Yuning Hou

A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases.

Background: CXCR2 plays an important role in various neutrophil-dominant inflammatory diseases. Results: A macromolecular signaling complex containing CXCR2, NHERF1, and phospholipase C (PLC)-β2 regulates neutrophil calcium mobilization, chemotaxis, and transepithelial migration. Conclusion: CXCR2·NHERF1·PLC-β2 macromolecular signaling complex is critical to neutrophil functions. Significance: CXCR2 macromolecular complex might be a potential therapeutic target for neutrophil infiltration-associated inflammatory diseases. Inflammation plays an important role in a wide range of human diseases such as ischemia-reperfusion injury, arteriosclerosis, cystic fibrosis, inflammatory bowel disease, etc. Neutrophilic accumulation in the inflamed tissues is an essential component of normal host defense against infection, but uncontrolled neutrophilic infiltration can cause progressive damage to the tissue epithelium. The CXC chemokine receptor CXCR2 and its specific ligands have been reported to play critical roles in the pathophysiology of various inflammatory diseases. However, it is unclear how CXCR2 is coupled specifically to its downstream signaling molecules and modulates cellular functions of neutrophils. Here we show that the PDZ scaffold protein NHERF1 couples CXCR2 to its downstream effector phospholipase C (PLC)-β2, forming a macromolecular complex, through a PDZ-based interaction. We assembled a macromolecular complex of CXCR2·NHERF1·PLC-β2 in vitro, and we also detected such a complex in neutrophils by co-immunoprecipitation. We further observed that the CXCR2-containing macromolecular complex is critical for the CXCR2-mediated intracellular calcium mobilization and the resultant migration and infiltration of neutrophils, as disrupting the complex with a cell permeant CXCR2-specific peptide (containing the PDZ motif) inhibited intracellular calcium mobilization, chemotaxis, and transepithelial migration of neutrophils. Taken together, our data demonstrate a critical role of the PDZ-dependent CXCR2 macromolecular signaling complex in regulating neutrophil functions and suggest that targeting the CXCR2 multiprotein complex may represent a novel therapeutic strategy for certain inflammatory diseases.

Structural Insights into Neutrophilic Migration Revealed by the Crystal Structure of the Chemokine Receptor CXCR2 in Complex with the First PDZ Domain of NHERF1.

Neutrophil plays an essential role in host defense against infection, but uncontrolled neutrophilic infiltration can cause inflammation and severe epithelial damage. We recently showed that CXCR2 formed a signaling complex with NHERF1 and PLC-2, and that the formation of this complex was required for intracellular calcium mobilization and neutrophilic transepithelial migration. To uncover the structural basis of the complex formation, we report here the crystal structure of the NHERF1 PDZ1 domain in complex with the C-terminal sequence of CXCR2 at 1.16 Å resolution. The structure reveals that the CXCR2 peptide binds to PDZ1 in an extended conformation with the last four residues making specific side chain interactions. Remarkably, comparison of the structure to previously studied PDZ1 domains has allowed the identification of PDZ1 ligand-specific interactions and the mechanisms that govern PDZ1 target selection diversities. In addition, we show that CXCR2 can bind both NHERF1 PDZ1 and PDZ2 in pulldown experiments, consistent with the observation that the peptide binding pockets of these two PDZ domains are highly structurally conserved. The results of this study therefore provide structural basis for the CXCR2-mediated neutrophilic migration and could have important clinical applications in the prevention and treatment of numerous neutrophil-dependent inflammatory disorders.

Emerging role of cystic fibrosis transmembrane conductance regulator - an epithelial chloride channel in gastrointestinal cancers

Cystic fibrosis transmembrane conductance regulator (CFTR), a glycoprotein with 1480 amino acids, has been well established as a chloride channel mainly expressed in the epithelial cells of various tissues and organs such as lungs, sweat glands, gastrointestinal system, and reproductive organs. Although defective CFTR leads to cystic fibrosis, a common genetic disorder in the Caucasian population, there is accumulating evidence that suggests a novel role of CFTR in various cancers, especially in gastroenterological cancers, such as pancreatic cancer and colon cancer. In this review, we summarize the emerging findings that link CFTR with various cancers, with focus on the association between CFTR defects and gastrointestinal cancers as well as the underlying mechanisms. Further study of CFTR in cancer biology may help pave a new way for the diagnosis and treatment of gastrointestinal cancers.

A critical role of CXCR2 PDZ-mediated interactions in endothelial progenitor cell homing and angiogenesis

Bone marrow-derived endothelial progenitor cells (EPCs) contribute to neovessel formation in response to growth factors, cytokines, and chemokines. Chemokine receptor CXCR2 and its cognate ligands are reported to mediate EPC recruitment and angiogenesis. CXCR2 possesses a consensus PSD-95/DlgA/ZO-1 (PDZ) motif which has been reported to modulate cellular signaling and functions. Here we examined the potential role of the PDZ motif in CXCR2-mediated EPC motility and angiogenesis. We observed that exogenous CXCR2 C-tail significantly inhibited in vitro EPC migratory responses and angiogenic activities, as well as in vivo EPC angiogenesis. However, the CXCR2 C-tail that lacks the PDZ motif (ΔTTL) did not cause any significant changes of these functions in EPCs. In addition, using biochemical assays, we demonstrated that the PDZ scaffold protein NHERF1 specifically interacted with CXCR2 and its downstream effector, PLC-β3, in EPCs. This suggests that NHERF1 might cluster CXCR2 and its relevant signaling molecules into a macromolecular signaling complex modulating EPC cellular functions. Taken together, our data revealed a critical role of a PDZ-based CXCR2 macromolecular complex in EPC homing and angiogenesis, suggesting that targeting this complex might be a novel and effective strategy to treat angiogenesis-dependent diseases.

Crystallographic analysis of NHERF1–PLCβ3 interaction provides structural basis for CXCR2 signaling in pancreatic cancer

The formation of CXCR2-NHERF1-PLCβ3 macromolecular complex in pancreatic cancer cells regulates CXCR2 signaling activity and plays an important role in tumor proliferation and invasion. We previously have shown that disruption of the NHERF1-mediated CXCR2-PLCβ3 interaction abolishes the CXCR2 signaling cascade and inhibits pancreatic tumor growth in vitro and in vivo. Here we report the crystal structure of the NHERF1 PDZ1 domain in complex with the C-terminal PLCβ3 sequence. The structure reveals that the PDZ1-PLCβ3 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four PLCβ3 residues contributing to specific interactions. We also show that PLCβ3 can bind both NHERF1 PDZ1 and PDZ2 in pancreatic cancer cells, consistent with the observation that the peptide binding pockets of these PDZ domains are highly structurally conserved. This study provides an understanding of the structural basis for the PDZ-mediated NHERF1-PLCβ3 interaction that could prove valuable in selective drug design against CXCR2-related cancers.

New Conformational State of NHERF1-CXCR2 Signaling Complex Captured by Crystal Lattice Trapping

NHERF1 is a PDZ adaptor protein that scaffolds the assembly of diverse signaling complexes and has been implicated in many cancers. However, little is known about the mechanism responsible for its scaffolding promiscuity or its ability to bind to multiple targets. Computational studies have indicated that PDZ promiscuity may be attributed to its conformational dynamics, but experimental evidence for this relationship remains very limited. Here we examine the conformational flexibility of the NHERF1 PDZ1 domain using crystal lattice trapping via solving PDZ1 structure of a new crystal form. The structure, together with prior PDZ1 structures of a different space group, reveals that 4 of 11 ligand-interacting residues undergo significant crystal packing-induced structural changes. Most of these residues correspond to the residues involved in allosteric transition when a peptide ligand binds. In addition, a subtle difference in ligand conformations causes the same peptide to bind in slightly different modes in different crystal forms. These findings indicate that substantial structural flexibility is present in the PDZ1 peptide-binding pocket, and the structural substate trapped in the present crystal form can be utilized to represent the conformational space accessible to the protein. Such knowledge will be critical for drug design against the NHERF1 PDZ1 domain, highlighting the continued need for experimentally determined PDZ1-ligand complexes.

Dysregulated Chemokine Signaling in Cystic Fibrosis Lung Disease: A Potential Therapeutic Target

CF lung disease is characterized by a chronic and non-resolving activation of the innate immune system with excessive release of chemokines/cytokines including IL-8 and persistent infiltration of immune cells, mainly neutrophils, into the airways. Chronic infection and impaired immune response eventually lead to pulmonary damage characterized by bronchiectasis, emphysema, and lung fibrosis. As a complete knowledge of the pathways responsible for the exaggerated inflammatory response in CF lung disease is lacking, understanding these pathways could reveal new therapeutic targets, and lead to novel treatments. Therefore, there is a strong rationale for the identification of mechanisms and pathways underlying the exaggerated inflammatory response in CF lung disease. This article reviews the role of inflammation in the pathogenesis of CF lung disease, with a focus on the dysregulated signaling involved in the overexpression of chemokine IL-8 and excessive recruitment of neutrophils in CF airways. The findings suggest that targeting the exaggerated IL-8/IL-8 receptor (mainly CXCR2) signaling pathway in immune cells (especially neutrophils) may represent a potential therapeutic strategy for CF lung disease.

Crystal structure of the NHERF1 PDZ2 domain in complex with the chemokine receptor CXCR2 reveals probable modes of PDZ2 dimerization.

The formation of CXCR2-NHERF1-PLCβ2 macromolecular complex in neutrophils regulates CXCR2 signaling and plays a key role in neutrophil chemotaxis and transepithelial neutrophilic migration. However, NHERF1 by itself, with only two PDZ domains, has a limited capacity in scaffolding the multiprotein-complex formation. Here we report the crystal structure of the NHERF1 PDZ2 domain in complex with the C-terminal CXCR2 sequence. The structure reveals that the PDZ2-CXCR2 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four CXCR2 residues contributing to specific interactions. The structure also reveals two probable modes of PDZ2 dimerization where the two canonical ligand-binding pockets are well separated and orientated in a unique parallel fashion. This study provides not only the structural basis for the PDZ-mediated NHERF1-CXCR2 interaction, but also an additional example of how PDZ domains may dimerize, which both could prove valuable in understanding NHERF1 complex-scaffolding function in neutrophils.

Abstract A94: CXCR2 macromolecular complex in pancreatic cancer: A potential therapeutic target in tumor growth.

Background: The signaling mediated by the CXC-chemokine receptor 2 (CXCR2) plays an important role in promoting the progression of pancreatic cancer, one of the most lethal malignancies. CXCR2 possesses a consensus PDZ motif at carboxyl termini. A variety of PDZ scaffold proteins have been documented to orchestrate the formation/regulation of multi-protein signaling complexes at plasma membrane. Therefore, the PDZ motif of CXCR2 could, theoretically, mediate potential interactions with certain PDZ scaffold proteins which might cluster CXCR2 with other relevant signaling molecules into macromolecular signaling complex to transduce signals into cell interior and thereafter regulate cell behavior with higher specificity and efficiency. Aim: The purpose of this study is to define the role of the potential PDZ-mediated CXCR2 macromolecular complex in pancreatic cancer progression and to explore novel therapeutic targets for pancreatic cancer. Methods: Human pancreatic cancer cells, HPAC and Colo357, were used in our study. GST pull-down assays were performed to detect the interaction between GST-PDZ scaffold fusion proteins and endogenous CXCR2 or PLC-β3 (one of the downstream molecules in CXCR2 signaling) in pancreatic cancer cells. Co-immunoprecipitation assays were performed to investigate the existence of endogenous CXCR2 macromolecular complex in pancreatic cancer cells. To evaluate the effect of NHERF1 knockdown on the formation of the CXCR2 macromolecular complex and cellular functions in pancreatic cancer cells, Lentivirus-based shRNAs targeting NHERF1 were generated and used to transduce the pancreatic cancer cells. MTT assay and invasion assay was used to assess the proliferation and invasiveness of pancreatic cancer cells, respectively. Recombinant Adeno-Associated Virus (rAAV) was used to transduce the HPAC cells with the exogenous CXCR2 C-tail gene (containing the PDZ motif), and the transduced HPAC cells were used in a subcutaneous xenograft model to assess the tumor growth in vivo. This in vivo human cancer mouse model was used to evaluate the therapeutic potential of disrupting CXCR2 PDZ motif-mediated interaction as a potential approach for pancreatic cancer treatment. Results: GST pull down assays demonstrated that both CXCR2 and PLC-β3 preferentially interacts with PDZ scaffold protein NHERF1 in a direct and PDZ motif-dependent manner. Co-immunoprecipitation assays revealed that an endogenous CXCR2 macromolecular complex (CXCR2-NHERF1-PLC-β3) exists in both Colo357 and HPAC cells. Furthermore, disrupting this complex, by gene delivery or peptide delivery of exogenous CXCR2 C-tail, significantly inhibits the proliferation and invasive potency of both Colo357 and HPAC cells. Knockdown of NHERF1 impedes formation of the CXCR2 macromolecular complex in pancreatic cancer cells, and significantly reduces cell proliferation and invasion. In addition, gene delivery of CXCR2 C-tail sequence by rAAV2 potently suppresses tumor growth in vivo. Conclusion: Our data demonstrated a physical and functional coupling of CXCR2 and its downstream effector PLC-β3 mediated by NHERF1, forming a macromolecular signaling complex for efficient and specific CXCR2 signaling in pancreatic cancer. Targeting the CXCR2 macromolecular complex may represent a novel and effective therapeutic strategy against pancreatic cancer. Citation Format: Shuo Wang, Yanning Wu, Yuning Hou, Marcello P. Castelvetere, Xiaoqing Guan, Jacob J. Oblak, Sanjeev Banerjee, Theresa M. Filtz, Fazul H. Sarkar, Chunying Li. CXCR2 macromolecular complex in pancreatic cancer: A potential therapeutic target in tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A94.