Activity-dependent cleavage of dyskinesia-related proline-rich transmembrane protein 2 (PRRT2) by calpain in mouse primary cortical neurons

Mutations of PRRT2 (proline-rich transmembrane protein 2) cause several neurological disorders, represented by paroxysmal kinesigenic dyskinesia (PKD), which is characterized by attacks of involuntary movements triggered by sudden voluntary movements. PRRT2 is reported to suppress neuronal excitation, but it is unclear how the function of PRRT2 is modulated during neuronal excitation. We found that PRRT2 is processed to a 12 kDa carboxy-terminal fragment (12K-CTF) by calpain, a calcium-activated cysteine protease, in a neuronal activity-dependent manner, predominantly via NMDA receptors or voltage-gated calcium channels. Furthermore, we clarified that 12K-CTF is generated by sequential cleavages at Q220 and S244. The amino-terminal fragment (NTF) of PRRT2, which corresponds to PKD-related truncated mutants, is not detected, probably due to rapid cleavage at multiple positions. Given that 12K-CTF lacks most of the proline-rich domain, this cleavage might be involved in the activity-dependent enhancement of neuronal excitation perhaps through transient retraction of PRRT2\u27s function. Therefore, PRRT2 might serve as a buffer for neuronal excitation, and lack of this function in PKD patients might cause neuronal hyperexcitability in their motor circuits

Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in the enteric nervous system

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7-a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system

Shared and Distinct Functions of the Transcription Factors IRF4 and IRF8 in Myeloid Cell Development

Interferon regulatory factor (IRF) 8 and IRF4 are structurally-related, hematopoietic cell-specific transcription factors that cooperatively regulate the differentiation of dendritic cells and B cells. Whilst in myeloid cells IRF8 is known to modulate growth and differentiation, the role of IRF4 is poorly understood. In this study, we show that IRF4 has activities similar to IRF8 in regulating myeloid cell development. The ectopic expression of IRF4 in myeloid progenitor cells in vitro inhibits cell growth, promotes macrophages, but hinders granulocytic cell differentiation. We also show that IRF4 binds to and activates transcription through the IRF-Ets composite sequence (IECS). Furthermore, we demonstrate that Irf8-/-Irf4-/- mice exhibit a more severe chronic myeloid leukemia (CML)-like disease than Irf8-/- mice, involving a disproportionate expansion of granulocytes at the expense of monocytes/macrophages. Irf4-/- mice, however, display no obvious abnormality in myeloid cell development, presumably because IRF4 is expressed at a much lower level than IRF8 in granulocyte-macrophage progenitors. Our results also suggest that IRF8 and IRF4 have not only common but also specific activities in myeloid cells. Since the expression of both the IRF8 and IRF4 genes is downregulated in CML patients, these results may add to our understanding of CML pathogenesis

Regulation of basophil and mast cell development by transcription factors

Basophils and mast cells play important roles in host defense against parasitic infections and allergic responses. Several progenitor populations, either shared or specific, for basophils and/or mast cells have been identified, thus elucidating the developmental pathways of these cells. Multiple transcription factors essential for their development and the relationships between them have been also revealed. For example, IRF8 induces GATA2 expression to promote the generation of both basophils and mast cells. The STAT5-GATA2 axis induces C/EBPα and MITF expression, facilitating the differentiation into basophils and mast cells, respectively. In addition, C/EBPα and MITF mutually suppress each other's expression. This review provides an overview of recent advances in our understanding of how transcription factors regulate the development of basophils and mast cells

Blockade of interaction of α9 integrin with its ligands hinders the formation of granulation in cutaneous wound healing

The wound healing is a complex process consisting of inflammatory reaction, proliferation of mesenchymal cells, and formation and contraction of granulation tissue. The integrin receptors play crucial roles in this process. Recently, α9 integrin has also been detected on keratinocytes within wound sites. However, its functional significance at various wound healing processes was not fully elucidated. To address the role of α9 integrin in wound healing process, we made a full-thickness skin excisional wound and treated mice with anti-α9 integrin antibody. It has been shown that wound healing process was divided into three distinct phases; first, re-epithelialization phase, second phase of granulation tissue formation, and followed by final phase of contraction of granulation tissue. We found that contraction of granulation tissue was not impaired by blocking the interaction of α9 integrin with its ligands, indicating that α9 integrin is not involved in myofibroblast differentiation. Of note, formation of granulation tissue as characterized by dense vimentin and CD31 positive area was impaired. The hindrance of granulation tissue formation is due to the inhibition of adhesion and migration of α9 integrin positive dermal fibroblasts. In conclusion, α9 integrin is involved in the formation of granulation tissue via regulating migration and adhesion of dermal fibroblasts in the full-thickness skin excisional wound model

Interleukin-17A Deficiency Accelerates Unstable Atherosclerotic Plaque Formation in Apolipoprotein E-Deficient Mice

Objective: Interleukin-17A (IL-17A), an inflammatory cytokine, has been implicated in atherosclerosis, in which inflammatory cells within atherosclerotic plaques express IL-17A. However, its role in the development of atheroscelrosis remains to be controversial. Methods and Results: To directly examine the role of IL-17A in atherosclerosis, we generated apolipoprotein E (ApoE)/IL-17A double-deficient (ApoE^[-/-]IL-17A^[-/-]) mice. Mice were fed with high-fat diet (HFD) for either 8 or 16 weeks, both starting at ages of 6-8 weeks. We found that splenic CD4+ T cells produced high amounts of IL-17A in ApoE^[-/-] mice after HFD feeding for 8 weeks. Atherosclerosis was significantly accelerated in HFD-fed ApoE^[-/-]IL-17A^[-/-] mice compared with ApoE^[-/-] mice. Splenic CD4+ T-cells of ApoE^[-/-]IL-17A^[-/-] mice after HFD feeding for 8 weeks, but not for 16 weeks, exhibited increased IFN-γ and decreased IL-5 production. Importantly, formation of vulnerable plaque as evidenced by reduced numbers of vascular smooth muscle cells and reduced type I collagen deposition in the plaque was detected in ApoE^[-/-]IL-17A^[-/-] mice after HFD feeding for 8 weeks. Conclusions: These results suggest that IL-17A regulates the early phase of atherosclerosis development after HFD feeding and plaque stability, at least partly if not all by modulating IFN-γ and IL-5 production from CD4+ T-cells

The Significance of SPP1 in Lung Cancers and Its Impact as a Marker for Protumor Tumor-Associated Macrophages

Macrophages are a representative cell type in the tumor microenvironment. Macrophages that infiltrate the cancer microenvironment are referred to as tumor-associated macrophages (TAMs). TAMs exhibit protumor functions related to invasion, metastasis, and immunosuppression, and an increased density of TAMs is associated with a poor clinical course in many cancers. Phosphoprotein 1 (SPP1), also known as osteopontin, is a multifunctional secreted phosphorylated glycoprotein. Although SPP1 is produced in a variety of organs, at the cellular level, it is expressed on only a few cell types, such as osteoblasts, fibroblasts, macrophages, dendritic cells, lymphoid cells, and mononuclear cells. SPP1 is also expressed by cancer cells, and previous studies have demonstrated correlations between levels of circulating SPP1 and/or increased SPP1 expression on tumor cells and poor prognosis in many types of cancer. We recently revealed that SPP1 expression on TAMs is correlated with poor prognosis and chemoresistance in lung adenocarcinoma. In this review, we summarize the significance of TAMs in lung cancers and discuss the importance of SPP1 as a new marker for the protumor subpopulation of monocyte-derived TAMs in lung adenocarcinoma. Several studies have shown that the SPP1/CD44 axis contribute to cancer chemoresistance in solid cancers, so the SPP1/CD44 axis may represent one of the most critical mechanisms for cell-to-cell communication between cancer cells and TAMs