Tomoko Ikeuchi

Pannexin 3 and connexin 43 modulate skeletal development through their distinct functions and expression patterns

ABSTRACT Pannexin 3 (Panx3) and connexin 43 (Cx43; also known as GJA1) are two major gap junction proteins expressed in osteoblasts. Here, we studied their functional relationships in skeletal formation by generating Panx3−/− and Panx3−/−;Cx43−/− mice and comparing their skeletal phenotypes with Cx43−/− mice. Panx3−/− mice displayed defects in endochondral and intramembranous ossification, resulting in severe dwarfism and reduced bone density. The skeletal abnormalities of Panx3−/−;Cx43−/− mice were similar to those in Panx3−/− mice. The gross appearance of newborn Cx43−/− skeletons showed no obvious abnormalities, except for less mineralization of the skull. In Panx3−/− mice, proliferation of chondrocytes and osteoblasts increased and differentiation of these cells was inhibited. Panx3 promoted expression of osteogenic proteins such as ALP and Ocn (also known as ALPL and BGLAP, respectively), as well as Cx43, by regulating Osx (also known as SP7) expression. Panx3 was induced in the early differentiation stage and reduced during the maturation stage of osteoblasts, when Cx43 expression increased in order to promote mineralization. Furthermore, only Panx3 functioned as an endoplasmic reticulum (ER) Ca2+ channel to promote differentiation, and it could rescue mineralization defects in Cx43−/− calvarial cells. Our findings reveal that Panx3 and Cx43 have distinct functions in skeletal formation. Summary: Panx3 and Cx43 are two important gap junction proteins expressed in osteoblasts. We find that Panx3 and Cx43 regulate skeletal formation through their distinct expression patterns and functions.

A vitamin D3 analog augmented interleukin-8 production by human monocytic cells in response to various microbe-related synthetic ligands, especially NOD2 agonistic muramyldipeptide.

Active metabolite vitamin D(3), 1α,25-dihydroxyvitamin D(3), is a pleiotropic factor and exhibits various physiological functions, including immunomodulating activities. In this study, the possible regulation of innate immune responses of human monocytic cells by a vitamin D(3) analog was examined. Human monocytic THP-1 cells were pre-treated with OCT, vitamin D(3) analog, 1α,25-dihydroxy-22-oxavitamin D(3), followed by stimulation with various chemically synthesized Toll-like receptors (TLR) and NOD1 and NOD2 ligands. OCT-treated cells produced more IL-8 than non-treated cells upon stimulation with various chemically-synthesized ligands: TLR2-agonistic lipopeptide (FSL-1), TLR3-agonistic poly I:C, TLR4-agonistic lipid A (E. coli-type LA-15-PP), NOD1-agonistic FK565 and NOD2-agonistic muramyldipeptide (MDP). Among the ligands, MDP was the highest inducer of IL-8 production in OCT-treated THP-1 cells, and IL-8 production increased depending on the treatment time until 72h. OCT up-regulated the expression of NOD2 in THP-1 cells, and OCT-treated cells exhibited higher activation of p38, JNK and ERK in the MAPK pathway, IκBα in the NF-κB pathway, and TAK1 upstream in response to MDP than non-treated cells. Analysis using siRNA against NOD2 and inhibitors of specific signal molecules indicated that the existence of NOD2 and activation of the above signaling molecules are required for enhanced production of IL-8 in OCT-treated THP-1 cells. These findings suggested that NOD2, NF-κB and MAPK pathways are involved in the activity of OCT to augment the response of human monocytic cells to MDP.

Identification of Peptides Derived from the C-terminal Domain of Fibulin-7 Active for Endothelial Cell Adhesion and Tube Formation Disruption.

Despite the research done on pathological angiogenesis, there is still a need for the development of new therapies against angiogenesis‐related diseases. Fibulin‐7 (Fbln7) is a member of the extracellular matrix fibulin protein family. The Fbln7 C‐terminal fragment, Fbln7‐C, binds to endothelial cells and inhibits their tube formation in culture. In this study, we screened 12 synthetic peptides, covering the fibulin‐globular domain of Fbln7‐C, to identify active sites for endothelial cell adhesion and in vitro antiangiogenic activity. Three peptides, fc10, fc11, and fc12, promoted Human Umbilical Vein Endothelial Cells (HUVECs) adhesion, and the morphology of HUVECs on fc10 was similar to that on Fbln7‐C. EDTA and the anti‐integrin β1 function‐blocking antibody inhibited HUVECs adhesion to both fc10 and fc12, and heparin inhibited HUVECs adhesion to both fc11 and fc12. fc10 and fc11 inhibited HUVECs tube formation. Our results suggest that three peptides from Fbln7‐C are biologically active for endothelial cell adhesion and disrupt the tube formation, suggesting a potential therapeutic use of these peptides for angiogenesis‐related diseases.

Cell adhesion protein fibulin-7 and its C-terminal fragment negatively regulate monocyte and macrophage migration and functions in vitro and in vivo

Fibulin‐7 (Fbln7) has been identified as the latest member of the fibulin family of secreted glycoproteins in developing teeth, functioning as a cell adhesion molecule and interacting with other matrix proteins, receptors, and growth factors. More recently, we have shown that the C‐terminal Fbln7 fragment (Fbln7‐C) has antiangiogenic activity in vitro. Fbln7 is also expressed in immune‐privileged tissues, such as eye and placenta, but its functional significance is unknown. In the current study, we show that human monocytes adhere to both full‐length Fbln7 (Fbln7‐FL) and Fbln7‐C, in part, via integrins α5β1 and α2β1. Morphologic studies and surface expression analyses of CD14, mannose receptor (CD206), major histocompatibility complex II, and CD11b receptors revealed that both Fbln7‐FL and Fbln7‐C inhibit M‐CSF‐induced monocyte differentiation. Fbln7‐C had significantly greater negative effects on cell spreading and stress fiber formation, including the production of IL‐6 and metalloproteinase‐1/‐9 compared with Fbln7‐FL. Furthermore, in an LPS‐induced systemic inflammation model, Fbln7‐C and Fbln7‐FL reduced the infiltration of immune cells, such as neutrophils and macrophages, to the inflamed peritoneum. Thus, these results suggest that Fbln7 and Fbln7‐C could modulate the activity of immune cells and have therapeutic potential for inflammatory diseases.—Sarangi, P. P., Chakraborty, P., Dash, S. P., Ikeuchi, T., de Vega, S., Ambatipudi, K., Wahl, L., Yamada, Y. Cell adhesion protein fibulin‐7 and its C‐terminal fragment negatively regulate monocyte and macrophage migration and functions in vitro and in vivo. FASEB J. 32, 4889–4898 (2018). www.fasebj.org

A dysbiotic microbiome triggers TH17 cells to mediate oral mucosal immunopathology in mice and humans

Combined patient and animal model studies implicate microbiota-triggered TH17 cells as disease drivers and therapeutic targets in periodontitis. Probing periodontitis pathology Periodontitis is a common inflammatory disease that can lead to bone loss. Dutzan et al. used a mouse model of ligature-induced periodontitis and samples from patients to demonstrate that TH17 cells drive disease. People naturally deficient in TH17 cells were less likely to develop periodontitis. These pathogenic cells were dependent on the microbiome, and targeting TH17 cells with a small-molecule inhibitor dampened inflammation and bone loss in mice. Their results reveal mechanisms behind the immunopathology and could lead to effective treatments for this disease. Periodontitis is one of the most common human inflammatory diseases, yet the mechanisms that drive immunopathology and could be therapeutically targeted are not well defined. Here, we demonstrate an expansion of resident memory T helper 17 (TH17) cells in human periodontitis. Phenocopying humans, TH17 cells expanded in murine experimental periodontitis through local proliferation. Unlike homeostatic oral TH17 cells, which accumulate in a commensal-independent and interleukin-6 (IL-6)–dependent manner, periodontitis-associated expansion of TH17 cells was dependent on the local dysbiotic microbiome and required both IL-6 and IL-23. TH17 cells and associated neutrophil accumulation were necessary for inflammatory tissue destruction in experimental periodontitis. Genetic or pharmacological inhibition of TH17 cell differentiation conferred protection from immunopathology. Studies in a unique patient population with a genetic defect in TH17 cell differentiation established human relevance for our murine experimental studies. In the oral cavity, human TH17 cell defects were associated with diminished periodontal inflammation and bone loss, despite increased prevalence of recurrent oral fungal infections. Our study highlights distinct functions of TH17 cells in oral immunity and inflammation and paves the way to a new targeted therapeutic approach for the treatment of periodontitis.

Abstract P19: Pannexin 3 Deficiency in Mice Delays the Wound Healing Process

PURPOSE: Pannexin 3 (Panx3) is a gap junction protein. We have previously shown that Panx3 plays multiple channel functions: 1) as a hemichannel to regulate intracellular ATP/cAMP levels between cells and the extracellular space, 2) as an ER calcium channel to regulate calcium flux within the cell, and 3) a gap junction to exchange ions and small molecules between cells. However, the role of Panx3 in skin tissue regeneration, proliferation, and/or differentiation is unclear. Here, we demonstrate that Panx3 plays a role in the skin wound healing process by controlling the inflammatory response, epidermal-mesenchymal transition (EMT), keratinocyte proliferation, and collagen deposition.