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Multiple sclerosis ( MS) is an incurable central nervous system autoimmune disease. Understanding MS pathogenesis is essential for the development of new MS therapies. In the present study, we identified a novel micro RNA (miR) that regulates experimental autoimmune encephalomyelitis ( EAE), an animal model of MS. Expression of miR223 was up-regulated specifically in spinal cords and lymphoid organs but not in other examined tissues. A global miR223 knockout (miR223−/−) in mice led to a significant delay in EAE onset, reduction in spinal cord lesion, and lessening of neurological symptoms. These protective effects could be reproduced in bone marrow chimeras reconstituted with miR223−/− haematopoietic stem cells. We also found that miR223 deficiency reduced T helper type 1 (Th1) and Th17 infiltration into spinal cords. To address underlying mechanisms, we investigated the role of miR223 in regulating the function, development and interaction of the major immune cells. Expression of the genes associated with dendritic cell ( DC) activation ( CD86 and MHC II) and Th1 and Th17 differentiation [interleukin-12 ( IL-12) and IL-23, respectively] was significantly decreased in the spleens of miR223−/− mice bearing EAE. The miR223−/− DCs expressed significantly lower levels of basal and lipopolysaccharide-induced IL-12 and IL-23 compared with the wild-type DCs. These data are consistent with the observed lower efficiency of miR223−/− DCs to support Th1 and Th17 differentiation from naive T cells over-expressing an EAE antigen-specific T-cell receptor. Our data suggest that miR223 promotes EAE, probably through enhancing DC activation and subsequently the differentiation of naive T cells toward Th1 and Th17 effector cells. [ABSTRACT FROM AUTHOR]

Cytoskeleton organization and lysosome secretion play an essential role in osteoclastogenesis and bone resorption. The cytoplasmic dynein is a molecular motor complex that regulates microtubule dynamics and transportation of cargos/organelles, including lysosomes along the microtubules. LIS1, NDE1, and NDEL1 belong to an evolutionary conserved pathway that regulates dynein functions. Disruption of the cytoplasmic dynein complex and deletion of LIS1 in osteoclast precursors arrest osteoclastogenesis. Nonetheless, the role of NDE1 and NDEL1 in osteoclast biology remains elusive. In this study, we found that knocking-down Nde1 expression by lentiviral transduction of specific shRNAs markedly inhibited osteoclastogenesis in vitro by attenuating the proliferation, survival, and differentiation of osteoclast precursor cells via suppression of signaling pathways downstream of M-CSF and RANKL as well as osteoclast differentiation transcription factor NFATc1. To dissect how NDEL1 regulates osteoclasts and bone homeostasis, we generated Ndel1 conditional knockout mice in myeloid osteoclast precursors (Ndel1ΔlysM) by crossing Ndel1-floxed mice with LysM-Cre mice on C57BL/6J background. The Ndel1ΔlysM mice developed normally. The µCT analysis of distal femurs and in vitro osteoclast differentiation and functional assays in cultures unveiled the similar bone mass in both trabecular and cortical bone compartments as well as intact osteoclastogenesis, cytoskeleton organization, and bone resorption in Ndel1ΔlysM mice and cultures. Therefore, our results reveal a novel role of NDE1 in regulation of osteoclastogenesis and demonstrate that NDEL1 is dispensable for osteoclast differentiation and function. [ABSTRACT FROM AUTHOR]

Leucine-rich repeat kinase-1 (Lrrk1) consists of ankyrin repeats (ANK), leucine-rich repeats (LRR), a GTPase-like domain of Roc (ROC), a COR domain, a serine/threonine kinase domain (KD), and WD40 repeats (WD40). Previous studies have revealed that knockout (KO) of Lrrk1 in mice causes severe osteopetrosis, and a human mutation of Lrrk1 leads to osteosclerotic metaphysial dysplasia. The molecular mechanism by which Lrrk1 regulates osteoclast function is unknown. In this study, we generated a series of Lrrk1 mutants and evaluated their ability to rescue defective bone resorption in Lrrk1-deficient osteoclasts by use of pit formation assays. Overexpression of Lrrk1 or LRR-truncated Lrrk1, but not ANK-truncated Lrrk1, WD40-truncated Lrrk1, Lrrk1-KD, or K651A mutant Lrrk1, rescued bone resorption function of Lrrk1 KO osteoclasts. We next examined whether RAC1/Cdc42 small GTPases are direct substrates of Lrrk1 in osteoclasts. Western blot and pull-down assays revealed that Lrrk1 deficiency in osteoclasts resulted in reduced phosphorylation and activation of RAC1/Cdc42. In vitro kinase assays confirmed that recombinant Lrrk1 phosphorylated RAC1-GST protein, and immunoprecipitation showed that the interaction of Lrrk1 with RAC1 occurred within 10 min after RANKL treatment. Overexpression of constitutively active Q61L RAC1 partially rescued the resorptive function of Lrrk1-deficient osteoclasts. Furthermore, lack of Lrrk1 in osteoclasts led to reduced autophosphorylation of p21 protein-activated kinase-1 at Ser 144 , catalyzed by RAC1/Cdc42 binding and activation. Our data indicate that Lrrk1 regulates osteoclast function by directly modulating phosphorylation and activation of small GTPase RAC1/Cdc42 and that its function depends on ANK, ROC, WD40, and kinase domains., (Copyright © 2016 the American Physiological Society.)

Background: Osteosclerotic metaphyseal dysplasia (OSMD) is a unique form of osteopetrosis characterised by severe osteosclerosis localised to the bone ends. The mode of inheritance is autosomal recessive. Its genetic basis is not known., Objective: To identify the disease gene for OSMD., Methods and Results: By whole exome sequencing in a boy with OSMD, we identified a homozygous 7 bp deletion (c.5938_5944delGAGTGGT) in the LRRK1 gene. His skeletal phenotype recapitulated that seen in the Lrrk1-deficient mouse. The shared skeletal hallmarks included severe sclerosis in the undermodelled metaphyses and epiphyseal margins of the tubular bones, costal ends, vertebral endplates and margins of the flat bones. The deletion is predicted to result in an elongated LRRK1 protein (p.E1980Afs*66) that lacks a part of its WD40 domains. In vitro functional studies using osteoclasts from Lrrk1-deficient mice showed that the deletion was a loss of function mutation. Genetic analysis of LRRK1 in two unrelated patients with OSMD suggested that OSMD is a genetically heterogeneous condition., Conclusions: This is the first study to identify the causative gene of OSMD. Our study provides evidence that LRRK1 plays a critical role in the regulation of bone mass in humans., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Thyroid hormone (TH) action is mediated through two nuclear TH receptors, THRα and THRβ. Although the role of THRα is well established in bone, less is known about the relevance of THRβ-mediated signaling in bone development. On ther basis of our recent finding that TH signaling is essential for initiation and formation of secondary ossification center, we evaluated the role of THRs in mediating TH effects on epiphysial bone formation. Two-day treatment of TH-deficient Tshr(-/-) mice with TH increased THRβ1 mRNA level 3.4-fold at day 7 but had no effect on THRα1 mRNA level at the proximal tibia epiphysis. Treatment of serum-free cultures of tibias from 3-day-old mice with T3 increased THRβ1 expression 2.1- and 13-fold, respectively, at 24 and 72 h. Ten-day treatment of Tshr(-/-) newborns (days 5-14) with THRβ1 agonist GC1 at 0.2 or 2.0 μg/day increased BV/TV at day 21 by 225 and 263%, respectively, compared with vehicle treatment. Two-day treatment with GC1 (0.2 μg/day) increased expression levels of Indian hedgehog (Ihh) 100-fold, osterix 15-fold, and osteocalcin 59-fold compared with vehicle at day 7 in the proximal tibia epiphysis. Gel mobility shift assay demonstrated that a putative TH response element in the distal promoter of mouse Ihh gene interacted with THRβ1. GC1 treatment (1 nM) increased Ihh distal promoter activity 20-fold after 48 h in chondroctyes. Our data suggest a novel role for THRβ1 in secondary ossification at the epiphysis that involves transcriptional upregulation of Ihh gene.

Although thyroid hormone (TH) is known to exert important effects on the skeleton, the nuclear factors constituting the TH receptor coactivator complex and the molecular pathways by which TH mediates its effects on target gene expression in osteoblasts remain poorly understood. A recent study demonstrated that the actions of TH on myoblast differentiation are dependent on diabetes- and obesity-related protein (DOR). However, the role of DOR in osteoblast differentiation is unknown. We found DOR expression increased during in vitro differentiation of bone marrow stromal cells into osteoblasts and also in MC3T3-E1 cells treated with TH. However, DOR expression decreased during cellular proliferation. To determine whether DOR acts as a modulator of TH action during osteoblast differentiation, we examined whether overexpression or knockdown of DOR in MC3T3-E1 cells affects the ability of TH to induce osteoblast differentiation by evaluating alkaline phosphatase (ALP) activity. ALP activity was markedly increased in DOR-overexpressing cells treated with TH. In contrast, loss of DOR dramatically reduced TH stimulation of ALP activity in MC3T3-E1 cells and primary calvaria osteoblasts transduced with lentiviral DOR shRNA. Consistent with reduced ALP activity, mRNA levels of osteocalcin, ALP, and Runx2 were decreased significantly in DOR shRNA cells. In addition, a common single nucleotide polymorphism (SNP), DOR1 found on the promoter of human DOR gene, was associated with circulating osteocalcin levels in nondiabetic subjects. Based on these data, we conclude that DOR plays an important role in TH-mediated osteoblast differentiation, and a DOR SNP associates with plasma osteocalcin in men.