TET enzymes regulate skeletal development through increasing chromatin accessibility of RUNX2 target genes.

The Ten-eleven translocation (TET) family of dioxygenases mediate cytosine demethylation by catalyzing the oxidation of 5-methylcytosine (5mC). TET-mediated DNA demethylation controls the proper differentiation of embryonic stem cells and TET members display functional redundancy during early gastrulation. However, it is unclear if TET proteins have functional significance in mammalian skeletal development. Here, we report that Tet genes deficiency in mesoderm mesenchymal stem cells results in severe defects of bone development. The existence of any single Tet gene allele can support early bone formation, suggesting a functional redundancy of TET proteins. Integrative analyses of RNA-seq, Whole Genome Bisulfite Sequencing (WGBS), 5hmC-Seal and Assay for Transposase-Accessible Chromatin (ATAC-seq) demonstrate that TET-mediated demethylation increases the chromatin accessibility of target genes by RUNX2 and facilities RUNX2-regulated transcription. In addition, TET proteins interact with RUNX2 through their catalytic domain to regulate cytosine methylation around RUNX2 binding region. The catalytic domain is indispensable for TET enzymes to regulate RUNX2 transcription activity on its target genes and to regulate bone development. These results demonstrate that TET enzymes function to regulate RUNX2 activity and maintain skeletal homeostasis.

Mechanical regulation of bone remodeling.

Bone remodeling is a lifelong process that gives rise to a mature, dynamic bone structure via a balance between bone formation by osteoblasts and resorption by osteoclasts. These opposite processes allow the accommodation of bones to dynamic mechanical forces, altering bone mass in response to changing conditions. Mechanical forces are indispensable for bone homeostasis; skeletal formation, resorption, and adaptation are dependent on mechanical signals, and loss of mechanical stimulation can therefore significantly weaken the bone structure, causing disuse osteoporosis and increasing the risk of fracture. The exact mechanisms by which the body senses and transduces mechanical forces to regulate bone remodeling have long been an active area of study among researchers and clinicians. Such research will lead to a deeper understanding of bone disorders and identify new strategies for skeletal rejuvenation. Here, we will discuss the mechanical properties, mechanosensitive cell populations, and mechanotransducive signaling pathways of the skeletal system.

Mechanical sensing protein PIEZO1 regulates bone homeostasis via osteoblast-osteoclast crosstalk.

Wolff's law and the Utah Paradigm of skeletal physiology state that bone architecture adapts to mechanical loads. These models predict the existence of a mechanostat that links strain induced by mechanical forces to skeletal remodeling. However, how the mechanostat influences bone remodeling remains elusive. Here, we find that Piezo1 deficiency in osteoblastic cells leads to loss of bone mass and spontaneous fractures with increased bone resorption. Furthermore, Piezo1-deficient mice are resistant to further bone loss and bone resorption induced by hind limb unloading, demonstrating that PIEZO1 can affect osteoblast-osteoclast crosstalk in response to mechanical forces. At the mechanistic level, in response to mechanical loads, PIEZO1 in osteoblastic cells controls the YAP-dependent expression of type II and IX collagens. In turn, these collagen isoforms regulate osteoclast differentiation. Taken together, our data identify PIEZO1 as the major skeletal mechanosensor that tunes bone homeostasis.

Paracrine and endocrine actions of bone-the functions of secretory proteins from osteoblasts, osteocytes, and osteoclasts.

The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes, and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin (SOST) that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influenceaA osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or "osteokines" from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin (OCN) secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2 (LCN2) is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain. We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.

Immune response of interferon-γ-inducible lysosomal thiol reductase (GILT) from Chinese sturgeon (Acipenser sinensis) to microbial invasion and its antioxdative activity in lipopolysaccharides-treated mammalian dentritic cells.

Interferon-γ-inducible lysosomal thiol reductase (GILT) plays an important role in the major histocompatibility complex-restricted antigen processing of endocytosed proteins via catalyzing the disulfide bond reduction in the endocytic pathway. Here, the cDNA of Chinese sturgeon (Acipenser sinensis) GILT (CsGILT) was cloned. It contained an open reading frame of 762 nucleotides encoding a protein of 254 amino acids with an estimated molecular weight of 28.1 kDa. The characteristic structural features, including a signature sequence CQHGX2ECX2NX4C, a CXXC motif, two potential N-glycosylation sites, and eight conserved cysteines were detected in the deduced amino acid sequence of CsGILT. CsGILT was widely expressed in Chinese sturgeon with the highest expression in the spleen, and CsGILT mRNA expression was significantly up-regulated when Chinese sturgeons were challenged with polyinosinic polycytidylic acid or Vibrio anguillarum. The recombinant CsGILT displayed obvious thiol reductase activity demonstrated by catalyzing the reduction of mouse IgG(H+L) by dithiothreitol into heavy chain and light chain. CsGILT also displayed significant antioxidant activity in mouse dentritic cells as indicated by its increasing GSH level and GSH/GSSG ratio, decreasing intracellular reactive oxygen species and nitric oxide levels and lipid peroxidation, as well as enhancing the activities of the antioxidative redox enzymes including catalase and superoxide dismutase. Our results suggested an important role for CsGILT in the immune response in Chinese sturgeon to pathogen invasion possibly via a conserved functional mechanism throughout vertebrate evolution, contributing to our understanding the immune biology and protection of Chinese sturgeon.

MHC class II alpha, beta and MHC class II-associated invariant chains from Chinese sturgeon (Acipenser sinensis) and their response to immune stimulation.

The major histocompatibility complex class II (MHC II) molecules play a vital role in adaptive immune response through presenting antigenic peptides to CD4+ T lymphocytes. To accomplish this physiologic function, the MHC class II-associated invariant chain interacts with the MHC II α/ß subunits and promotes their correct assembly and efficient traffic. Here, we isolated the cDNAs of MHC II α, ß and MHC II-associated invariant chains (designated as CsMHC II α, CsMHC II ß, and CsMHC II γ) from Chinese sturgeon (Acipenser sinensis). The CsMHC II α, ß, and γ mRNAs were widely expressed in Chinese sturgeon, and the highest expression was found in spleen for CsMHC II α and ß chains, while in head kidney for CsMHC II γ chain. Stimulation to Chinese sturgeon with inactivated trivalent bacterial vaccine or polyinosinic polycytidylic acid (poly(I:C)) up-regulated the expressions of CsMHC II α, and ß mRNAs, and their transcripts were overall more quickly up-regulated by poly(I:C) than by bacterial vaccine. Poly(I:C) induced higher CsMHC II γ expression than bacterial vaccine in intestine and spleen, while lower than bacterial vaccine in head kidney and liver. When co-expressed in mouse dendritic cells, the CsMHC II γ chain bound to both the MHC II α and ß chains. Furthermore, the over-expressed CsMHC II γ chain, not CsMHC II α or CsMHC II ß chain, activated NF-κB and STAT3 in mouse dendritic cells, and induced TNF-α and IL-6 expressions as well. This activity was nearly abolished by mutation of the Ser29/Ser34 to Ala29/Ala34 in CsMHC II γ. These results suggested that CsMHC II α, ß, and γ chains might play important role in immune response to pathogen microbial infection of Chinese sturgeon possibly via a conserved functional mechanism throughout vertebrate evolution, which might contribute to our understanding the immune biology of sturgeons.

Three ferritin subunit analogs in Chinese giant salamander (Andrias davidianus) and their response to microbial stimulation.

Ferritin, an evolutionarily conserved iron-binding protein, plays important roles in iron storage and detoxification and in host immune response to invading stimulus as well. In the present study, we identified three ferritin subunit analog cDNAs from Chinese giant salamander (Andrias davidianus). All the three ferritin subunit cDNAs had a putative iron responsive element in the 5'-untranslated region. Two deduced ferritin subunits (designated as cgsFerH and cgsFerM) had the highest identity of 90% to H type subunit of vertebrate ferritins, while another deduced ferritin subunit (designated as cgsFerL) had the highest identity of 84% to L type subunit of vertebrate ferritins. The Chinese giant salamander ferritin (cgsFer) was widely expressed in various tissues, with highest expression for cgsFerH and cgsFerL in liver and highest expression for cgsFerM in spleen. Infection of Chinese giant salamander with A. davidianus ranavirus showed significant induction of cgsFer expression. Both lipopolysaccharide and iron challenge drastically augmented cgsFer expression in the splenocytes and hepatocytes from Chinese giant salamander. In addition, recombinant cgsFers bound to ferrous iron in a dose-dependent manner, with significant ferroxidase activity. Furthermore, the recombinant cgsFer inhibited the growth of the pathogen Vibrio anguillarum. These results indicated that cgsFer was potential candidate of immune molecules involved in acute phase response to invading microbial pathogens in Chinese giant salamander possibly through its regulatory roles in iron homeostasis.