostic biomarker in node-negative breast cancer tumors: A multicentric 2004 national PHRC study,” International Journal of Cancer, 2012. 131(2): p. 426–37. [3] Pitcher, J., et al., “Disruption of neuronal CXCR4 function by opioids: preliminary evidence of ferritin heavy chain as a potential etiological agent in neuroAIDS,” Journal of Neuroimmunology, 2010. 224(1-2): p. 66–71. [4] Rosen, H. R., “Placental isoferritin-associated p43 in pregnancy and breast cancer: minireview,” Neoplasma, 1996. 43(6): p. 357–62.

Expression and function of CXCR4 in human salivary gland cancers

Salivary gland cancers (SGCs) frequently metastasize to cervical lymph nodes and distant organs. Currently, the mechanisms responsible for the metastatic behavior of SGC cells are not fully understood. We previously demonstrated that the stromal cell-derived factor-1 (SDF-1; also known as CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral squamous cell carcinoma. In the present study, we investigated the role of CXCR4 in the metastatic behavior of SGCs. We examined the expression of CXCR4 mRNA and protein in human SGC cell lines by quantitative RT-PCR and western blotting, respectively. The expression of CXCR4 mRNA and protein were frequently upregulated in 5 out of 6 SGC cell lines. Functional CXCR4 expression was demonstrated by the ability of these SGC cell lines to migrate toward an SDF-1 gradient. SDF-1 rapidly activated extracellular signal-regulated kinase (ERK)1/2 in SGC cell lines. Immunohistochemical analysis revealed that CXCR4 protein expression was detected in either the nucleus or cytoplasm of cancer cells in 16 out of 20 tissues of adenoid cystic carcinoma (ACC) and in 4 out of 6 tissues of mucoepidermoid carcinoma, which are representative of SGC. Furthermore, ACC cell lines exhibited dramatic metastasis to the lung following intravenous inoculation, whereas AMD3100, a CXCR4 antagonist, significantly inhibited lung metastasis of the cells, ameliorated body weight loss and improved the survival rate of tumor-bearing nude mice. These results indicate that CXCR4 expression contributes to the metastatic potential of SGCs.

The CXCR3(+)CD56Bright Phenotype Characterizes a Distinct NK Cell Subset with Anti-Fibrotic Potential That Shows Dys-Regulated Activity in Hepatitis C

BackgroundIn mouse models, natural killer (NK) cells have been shown to exert anti-fibrotic activity via killing of activated hepatic stellate cells (HSC). Chemokines and chemokine receptors critically modulate hepatic recruitment of NK cells. In hepatitis C, the chemokine receptor CXCR3 and its ligands have been shown to be associated with stage of fibrosis suggesting a role of these chemokines in HCV associated liver damage by yet incompletely understood mechanisms. Here, we analyzed phenotype and function of CXCR3 expressing NK cells in chronic hepatitis C.MethodsCirculating NK cells from HCV-infected patients (n = 57) and healthy controls (n = 27) were analyzed with respect to CXCR3 and co-expression of different maturation markers. Degranulation and interferon-γ secretion of CXCR3(+) and CXCR3(−) NK cell subsets were studied after co-incubation with primary human hepatic stellate cells (HSC). In addition, intra-hepatic frequency of CXCR3(+) NK cells was correlated with stage of liver fibrosis (n = 15).ResultsWe show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. In healthy controls CXCR3(+)CD56Bright NK cells displayed strongest activity against HSC. Chronic hepatitis C was associated with a significantly increased frequency of CXCR3(+)CD56Bright NK cells which showed impaired degranulation and impaired IFN-γ secretion in response to HSC. Of note, we observed intra-hepatic accumulation of this NK cell subset in advanced stages of liver fibrosis.ConclusionWe show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. Intra-hepatic accumulation of the functionally impaired CXCR3(+)CD56Bright NK cell subset might be involved in HCV-induced liver fibrosis.

Expression and function of the SDF-1 chemokine receptors CXCR4 and CXCR7 during mouse limb muscle development and regeneration

The chemokine, SDF-1/CXCL12, and its receptor, CXCR4, have been implied to play major roles during limb myogenesis. This concept was recently challenged by the identification of CXCR7 as an alternative SDF-1 receptor, which can either act as a scavenger receptor, a modulator of CXCR4, or an active chemokine receptor. We have now re-examined this issue by determining whether SDF-1 would signal to C2C12 myoblasts and subsequently influence their differentiation via CXCR4 and/or CXCR7. In addition, we have analyzed CXCR7, CXCR4, and SDF-1 expression in developing and injured mouse limb muscles. We demonstrate that in undifferentiated C2C12 cells, SDF-1-dependent cell signaling and resulting inhibitory effects on myogenic differentiation are entirely mediated by CXCR4. We further demonstrate that CXCR7 expression increases in differentiating C2C12 cells, which in turn abrogates CXCR4 signaling. Moreover, consistent with the view that CXCR4 and CXCR7 control limb myogenesis in vivo by similar mechanisms, we found that CXCR4 expression is the highest in late embryonic hindlimb muscles and drops shortly after birth when secondary muscle growth terminates. Vice versa, CXCR7 expression increased perinatally and persisted into adult life. Finally, underscoring the role of the SDF-1 system in muscle regeneration, we observed that SDF-1 is continuously expressed by endomysial cells of postnatal and adult muscle fibers. Analysis of dystrophin-deficient mdx mice additionally revealed that muscle regeneration is associated with muscular re-expression of CXCR4. The apparent tight control of limb muscle development and regeneration by CXCR4 and CXCR7 points to these chemokine receptors as promising therapeutic targets for certain muscle disorders.

C‐X‐C chemokine receptor 7

AbstractBACKGROUND:C‐X‐C chemokine receptor 4 (CXCR4) and CXCR7 are 7‐transmembrane chemokine receptors of the stroma‐derived factor (SDF‐1). CXCR4, but not CXCR7, has been examined in bladder cancer (BCa). This study examined the functional and clinical significance of CXCR7 in BCa.METHODS:CXCR4 and CXCR7 levels were measured in BCa cell lines, tissues (normal = 25; BCa = 44), and urine specimens (n = 186) by quantitative polymerase chain reaction and/or immunohistochemistry. CXCR7 function in BCa cells were examined by transient transfections using a CXCR7 expression vector or small interfering RNA.RESULTS:In BCa cell lines, CXCR7 messenger RNA levels were 5‐ to 37‐fold higher than those for CXCR4. Transient overexpression of CXCR7 in BCa cell lines promoted growth and chemotactic motility. CXCR7 colocalized and formed a functional complex with epidermal growth factor receptor, phosphoinositide 3‐kinase/Akt, Erk, and src and induced their phosphorylation. CXCR7 also induced up‐regulation of cyclin‐D1 and bcl‐2. Suppression of CXCR7 expression reversed these effects and induced apoptosis. CXCR7 messenger RNA levels and CXCR7 staining scores were significantly (5‐ to 10‐fold) higher in BCa tissues than in normal tissues (P < .001). CXCR7 expression independently associated with metastasis (P = .019) and disease‐specific mortality (P = .03). CXCR7 was highly expressed in endothelial cells in high‐grade BCa tissues when compared to low‐grade BCa and normal bladder. CXCR7 levels were elevated in exfoliated urothelial cells from high‐grade BCa patients (P = .0001; 90% sensitivity; 75% specificity); CXCR4 levels were unaltered.CONCLUSIONS:CXCR7 promotes BCa cell proliferation and motility plausibly through epidermal growth factor receptor receptor and Akt signaling. CXCR7 expression is elevated in BCa tissues and exfoliated cells and is associated with high‐grade and metastasis. Cancer 2013. © 2012 American Cancer Society.

Proper desensitization of CXCR4 is required for lymphocyte development and peripheral compartmentalization in mice

AbstractDesensitization controls G protein–dependent signaling of chemokine receptors. We investigate the physiologic implication of this process for CXCR4 in a mouse model harboring a heterozygous mutation of the Cxcr4 gene, which engenders a desensitization-resistant receptor. Such anomaly is linked to the warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, a human rare combined immunodeficiency. Cxcr4+/mutant(1013) mice display leukocytes with enhanced responses to Cxcl12 and exhibit leukopenia as reported in patients. Treatment with CXCL12/CXCR4 antagonists transiently reverses blood anomalies, further demonstrating the causal role of the mutant receptor in the leukopenia. Strikingly, neutropenia occurs in a context of normal bone marrow architecture and granulocyte lineage maturation, indicating a minor role for Cxcr4-dependent signaling in those processes. In contrast, Cxcr4+/1013 mice show defective thymopoiesis and B-cell development, accounting for circulating lymphopenia. Concomitantly, mature T and B cells are abnormally compartmentalized in the periphery, with a reduction of primary follicles in the spleen and their absence in lymph nodes mirrored by an unfurling of the T-cell zone. These mice provide a model to decipher the role of CXCR4 desensitization in the homeostasis of B and T cells and to investigate which manifestations of patients with WHIM syndrome may be overcome by dampening the gain of CXCR4 function.

Expression of CXCR7, CXCR4, CXCR3 and their chemokine ligands in glioblastoma.

2043 Background: The chemokine receptors CXCR4 and CXCR7, and their shared ligand CXCL12/SDF-1, have been implicated in multiple aspects of tumorigenesis, including tumor cell proliferation, angiogenesis, vasculogenesis, and metastasis. Recently, a role for CXCR4 and CXCR7 has been postulated in the control of glioblastoma growth and vasculogenesis. We set out to define the expression patterns of these proteins on primary glioblastoma tumors and associated vasculature. Methods: First, a tumor microarray containing grades 1, 3, and 4 glioma samples were analyzed by immunohistochemical techniques for the expression of CXCR7. Second, two glioblastoma microarrays were analyzed by the same technique for CXCR7, CXCR4, and their shared chemokine ligand CXCL12. The same microarrays were also probed for the expression of the additional CXCR7 ligand, CXCL11/ITAC, as well as the chemokine receptor CXCR3, which is also a receptor for CXCL11. Tissues were scored in a blinded fashion by a certified neuropathologist for both the intensity of signal and location of signal for all proteins tested. Results: CXCR7 expression was largely dependent on tumor grade, as the receptor was usually absent on most grade 1 gliomas but was moderately-to-highly expressed in the majority of anaplastic astrocytomas and glioblastomas. CXCR7 expression on tumor-associated vasculature was seen in all samples and its expression level also increased with glioma grade. In contrast to CXCR7, CXCR4 expression in glioblastoma was usually limited to the glioma cells, with only infrequent expression on the vasculature. Conversely, CXCL12 expression in glioblastoma was usually limited to the vasculature. CXCR3 was detected in a subset of glioblastomas, and only on the glioma cells. The CXCR7 and CXCR3 ligand CXCL11 was, like CXCR7, expressed on both the glioma cells and vasculature. Conclusions: These results, in light of prior evidence for CXCR7 in tumor growth, suggest a positive role for CXCR7 in the development of glioblastoma. Because CXCR7 colocalizes with its ligands, CXCL12 and CXCL11, as well as their other receptors, CXCR4 and CXCR3, the function of CXCR7 in glioblastoma may lie in the regulation of both the CXCR4/CXCL12 and CXCR3/CXCL11 axes.

Single and Coexpression of CXCR4 and CXCR5 Identifies CD4 T Helper Cells in Distinct Lymph Node Niches during Influenza Virus Infection

Influenza virus infection results in strong, mainly T-dependent, extrafollicular and germinal center B cell responses, which provide lifelong humoral immunity against the homotypic virus strain. Follicular T helper cells (T(FH)) are key regulators of humoral immunity. Questions remain regarding the presence, identity, and function of T(FH) subsets regulating early extrafollicular and later germinal center B cell responses. This study demonstrates that ICOS but not CXCR5 marks T cells with B helper activity induced by influenza virus infection and identifies germinal center T cells (T(GC)) as lymph node-resident CD4(+) ICOS(+) CXCR4(+) CXCR5(+) PSGL-1(lo) PD-1(hi) cells. The CXCR4 expression intensity further distinguished their germinal center light and dark zone locations. This population emerged strongly in regional lymph nodes and with kinetics similar to those of germinal center B cells and were the only T(FH) subsets missing in influenza virus-infected, germinal center-deficient SAP(-/-) mice, mice which were shown previously to lack protective memory responses after a secondary influenza virus challenge, thus indicting the nonredundant functions of CXCR4- and CXCR5-coexpressing CD4 helper cells in antiviral B cell immunity. CXCR4-single-positive T cells, present in B cell-mediated autoimmunity and regarded as "extrafollicular" helper T cells, were rare throughout the response, despite prominent extrafollicular B cell responses, revealing fundamental differences in autoimmune- and infection-induced T-dependent B cell responses. While all ICOS(+) subsets induced similar antibody levels in vitro, CXCR5-single-positive T cells were superior in inducing B cell proliferation. The regulation of T cell localization, marked by the single and coexpression of CXCR4 and CXCR5, might be an important determinant of T(FH) function.

KSHV infection of endothelial cells manipulates CXCR7-mediated signaling: implications for Kaposi’s Sarcoma progression and intervention

CXCR7 was recently characterized as an alternative receptor for the chemokine CXCL12/SDF-1, previously thought to bind and signal exclusively through CXCR4. We recently identified CXCR7 as a key cellular factor in the endothelial cell (EC) dysfunction associated with KSHV infection. CXCL12 signaling is critically associated with tumor growth, angiogenesis and metastasis in several diverse tumors and is one of the most studied chemokine/chemokine receptor interactions in cancer systems. The tumorigenic activity of the CXCL12 signaling axis offers an attractive target for therapeutic intervention in multiple cancers including Kaposi’ sS arcoma (KS). However, most of the research to date was based on the assumption that CXCR4 was the sole CXCL12 receptor, and thus focused on the development of CXCR4-targeted treatments. CXCR4 participates in important homeostatic functions including hematopoiesis and mucosal immunity, while CXCR7 is rarely expressed in normal adult cells. As a result, CXCR7 may be a more specific chemotherapeutic target for tumor cells and tumor-associated vasculature with fewer adverse effects than treatments targeting CXCR4. CXCR7 is poorly studied throughout the cancer literature and although CXCR7 expression has been found in tumor-associated vasculature, no studies comprehensively examine the biology of CXCR7 in EC and its implications for tumor biology. We seek to define th er ole of CXCR7-mediated CXCL12 signaling in EC biology, and in the context of KSHV infection, in order to determine potential contributions of CXCR7 signaling to KSHV-mediated EC transformation and KS tumorigenesis. We demonstrate that CXCR7 is strongly expressed on LANA+ spindle cells in KS biopsy tissue at all stages of tumor progression. We further demonstrate that CXCR7 induction by KSHV in vitro is specific to lymphatic EC lineages and occurs coincident with the acquisition of spindle morphology. Detailed examination of CXCR7 functions in EC biology reveals multiple roles for CXCR7 that could impact KS tumorigenesis, including effects on cellular proliferation, junctional integrity, cell survival and metastatic capacity. Specifically, we determine that CXCR7 expression results in a loss of PECAM/CD31 expression, perturbing the formation and maintenance of EC monolayers. Moreover, CXCR7+ EC display significant SDF-1 dependent hypermotility, as measured via Electrical Cell-Substrate Impedence Sensing (ECIS). We also demonstrate that SDF-1 signaling through CXCR7 expression is enhanced in EC undergoing anchorage-deprivation, affecting EC cell survival and invasion into SDF-1 rich niches. Taken together, these results demonstrate that CXCR7 is a novel KSHV-induced oncogene with the capacity to influence multiple aspects of KS pathogenesis including tumor growth, seeding and metastasis.

Abstract 538: ROS differentially regulates prostate cancer cell survival

Abstract In vitro and in vivo studies have shown that ROS accumulation correlates with increasing aggressiveness of prostate cancer cells, and increased expression of oncogenic molecules, such as CXCR4. CXCR4 is a G-Protein Coupled Receptor that is heavily implicated in the metastatic dissemination of prostate tumor cells to bone marrow. In cancer and other cell models, CXCR4 expression and activity increases in response to hypoxic environments and/or oxidative stress. Cells react by inducing expression of a HIF1α transcription factor, which enhances expression of targeted genes involved in adjusting to oxidative stress and cell survival. HIF1α has been shown to directly induce the expression of CXCR4 in prostate cancer cells. Therefore, generation of ROS in tumor tissues may critically influence CXCR4-mediated expression and functions, ultimately encouraging cancer progression. In this study, we elucidate a mechanism by which ROS induces CXCR4 expression and function in prostate cancer cells. We initially investigated the role of ROS on the phosphorylation of AKT (p-AKT) and ERK1/2 (p-ERK1/2) in DU145 and 22Rv1 prostate cancer cell lines. Using H2O2 as our model of ROS, we found that H2O2 induced a gradual expression of p-AKT and p-ERK1/2 by western blot analysis, which was abrogated by the ROS scavenger N-acetyl cysteine (NAC). It has been shown that PTEN is a negative regulator of AKT and ERK1/2 signaling, and that ROS accumulation inactivates PTEN phosphatase function; hence, we determined whether the increase in p-AKT and p-ERK1/2 expression was due to loss of PTEN function. By performing a phosphatase assay, we found that H2O2 inhibited PTEN catalytic activity compared to untreated cells, while NAC abrogated these effects in both cell lines. The increase in ROS and loss of PTEN has been shown to correlate with CXCR4-mediated function; therefore, we determined the role of H2O2 on CXCR4 expression. We found that H2O2 induced CXCR4 expression in DU145 cells, but not in 22Rv1 cells. Furthermore, we investigated whether ROS-mediated induction of CXCR4 was HIF1α dependent. Interestingly, H2O2 induced expression of HIF1α in DU145 cells, which was abrogated by siRNA for HIF1α. HIF1α expression decreased in 22Rv1 cells, which correlated with apoptosis, as confirmed by Annexin-V analysis. Finally, we observed that the H2O2-mediated increase in CXCR4 enhanced transendothelial invasion and migration in DU145 cells in a CXCR4 dependent manner. Our study suggests that ROS differentially regulates CXCR4 expression and function in prostate cancer cells, suggesting that ROS plays a differential role on heterogeneous tumor mass. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 538. doi:1538-7445.AM2012-538

CX3CR1 regulates intestinal macrophage homeostasis, bacterial translocation and colitogenic TH17 responses in mice

The two most common forms of inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis, affect approximately 1 million people in the United States. Uncontrolled APC reactivity toward commensal bacteria is implicated in the pathogenesis of the disease. A number of distinct APC populations exist in the intestinal lamina propria (LP), but their relative contributions to inflammation remain unclear. Here, we demonstrate important roles for the chemokine receptor CX3CR1 in maintaining LP macrophage populations, preventing translocation of commensal bacteria to mesenteric lymph nodes (mLNs), and limiting colitogenic Th17 responses. CX3CR1 was expressed in resident LP macrophages but not DCs. LP macrophages were decreased in two strains of CX3CR1‐knockout mice and in mice deficient in CX3CL1. All these knockout strains displayed markedly increased translocation of commensal bacteria to mLNs. Additionally, the severity of DSS‐induced colitis was dramatically enhanced in the knockout mice as compared with controls. Disease severity could be limited by either administration of neutralizing IL‐17A antibodies or transfer of CX3CR1‐sufficient macrophages. Our data thus suggest key roles for the CX3CR1/CX3CL1 axis in the intestinal mucosa; further clarification of CX3CR1 function will likely direct efforts toward therapeutic intervention for mucosal inflammatory disorders.

Biomechanical stress and estrogen impact on human osteoblasts

Biomechanical stress and estrogen impact on human osteoblasts

Novel Roles for the E3 Ubiquitin Ligase Atrophin-interacting Protein 4 and Signal Transduction Adaptor Molecule 1 in G Protein-coupled Receptor Signaling

The CXCL12/CXCR4 signaling axis plays an important role in human health and disease; however, the molecular mechanisms mediating CXCR4 signaling remain poorly understood. Ubiquitin modification of CXCR4 by the E3 ubiquitin ligase AIP4 is required for lysosomal sorting and degradation, which is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. CXCR4 sorting is regulated by an interaction between endosomal localized arrestin-2 and STAM-1, an ESCRT-0 component. Here, we report a novel role for AIP4 and STAM-1 in regulation of CXCR4 signaling that is distinct from their function in CXCR4 trafficking. Depletion of AIP4 and STAM-1 by siRNA caused significant inhibition of CXCR4-induced ERK-1/2 activation, whereas overexpression of these proteins enhanced CXCR4 signaling. We further show that AIP4 and STAM-1 physically interact and that the proline-rich region in AIP4 and the SH3 domain in STAM-1 are essential for the interaction. Overexpression of an AIP4 catalytically inactive mutant and a mutant that shows poor binding to STAM-1 fails to enhance CXCR4-induced ERK-1/2 signaling, as compared with wild-type AIP4, suggesting that the interaction between AIP4 and STAM-1 and the ligase activity of AIP4 are essential for ERK-1/2 activation. Remarkably, a discrete subpopulation of AIP4 and STAM-1 resides in caveolar microdomains with CXCR4 and appears to mediate ERK-1/2 signaling. We propose that AIP4-mediated ubiquitination of STAM-1 in caveolae coordinates activation of ERK-1/2 signaling. Thus, our study reveals a novel function for ubiquitin in the regulation of CXCR4 signaling, which may be broadly applicable to other G protein-coupled receptors.

Regulation of CXCR4 conformation by the small GTPase Rac1: implications for HIV infection

AbstractThe chemokine receptor CXCR4 is a critical regulator of cell migration and serves as a coreceptor for HIV-1. The chemokine stromal cell derived factor-1, also known as CXCL12, binds to CXCR4 and exerts its biologic functions partly through the small guanosine triphosphate hydrolase (GTPase) Rac1 (ras-related C3 botulinum toxin substrate 1). We show in different cell types, including CD34+ hematopoietic stem and progenitor cells, that inhibition of Rac1 causes a reversible conformational change in CXCR4, but not in the related receptors CXCR7 or CCR5. Biochemical experiments showed that Rac1 associates with CXCR4. The conformational change of CXCR4 on Rac1 inhibition blocked receptor internalization and impaired CXCL12-induced Gαi protein activation. Importantly, we found that the conformation adopted by CXCR4 after Rac1 inhibition prevents HIV-1 infection of both the U87-CD4-CXCR4 cell line and of primary peripheral blood mononuclear cells. In conclusion, our data show that Rac1 activity is required to maintain CXCR4 in the responsive conformation that allows receptor signaling and facilitates HIV-1 infection; this implies that Rac1 positively regulates CXCR4 function and identifies the Rac1-CXCR4 axis as a new target for preventing HIV-1 infection.