SH3BP2 Gain-Of-Function Mutation Exacerbates Inflammation and Bone Loss in a Murine Collagen-Induced Arthritis Model

Objective

SH3BP2 is a signaling adapter protein which regulates immune and skeletal systems. Gain-of-function mutations in SH3BP2 cause cherubism, characterized by jawbone destruction. This study was aimed to examine the role of SH3BP2 in inflammatory bone loss using a collagen-induced arthritis (CIA) model.

Methods

CIA was induced in wild-type (Sh3bp2^+/+) and heterozygous P416R SH3BP2 cherubism mutant knock-in (Sh3bp2^KI/+) mice, an SH3BP2 gain-of-function model. Severity of the arthritis was determined by assessing the paw swelling and histological analyses of the joints. Micro-CT analysis was used to determine the levels of bone loss. Inflammation and osteoclastogenesis in the joints were evaluated by quantitating the gene expression of inflammatory cytokines and osteoclast markers. Furthermore, involvement of the T- and B-cell responses was determined by draining lymph node cell culture and measurement of the serum anti-mouse type II collagen antibody levels, respectively. Finally, roles of the SH3BP2 mutation in macrophage activation and osteoclastogenesis were determined by evaluating the TNF-α production levels and osteoclast formation in bone marrow-derived M-CSF-dependent macrophage (BMM) cultures.

Results

Sh3bp2^KI/+ mice exhibited more severe inflammation and bone loss, accompanying an increased number of osteoclasts. The mRNA levels for TNF-α and osteoclast marker genes were higher in the joints of Sh3bp2^KI/+ mice. Lymph node cell culture showed that lymphocyte proliferation and IFN-γ and IL-17 production were comparable between Sh3bp2^+/+ and Sh3bp2^KI/+ cells. Serum anti-type II collagen antibody levels were comparable between Sh3bp2^+/+ and Sh3bp2^KI/+ mice. In vitro experiments showed that TNF-α production in Sh3bp2^KI/+ BMMs is elevated compared with Sh3bp2^+/+ BMMs and that RANKL-induced osteoclastogenesis is enhanced in Sh3bp2^KI/+ BMMs associated with increased NFATc1 nuclear localization.

Conclusion

Gain-of-function of SH3BP2 augments inflammation and bone loss in the CIA model through increased macrophage activation and osteoclast formation. Therefore, modulation of the SH3BP2 expression may have therapeutic potential for the treatment of rheumatoid arthritis.     

A Dictyostelium cellobiohydrolase orthologue that affects developmental timing

Dictyostelium discoideum is a facultative multicellular amoebozoan with cellulose in the stalk and spore coat of its fruiting body as well as in the extracellular matrix of the migrating slug. The organism also harbors a number of cellulase genes. One of them, cbhA, was identified as a candidate cellobiohydrolase gene based on the strong homology of its predicted protein product to fungal cellobiohydrolase I (CBHI). Expression of the cbhA was developmentally regulated, with strong expression in the spores of the mature fruiting body. However, a weak but detectable level of expression was observed in the extracellular matrix at the mound — tipped finger stages, in prestalk O cells, and in the slime sheath of the migrating slug — late culminant stages. A null mutant of the cbhA showed almost normal morphology. However, the developmental timing of the mutant was delayed by 2–4 h. When a c-Myc epitope-tagged CbhA was expressed, it was secreted into the culture medium and was able to bind crystalline cellulose. The CbhA-myc protein was glycosylated, as demonstrated by its ability to bind succinyl concanavalin A-agarose. Moreover, conditioned medium from the cbhA-myc ^oe strain displayed 4-methylumbelliferyl β-d-cellobioside (4-MUC) digesting activity in Zymograms in which conditioned medium was examined via native-polyacrylamide gel electrophoresis or spotted on an agar plate containing 4-MUC, one of the substrates of cellobiohydrolase. Taken together, these findings indicate that Dictyostelium CbhA is an orthologue of CBH I that is required for a normal rate of development.     

JSAP1, a Novel Jun N-Terminal Protein Kinase (JNK)-Binding Protein That Functions as a Scaffold Factor in the JNK Signaling Pathway

The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38alpha), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.     

Study of the structure-activity relationship of polymyxin analogues

A structure-activity relationship study on three classes of polymyxin analogues focusing on hydrophobicity was conducted.     

Excitatory Neuronal Hubs Configure Multisensory Integration of Slow Waves in Association Cortex

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Crosstalk in skin: melanocytes,keratinocytes, stem cells,and melanoma

In the vertebrate embryo, melanocytes arise from the neural crest, migrate to and colonize the basal layer within the skin and skin appendages. Post-migratory melanocytes are securely attached to the basement membrane, and their morphology, growth, adhesion, and migration are under control of neighboring keratinocytes. Melanoma is a malignant tumor originated from melanocytes or their progenitor cells. During melanocyte transformation and melanoma progression, melanocytes lose their interactions with keratinocytes, resulting in uncontrolled proliferation and invasion of the malignant cells. Melanoma cells at the advanced stages often lack melanocytic features and resemble multipotent progenitors, which are a potential melanocyte reservoir in human skin. In this mini-review, we will summarize findings on cell-cell interactions that are responsible for normal melanocyte homeostasis, stem cell self-renewal, and differentiation. Our ultimate goal is to define molecules and pathways, which are essential for normal cell-cell interactions but deregulated in melanoma formation and progression.     

Kinetics of hydrolysis, oxidation, and adsorption during olive oil degradation by activated sludge

Kinetics of the degradation of olive oil by an acclimated activated sludge were studied. Kinetic constants for the lipid removal from the mixed liquor and for that from the supernatant and for the hydrolysis step were evaluated using Michelis-Menten equations. The maximum specific reactions rates (v(max)) and the saturation constants (K(m)) were v(max) = 1.20 mg lipid mg(-1) MLVSS day(-1) and K(m) = 1290 mg/L for lipid removal from the mixed liquor; v(max) = 1.54 mg lipid mg(-1) MLVSS day(-1) and K(m) = 801 mg/L for that from the supernatant; v(max) = 1.57 mg olive oil mg(-1) MLVSS day(-1) and K(m) = 1750 mg/L for the hydrolysis of olive oil (where MLVSS refers to mixed liquor volatile suspended solids). The adsorption of olive oil by the activated sludge contributed to the lipid removal from the supernatant. The specific rate of this adsorption was also estimated. The hydrolysis, rather than the oxidation of free fatty acids, was the rate limiting step in the degradation of olive oil when the concentration of olive oil was lower than about 800 mg/L.     

The macula densa site of avian kidney

Summary The macula densa (MD) site of the kidney, at which the distal tubule is attached to the vascular pole of the glomerulus, was examined with the light microscope in domestic fowl and Japanese quail; and in the fowl also with the electron microscope. The characteristics of mammalian MD cells, as reported in the literature, are compared with those of the cells in the avian MD site. The avian cells possess some of the characteristics of mammalian MD cells and they are distinguishable from the cells in adjacent portions of the distal tubule. The Golgi system in the avian cells is apical to the nucleus, unlike in mammals where its location is basal. The cells in the avian MD sites can be considered as structurally transitional between the typical MD cells in mammals and the ordinary cells of the distal tubule. These findings are discussed in relation to the function of the avian kidney and to its control by the renin mechanism.* This investigation was supported in part by U.S. Public Health Service Research Grant HE 10338 from the National Heart Institute. We thank Mr. T. Takizawa for technical assistance. A preliminary report of this study was given at the 13th Annual Meeting of Japanese Society of Nephrology in Kyoto, 1970.     

Dietary intake of the citrus flavonoid hesperidin affects stress-resilience and brain kynurenine levels in a subchronic and mild social defeat stress model in mice

ABSTRACT

Depressive disorders are partly caused by chronic inflammation through the kynurenine (KYN) pathway. Preventive intervention using anti-inflammatory reagents may be beneficial for alleviating the risk of depression. In this study, we focused on the Japanese local citrus plant, Citrus tumida hort. ex Tanaka (C. tumida; CT), which contains flavonoids such as hesperidin that have anti-inflammatory actions. The dietary intake of 5% immature peels of CT fruits slightly increased stress resilience in a subchronic and mild social defeat (sCSDS) model in mice. Moreover, the dietary intake of 0.1% hesperidin significantly increased stress resilience and suppressed KYN levels in the hippocampus and prefrontal cortex in these mice. In addition, KYN levels in the hippocampus and prefrontal cortex were significantly correlated with the susceptibility to stress. In conclusion, these results suggest that dietary hesperidin increases stress resilience by suppressing the augmentation of KYN signaling under sCSDS.