Analysis of CCR2 splice variant expression patterns and functional properties.

BACKGROUND: C-C motif chemokine receptor 2 (CCR2), the main receptor for monocyte chemoattractant protein-1 (MCP-1), is expressed on immune cells, including monocytes, macrophages, and activated T cells, and mediates cell migration toward MCP-1 in inflammation-related diseases. The CCR2 gene encodes two isoforms: CCR2A and CCR2B. The CCR2B open reading frame is localized in a single exon, similar to other chemokine receptors, and CCR2A and CCR2B feature different amino acid sequences in their C-terminal intracellular loops due to alternative splicing. Most biochemical studies on CCR2-related cellular responses in the immune system have focused on CCR2B, with few reports focused on CCR2A. Understanding the functional properties of CCR2A in cellular responses may elucidate the roles played by MCP-1 and CCR2 in pathophysiological responses. RESULTS: CCR2 gene expression analysis in several cell types revealed that most adherent cells only expressed CCR2A, whereas CCR2B expression was dominant in monocytic cells. The C-terminal Helix 8 region of CCR2A contains few basic amino acids, which may be unfavorable for cell surface localization, as confirmed with the HiBiT assay. CCR2B contains many C-terminal Ser/Thr residues, similar to other chemokine receptors, which may be phosphorylated by G protein-coupled receptor kinases (GRKs) to promote ß-arrestin recruitment and subsequent endocytosis. By contrast, CCR2A contains few C-terminal Ser/Thr residues, which are unlikely to be phosphorylated by GRKs. CCR2A localized on the cell surface is resistant to internalization, despite the interaction between Gß and GRKs induced by ligand binding with CCR2A. CCR2A induced cellular responses at a relatively higher degree than CCR2B, although both receptors mediated signaling events through Gαq and Gαi. HeLa cells lacking CCR2A showed slowed growth compared with parent cells, regardless of MCP-1 stimulation, and their chemotactic activity toward MCP-1, in addition to basal motility, was significantly impaired. CONCLUSION: MCP-1 and CCR2 may play pivotal roles in cancer progression by recruiting macrophages into cancer tissue. This study demonstrates that CCR2A but not CCR2B is expressed in solid cancer-derived cells. CCR2A is resistant to internalization by ß-arrestin due to a distinct C-terminal region from CCR2B, which enhances MCP-1-stimulated responses, indicating that CCR2A may play essential roles in solid cancer progression.

Establishment of a NanoBiT-Based Cytosolic Ca2+ Sensor by Optimizing Calmodulin-Binding Motif and Protein Expression Levels.

Cytosolic Ca2+ levels ([Ca2+]c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca2+]c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca2+]c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca2+]c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca2+-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca2+]c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca2+]c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca2+]c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca2+]c in single cells and animal models.

Inhibition of RANKL-induced osteoclast differentiation through the downregulation of c-Fos and NFATc1 by Eremochloa ophiuroides (centipedegrass) extract.

Osteoclasts, derived from hematopoietic stem cells, are specialized macrophages and have a homeostatic role in skeletal modeling and remodeling with bone-forming osteoblasts. However, excessive osteoclast activity induces bone diseases, including osteoporosis, periodontitis and rheumatoid arthritis. Natural substances have received attention as therapeutic drugs in human diseases. In the current study, cells isolated from mouse bone marrow, and a mouse model, were used to determine the effect of centipedegrass extract (CGE) on osteoclasts. Multiple concentrations of CGE were administered to bone marrow cells for 24­72 hours and, for the in vivo study, mice were treated with CGE for 8 days. The effects of CGE on transcription and translation of osteoclast-associated molecules were then determined using reverse transcription-polymerase chain reaction and immunoblotting, respectively. In the present study it was shown that CGE extracted from Eremochloa ophiuroides (centipedegrass) inhibited receptor activator of nuclear factor κ­B ligand (RANKL)­mediated osteoclast differentiation in bone marrow macrophages, without cytotoxicity, in a dose­dependent manner. CGE decreased the expression levels of osteoclast­specific genes, including matrix metalloproteinase­9, osteoclast­associated immunoglobulin­like receptor and cathepsin K, however, CGE had no inhibitory effect on the expression levels of mitogen­activated protein kinases, nuclear factor­κB and Akt. Furthermore, the protein and RNA levels of RANKL­induced c­Fos and nuclear factor of activated T-cell cytoplasmic 1 were suppressed by CGE. These results indicated that CGE may serve as a useful drug in the prevention of bone loss.

Novel Radiolytic Rotenone Derivative, Rotenoisin B with Potent Anti-Carcinogenic Activity in Hepatic Cancer Cells.

Rotenone, isolated from roots of derris plant, has been shown to possess various biological activities, which lead to attempting to develop a potent drug against several diseases. However, recent studies have demonstrated that rotenone has the potential to induce several adverse effects such as a neurodegenerative disease. Radiolytic transformation of the rotenone with gamma-irradiation created a new product, named rotenoisin B. The present work was designed to investigate the anticancer activity of rotenoisin B with low toxicity and its molecular mechanism in hepatic cancer cells compared to a parent compound, rotenone. Our results showed rotenoisin B inhibited hepatic cancer cells' proliferation in a dose dependent manner and increased in apoptotic cells. Interestingly, rotenoisin B showed low toxic effects on normal cells compared to rotenone. Mitochondrial transmembrane potential has been decreased, which leads to cytochrome c release. Down regulation of anti-apoptotic Bcl-2 levels as well as the up regulation of proapoptotic Bax levels were observed. The cleaved PARP (poly ADP-ribose polymerase) level increased as well. Moreover, phosphorylation of extracellular signal regulated kinase (ERK) and p38 slightly up regulated and intracellular reactive oxygen species (ROS) increased as well as cell cycle arrest predominantly at the G2/M phase observed. These results suggest that rotenoisin B might be a potent anticancer candidate similar to rotenone in hepatic cancer cells with low toxicity to normal cells even at high concentrations compared to rotenone.

Centipedegrass extract induces apoptosis through the activation of caspases and the downregulation of PI3K/Akt and MAPK phosphorylation in leukemia cells.

Acute lymphoblastic leukemia (ALL), which involves the blood and bone marrow, is the most common type of cancer in children younger than 5 years of age. Previous studies have investigated the effects of centipedegrass extract (CGE), which is mainly composed of maysin and its derivatives, and have demonstrated that it has various biological activities, including antioxidant and anti­inflammatory activities, pancreatic lipase inhibitory activity, anti-adipogenic activity and insecticidal activity. To the best of our knowledge, this study is the first to investigate the anticancer effects of CGE in ALL cell lines and to elucidate the mechanisms underlying these effects. Cell viability was measured by thiazolyl blue tetrazolium blue (MTT) assay. Apoptosis, cell cycle progression and mitochondrial membrane potential (∆Ψm) were determined by flow cytometry. The effects of CGE on the phosphatidylinositol 3­kinase (PI3K)/Akt pathway and mitogen­activated protein kinases (MAPKs) were assessed by immunoblotting. PI3K, MAPK and caspase inhibitors were used to further confirm the molecular mechanisms involved. Our results clearly demonstrated that the proliferation of the ALL cells was significantly inhibited by CGE in a dose­dependent manner. Apoptosis was accompanied by the induction of significant G1 cell cycle arrest. The resulting alteration of the ∆Ψm increased the activity of caspase­3/7. The induction of apoptosis was enhanced by the combined treatment of CGE with a PI3K inhibitor or an extracellular signal-regulated kinase (ERK) inhibitor, whereas the CGE­induced apoptosis was inhibited in the presence of caspase inhibitors, such as z­VAD­fmk and z­IETD­fmk. Furthermore, CGE inhibited PI3K activity by decreasing the levels of phosphorylated (p­)Akt, p­BAD, and Bcl­2 together with the levels of MAPKs, including p­ERK and p­JNK, but demonstrated no effects on p38 MAPK. Thus, our data suggest that CGE may be a novel natural compound with potential for use as an antitumor agent in ALL.