Regulation of CXCR4 signaling

The chemokine receptor CXCR4 belongs to the large superfamily of G protein-coupled receptors, and is directly involved in a number of biological processes including organogenesis, hematopoiesis, and immune response. Recent evidence has highlighted the role of CXCR4 in a variety of diseases including HIV, cancer, and WHIM syndrome. Importantly, the involvement of CXCR4 in cancer metastasis and WHIM syndrome appears to be due to dysregulation of the receptor leading to enhanced signaling. Herein we review what is currently known regarding the regulation of CXCR4 and how dysregulation contributes to disease progression.     

Regulation of CXCR4 signaling

The chemokine receptor CXCR4 belongs to the large superfamily of G protein-coupled receptors, and is directly involved in a number of biological processes including organogenesis, hematopoiesis, and immune response. Recent evidence has highlighted the role of CXCR4 in a variety of diseases including HIV, cancer, and WHIM syndrome. Importantly, the involvement of CXCR4 in cancer metastasis and WHIM syndrome appears to be due to dysregulation of the receptor leading to enhanced signaling. Herein we review what is currently known regarding the regulation of CXCR4 and how dysregulation contributes to disease progression.     

Membrane Tension Gates ERK-Mediated Regulation of Pluripotent Cell Fate

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The Role of CXCR3 in DSS-Induced Colitis

Inflammatory bowel disease (IBD) is a group of disorders that are characterized by chronic, uncontrolled inflammation in the intestinal mucosa. Although the aetiopathogenesis is poorly understood, it is widely believed that IBD stems from a dysregulated immune response towards otherwise harmless commensal bacteria. Chemokines induce and enhance inflammation through their involvement in cellular trafficking. Reducing or limiting the influx of these proinflammatory cells has previously been demonstrated to attenuate inflammation. CXCR3, a chemokine receptor in the CXC family that binds to CXCL9, CXCL10 and CXCL11, is strongly overexpressed in the intestinal mucosa of IBD patients. We hypothesised that CXCR3 KO mice would have impaired cellular trafficking, thereby reducing the inflammatory insult by proinflammatory cell and attenuating the course of colitis. To investigate the role of CXCR3 in the progression of colitis, the development of dextran sulfate sodium (DSS)-induced colitis was investigated in CXCR3^−/− mice over 9 days. This study demonstrated attenuated DSS-induced colitis in CXCR3^−/− mice at both the macroscopic and microscopic level. Reduced colitis correlated with lower recruitment of neutrophils (p = 0.0018), as well as decreased production of IL-6 (p<0.0001), TNF (p = 0.0038), and IFN-γ (p = 0.0478). Overall, our results suggest that CXCR3 plays an important role in recruiting proinflammatory cells to the colon during colitis and that CXCR3 may be a therapeutic target to reduce the influx of proinflammatory cells in the inflamed colon.     

Novel CXCR3 antagonists with a piperazinyl-piperidine core

High-throughput screening of an encoded combinatorial aryl piperazine library led to the identification of a novel series of potent piperazinyl-piperidine based CXCR3 antagonists. Analogs of the initial hit were synthesized via solid and solution phase methods to probe the influence of structure on the CXCR3 binding of these molecules. Various functional groups were found to contribute to the overall potency and essential molecular features were identified.     

Dual Targeting of the Chemokine Receptors CXCR4 and ACKR3 with Novel Engineered Chemokines

The chemokine CXCL12 and its G protein-coupled receptors CXCR4 and ACKR3 are implicated in cancer and inflammatory and autoimmune disorders and are targets of numerous antagonist discovery efforts. Here, we describe a series of novel, high affinity CXCL12-based modulators of CXCR4 and ACKR3 generated by selection of N-terminal CXCL12 phage libraries on live cells expressing the receptors. Twelve of 13 characterized CXCL12 variants are full CXCR4 antagonists, and four have K_d values <5 nm. The new variants also showed high affinity for ACKR3. The variant with the highest affinity for CXCR4, LGGG-CXCL12, showed efficacy in a murine model for multiple sclerosis, demonstrating translational potential. Molecular modeling was used to elucidate the structural basis of binding and antagonism of selected variants and to guide future designs. Together, this work represents an important step toward the development of therapeutics targeting CXCR4 and ACKR3.     

活化的T细胞核内因子（NFAT）的调节及其在神经系统中的功能

活化的T细胞核内因子（nuclear factor of activated T-cells, NFAT）作为细胞信号转导通路中的一类重要的转录因子参与细胞功能的调节. NFAT的活化主要是通过细胞内钙/钙调神经磷酸酶（Ca2+/calcineurin）的刺激启动,它脱磷酸后发生核转位并与DNA的特定序列结合,同时通过与其它转录因子的协同作用,调节目的基因的特定表达. NFAT在免疫系统中所调节的基因表达已经得到了充分的研究. 近年实验研究发现,NFAT的转录因子家族在脊椎动物的神经系统中也发挥着非常重要的作用. 本文综述了NFAT家族蛋白的分类、结构、磷酸酶与激酶对其出入核的调节及在神经系统中的研究进展,使得能够更加全面地认识calcineurin/NFAT信号通路的作用. 此外,由于环孢菌素A（cyclosporin A）等药物在神经系统应用的局限性,对于NFAT调节深入研究,也将为筛选或者开发更为高效、低毒药物提供新的思路.     

A Novel Role for p115RhoGEF in Regulation of Epithelial Plasticity

Epithelial plasticity plays a critical role during physiological processes, such as wound healing and tissue regeneration, and dysregulation of epithelial plasticity can lead to pathological conditions, such as cancer. Cell-cell junctions are a critical feature of epithelial cells and loss of junctions is associated with acquisition of mesenchymal features, such as enhanced protrusion and migration. Although Rho has been implicated in regulation of junctions in epithelial cells, the role of Rho signaling in the regulation of epithelial plasticity has not been understood. We show that members of the RGS RhoGEFs family play a critical role in regulation of epithelial cell-cell junctions in breast epithelial cells. We identify a novel role for p115RhoGEF in regulation of epithelial plasticity. Loss of p115RhoGEF leads to decreased junctional E-cadherin and enhanced protrusiveness and migration. Conversely, overexpression of p115RhoGEF enhanced junctional E-cadherin and inhibited cell protrusion and migration. siRNA screen of 23 Rho effectors showed that members of the Diaphanous-Related Formin (DRF) family are required for p115RhoGEF-mediated changes in epithelial plasticity. Thus, our data indicates a novel role for p115RhoGEF in regulation of epithelial plasticity, which is dependent on Rho-DRF signaling module.     

Role for CD74 and CXCR4 in clathrin-dependent endocytosis of the cytokine MIF

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that plays a role in innate and adaptive immunity. Depending on the cellular context and disease state, MIF signaling is mediated by its receptors CXCR2, CXCR4 and/or CD74. Although it is known that MIF is endocytosed, the exact mechanism has remained unknown. In exploring the mechanism of MIF endocytosis with biologically active Alexa(546)MIF, pathway-specific inhibitors (monodansylcadaverine, MDC; chlorpromazine, CPZ; dynasore; dominant-negative dynamin, bafilomycin, nocodazole) and receptor overexpression and blockade approaches, we identified a clathrin/dynamin-dependent endocytosis pathway as the main track for MIF internalization. MIF endocytosis was rapid and colocalization with both early and late endosomal vesicles in a microtubule- and acidification-dependent manner was observed. LDL endocytosis (which is clathrin-mediated) served as a control and was similarly inhibited by MDC or dynasore. When MIF endocytosis was compared to that of transferrin, acetylated LDL, and choleratoxin B (the latter internalized by a clathrin-independent pathway) by colocalization studies, the MIF internalization pathway clearly resembled that of LDL but also shared early trafficking with transferrin, whereas no colocalization with choleratoxin was noted. To identify the receptors involved in MIF endocytosis, we focused on CD74 and CXCR4 which form a heteromeric complex. Ectopic overexpression of CD74 in HEK293 and HeLa cells, which do not endogenously express CD74, led to a marked acceleration of MIF endocytosis while pharmacological blockade of CXCR4, which is endogenously expressed on these cells, with AMD3100 led to a 20% reduction of MIF endocytosis in HEK293-CD74 transfectants, whereas in untransfected cells, a blockade of 40% was observed. Of note, both CD74 and CXCR4 strongly colocalize with Alexa(546)MIF both on the plasma membrane and in endosomal compartments. Moreover, MIF-stimulated AKT signaling, which was previously shown to involve both CD74 and CXCR4, was reduced by endocytosis inhibitors, indicating that MIF signaling is at least in part due to endosomal signaling mechanisms. Thus, MIF uptake follows a rapid LDL-like, clathrin- and dynamin-dependent endocytosis pathway, which is dependent on the receptors CD74 and CXCR4 and leads to the initiation of endosomal signaling responses.     

Novel N-substituted benzimidazole CXCR4 antagonists as potential anti-HIV agents

The lead optimization of a series of N-substituted benzimidazole CXCR4 antagonists is described. Side chain modifications and stereochemical optimization led to substantial improvements in potency and protein shift to afford compounds with low nanomolar anti-HIV activity.     

Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells

Chemokines are secreted into the tumor microenvironment by tumor-infiltrating inflammatory cells as well as by tumor cells. Chemokine receptors mediate agonist-dependent cell responses, including migration and activation of several signaling pathways. In the present study we show that several human melanoma cell lines and melanoma cells on macroscopically infiltrated lymph nodes express the chemokine receptors CXCR3 and CXCR4. Using the highly invasive melanoma cell line BLM, we demonstrate that the chemokine Mig, a ligand for CXCR3, activates the small GTPases RhoA and Rac1, induces a reorganization of the actin cytoskeleton, and triggers cell chemotaxis and modulation of integrin VLA-5- and VLA-4-dependent cell adhesion to fibronectin. Furthermore, the chemokine SDF-1alpha, the ligand of CXCR4, triggered modulation of beta(1) integrin-dependent melanoma cell adhesion to fibronectin. Additionally, Mig and SDF-1alpha activated MAPKs p44/42 and p38 on melanoma cells. Expression of functional CXCR3 and CXCR4 receptors on melanoma cells indicates that they might contribute to cell motility during invasion as well as to regulation of cell proliferation and survival.     

A minimalistic 3D pharmacophore model for cyclopentapeptide CXCR4 antagonists

Because of its involvement in HIV entry, the chemokine receptor CXCR4 is an attractive target for antiretroviral drugs. Despite the large number of CXCR4 inhibitors studied, the 3D pharmacophore for binding to CXCR4 remains elusive, mainly as a result of conformational flexibility inherent in the identified ligands. In the present study, an exhaustive systematic exploration of the conformational space for a series of analogs of FC131, a cyclopentapeptide CXCR4 antagonist, has been performed. By comparing the resulting low-energy conformations using different sets of atoms, specific conformational features common only to the high/medium affinity compounds were identified. These features included the spatial arrangement of three pharmacophoric side chains as well as the orientation of a specific backbone amide bond. Together these features represent a minimalistic 3D pharmacophore model for binding of the cyclopentapeptide antagonists to CXCR4. The model enables rationalization of the experimental affinity data for this class of compounds as well as for the peptidomimetic KRH-1636.