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Bone erosions in rheumatoid arthritis: recent developments in pathogenesis and therapeutic implications.

Bone erosions develop early in the course of rheumatoid arthritis (RA) and deteriorate progressively, causing joint damage and resulting in impaired functional capacity of patients. During the last years, considerable number of studies has increased our understanding of the pathogenetic mechanisms mediating the development of bone erosions in RA. Increased production of RANKL and other cytokines, dysregulation of innate immune mechanisms, autoantibodies specific to RA and alterations of microRNA expression stimulate differentiation and function of osteoclasts, which are responsible for the development of bone erosions. Besides, increased levels of cytokines, overproduction of antagonists of the canonical Wnt signaling pathway and deficient production of bone morphogenetic proteins result in impaired osteoblast differentiation and function, undermining the capacity of bone erosions to repair. Disease-modifying antirheumatic drugs, synthetic or biological, currently used in the treatment of RA, can halt the progression of bone erosions and may even lead to partial repair, although complete repair is unattainable. Targeting pathogenetic mechanisms participating in the erosive process may add to the therapeutic effect of DMARDs and help in the prevention or repair of bone erosions. However, more studies are still needed to confirm whether such therapeutic strategies are effective.

The purpose of this article is to review the literature on the pathogenetic mechanisms leading to the development of bone erosions in RA, as well as the implications on the treatment of this disease.

Osteoclasts are the cells responsible for bone erosions in RA
Chronic inflammation of synovial membrane (synovium) in RA results in hyperplasia and formation of pannus, a thickened, hypertrophied synovial membrane that invades the adjacent cartilage and bone and causes cartilage degradation and bone erosions. Thus, bone erosions in RA are typically located at the interface between pannus and bone at the joint margins and subchondral bone. Osteoclasts are the cells responsible for the development of bone erosions. Using Tartrate-Resistant Acid Phosphatase (TRAP) and calcitonin receptor as markers of osteoclasts, Gravallese et al. (1998) identified these cells at the pannus-bone interface where they form resorption pits leading eventually to bone erosions 5 .
Imaging studies have demonstrated that bone marrow edema in Magnetic Resonance Imaging (MRI) often precedes the development of bone erosions in RA 6-8 . Histologic analysis has shown that bone erosions in MRI in RA correspond to a localized destruction of cortical bone adjacent to replacement of bone marrow fat by inflammatory cells. In addition, MRI bone marrow edema is due to infiltration of bone marrow by inflammatory cells, making the term "osteitis" more appropriate than "bone marrow edema" 9 . In another study, histologic examination of bone samples with MRI bone edema in RA identified infiltration of bone marrow by osteoclasts, macrophages, plasma cells, as well as CD8+ T cells and B cells 10 . Therefore, infiltration of bone adjacent to inflamed synovium by inflammatory cells and osteoclasts leads to increased bone resorption and eventually development of bone erosions.
There are a number of pathogenetic mechanisms leading to osteoclast activation and development of bone erosions in RA and they will be addressed in the following sections of this article.

Receptor activator of nuclear factor kappa-B ligand (RANKL) stimulates osteoclasts in RA
Receptor activator of nuclear factor kappa-B ligand (RANKL) is a cytokine belonging to the Tumor Necrosis Factor (TNF) superfamily, which binds to receptor activator of nuclear factor kappa-B (RANK) on osteoclasts and osteoclast precursors and is a key factor for osteoclast differentiation and activation. Osteoprotegerin (OPG) is a soluble decoy receptor of RANKL, which binds to RANKL and inhibits activation of RANK. Inflamed synovium and pannus in RA produce significantly higher levels of RANKL and lower levels of OPG in comparison to healthy synovium [11][12][13] . The cells responsible for the increased expression of RANKL in the inflamed synovial membrane are Fibroblast-like Synoviocytes (FLS) and T lymphocytes. The increased RANKL/OPG ratio results in increased osteoclast differentiation and activation at the synovium-bone interface and the development of bone erosions in RA 13,14 .

The role of cytokines in osteoclast activation in RA
The central role increased cytokine production plays in osteoclastogenesis and bone erosion formation in RA is well known. However, during the last years our knowledge concerning cytokine-induced osteoclastogenesis in RA and implicated signaling pathways is constantly expanding.
Tumor necrosis factor (TNF) is a cytokine playing a principal role in the pathogenesis of RA and it is highly expressed by peripheral blood monocytes and synovial macrophages in patients with RA 15 . TNF stimulates osteoclast differentiation (osteoclastogenesis) both indirectly by enhancing RANK signaling pathway and directly by acting on osteoclast precursors. Firstly, TNF upregulates RANK expression in osteoclast precursors 16 , as well as RANKL production by fibroblast-like synoviocytes (FLS) 17 . Moreover, TNF stimulates the expression of nuclear factor of activated T cells (NFATc1) and B lymphocyte-induced maturation protein-1 (Blimp1) in osteoclast precursors. NFATc1 and Blimp1 are transcription factors which are induced by RANKL and enhance ostoclastogenesis 18,19 . On the other hand, TNF acts directly on osteoclast precursors and facilitates their differentiation 20,21 . Furthermore, TNF stimulates the expression of macrophage colony-stimulating factor (M-CSF) by bone marrow stromal cells 22 , as well as the expression of c-fms (the receptor for M-CSF) by osteoclast precursors 23 . M-CSF plays an important role in osteoclast differentiation.
Interleukin-1 (IL-1) is another cytokine participating in RA pathogenesis and is produced by peripheral blood monocytes and synovial macrophages in RA synovium 15 . IL-1 stimulates multinucleation and activation of osteoclasts. Besides, IL-1 acts synergistically with TNF in order to promote osteoclast differentiation. TNF enhances IL-1 expression and the latter induces RANKL expression by bone marrow stromal cells 24 .
IL-6 also plays part in the pathogenesis of RA and increased osteoclast activity. It is expressed by FLS and synovial macrophages of the RA synovium 25,26 and mediates RANKL induction by TNF and IL-17, since it directly stimulates RANKL expression by FLS 17 .
IL-17 is produced by T helper cells type 17 (Th17 cells) and plays an important role in RA pathogenesis and increased osteoclastogenesis. IL-17 stimulates osteoclastogenesis indirectly by enhancing RANK signaling, since it induces RANKL expression on osteoblastic cells 27 as well as RANK expression on osteoclast precursors 28 . IL-17 also enhances osteoclast differentiation and function by inducing prostaglandin-E 2 (PGE 2 ) expression on osteoblasts 29 . Besides, IL-17 acts directly on osteoclast precursors in order to promote osteoclastogenesis 30 . Furthermore, IL-17 enhances the production of cytokines TNF and IL-1 by macrophages 31 and IL-6 by fibroblasts 32 .
IL-15, IL-33 and IL-34 also promote osteoclastogenesis in RA. IL-15 is produced in inflamed synovium in RA, it induces cytokine production by T cells 33 and it enhances RANKLdependent and T cell-dependent osteoclastogenesis 34,35 . IL-33 is expressed by synovial fibroblasts in RA 36 and stimulates osteoclastogenesis 37 . The expression of IL-33 is induced by TNF and IL-1 36 . IL-34 is produced by FLS in RA and facilitates chemotactic migration of peripheral blood mononuclear cells and RANKL-dependent osteoclast differentiation. The expression of IL-34 by FLS is induced by TNF 38 .

Innate immune mechanisms and osteoclast activation in RA
During last years, the role of innate immune mechanisms in increased osteoclastogenesis and bone erosion formation in RA has come into light. These mechanisms include the ITAM signaling pathway and Toll-like receptors.
Τhe immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway Τhe Immunoreceptor Tyrosine-Based Activation Motif (ITAM) is a common sequence of four amino acids repeated twice in the cytoplasmic domains of transmembrane proteins which serve as signaling adaptors in innate and adaptive immune cells and in osteoclasts. The motif contains two tyrosine residues which are phosphorylated when the associated receptors bind to their ligands. The phosphorylated tyrosine residues form docking sites for tyrosine kinases and other proteins which activate intracellular signaling cascades 52 . The main ITAM-bearing signaling adaptors expressed by osteoclast precursors are DAP12 (DNAX-activating protein 12) and FcRγ (Fc receptor common γ subunit). Receptors associated with DAP12 are TREM2 (triggering receptor expressed on myeloid cells-2), MDL-1 (Myeloid DAP12-associated lectin 1) and Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15), whereas receptors associated with FcRγ are OSCAR (Osteoclast activating receptor), PIR-A (Paired immunoglobulin-like receptor A) and FcR (Fc receptors). The ITAM signaling pathway in osteoclast precursors acts synergistically with the RANK signaling pathway in order to induce osteoclast differentiation and activation. The activation of ITAM-signaling adaptors leads to an increase of cytosolic concentration of calcium, which is necessary for the activation of NFATc1 -a transcription factor of the RANK pathway 53 .
The ITAM signaling pathway is activated in RA. Herman et al. demonstrated that OSCAR was expressed by osteoclasts at the sites of bone erosions in the joints of patients with RA. OSCAR was also expressed by mononuclear cells adjacent to synovial microvessels and by peripheral blood monocytes (PBMCs). The expression of OSCAR by PBMCs in RA patients was significantly higher compared to healthy controls and was correlated to disease activity. Monocytes with higher OSCAR expression were more likely to differentiate into osteoclasts. The expression of OSCAR by monocytes was induced by TNF. Thus, the activation of the ITAM signaling pathway in RA leads to increased osteoclastogenesis 54 . In another study, Crotti et al. found that the percentage of cells expressing OSCAR, FcRγ, DAP12 and TREM2 was significantly higher in active RA synovium compared to healthy synovium. These cells were mostly macrophage-like cells 55 .

Toll-like receptors (TLR)
Toll-like receptors play an important role in innate immunity, since they are expressed by innate immune cells and are activated by pathogen-associated molecular patterns. TLRs are also expressed by osteoblasts and osteoclasts and participate in osteoclastogenesis. Activation of TLRs in osteoblasts induces the expression of RANKL and TNF, leading to increased osteoclastogenesis. Activation of TLRs in osteoclast precursors in early stages of differentiation inhibits osteoclastogenesis, whereas in cells having started osteoclastic differentiation, TLR activation enhances osteoclastogenesis. TLRs also promote the survival of mature osteoclasts 56 .
The expression of Toll-like Receptor-5 (TLR5) in RA synovial tissue and fluid macrophages and in RA peripheral blood monocytes is significantly higher compared to control monocytes. Besides, the expression of TLR5 correlates with RA disease activity. TLR5 activation in RA monocytes leads to increased TNF expression, while TLR5 expression is enhanced by TNF (positive feedback regulation). Moreover, TLR5 expression in RA macrophages is induced by IL-8 and IL-17 57 . Another study demonstrated that ligation and activation of TLR5 in monocytes from RA patients results in stimulation of RANK expression and increased differentiation into mature osteoclasts. Furthermore, stimulation of TLR5 in a mouse model of RA increased TNF production, joint inflammation, osteoclast infiltration and bone erosions of the inflamed joints. On the other hand, treatment with anti-TLR5 antibody reduced joint inflammation and bone erosions in the RA mouse model 58) . Thus, it appears that increased expression of TLR5 in RA participates to osteoclastogenesis and formation of bone erosions.
TLR4 is another Toll-like receptor whose role in RA pathogenesis and osteoclastogenesis is being investigated. Patients with RA have increased levels of endogenous TLR4 ligands, such as proteins S100A8, S100A9, tenascin-C and HMGB-1 (high mobility group box chromosomal protein 1), in peripheral blood and in synovial fluid [59][60][61][62][63] . Furthermore, stimulation of TLR4 in RA synovial fibroblasts induces RANKL expression by them 64 . Animal models of RA exhibit increased expression of TLR4 in synovial membrane of the inflamed joints, while deletion of TLR4 gene results in reduced joint inflammation, reduced osteoclast infiltration and reduced bone erosions of the joints [65][66][67] . In addition, mouse RA models exhibit increased expression of endogenous TLR4 ligands, which enhance osteoclastogenesis via TNF and IL-6. Treatment with anti-TLR4 antibody reduced joint inflammation and osteoclast infiltration of the joints of the RA mouse model 68 .
Therefore, apart from TLR5, TLR4 also seems to participate in bone erosion formation in RA. However, more studies are needed to elucidate the role of Toll-like receptors in osteoclastogenesis in RA and the pathogenetic mechanisms that lead to their activation.

Autoantibody-mediated osteoclastogenesis in RA
Anti-citrullinated protein antibodies (ACPA) Anti-Citrullinated Protein Antibodies (ACPA) are detected in patients with RA and are useful in RA diagnosis, since they are characterised by high specificity. Besides, positive ACPA in RA patients are predictive for the development of severe disease and of bone erosions in the joints 69 . Furthermore ACPA positive healthy individuals, without clinical signs of RA, exhibit bone loss periarticularly. Considering that ACPA positivity precedes clinical onset of the disease by years and that bone erosions may be detected in RA patients within the first few weeks after diagnosis, it may be speculated that osteoclast activation and bone resorption commence before clinical onset of RA and are caused not only by synovitis and cytokines but also by autoantibodies like ACPA 70 .
Harre et al. investigated the effect of ACPA on osteoclastogenesis. They demonstrated that osteoclasts and osteoclast precursor cells express enzymes causing protein citrullination (Peptidylarginine Deiminases, PAD) and that citrullination of vimentin in osteoclast precursors is increased during their differentiation. ACPA with specificity to citrullinated vimentin are detected in serum of RA patients and bind to the surface of osteoclast precursors. Harre et al. showed that binding of citrullinated vimentin-specific ACPA to osteoclast precursors stimulates osteoclastogenesis and bone resorptive activity of osteoclasts. They also elucidated the mechanism for this effect; ACPA induce TNF expression by osteoclast precursors, which acts in an autocrine way to enhance osteoclastogenesis. In addition, ACPA induce the expression of RANK and the receptor of M-CSF by osteoclast precursors 71 . The induction of TNF production by monocytes and macrophages caused by ACPA has been demonstrated by another study as well 72 .
In a recent study, Krishnamurthy et al. demonstrated that ACPA specific to citrullinated vimentin and enolase promote osteoclastogenesis, whereas ACPA specific to other citrullinated epitopes fail to do so. They also showed that ACPA induce the expression of IL-8 by osteoclast precursor cells, which acts in an autocrine way in order to facilitate osteoclastogenesis. Inhibition of IL-8 with an anti-IL-8 antibody neutralized this effect of ACPA, meaning that IL-8 probably mediates the osteoclastogenic effect of ACPA 73 .
Further on, in another report it has been manifested that in ACPA-negative patients higher femoral z-scores were observed, as well as lower osteoporosis prevalence 74 . On the other hand, it has been reported that in ACPA-positive patients lower BMI and disease duration were observed. Finally, lumbar z-score was higher in ACPA-negative patients with a marginal significance, as well as, an inverse correlation between ACPA titer and femoral neck, total femur, and lumbar Z-score were observed 74 .
Therefore, increasing evidence indicates a role for ACPA in osteoclastogenesis, bone erosion formation and bone loss in RA. Further studies are needed to show if these findings may have an application in clinical practice.

Autoantibody immune complexes
Besides ACPA, it seems that autoantibody immune complexes (complexes derived from autoantibodies binding to their antigen) also promote osteoclastogenesis in RA. Osteoclast precursor cells and mature osteoclasts express fragment crystallizable (Fc) γ receptors (FcγR) in the cell surface, which recognize and bind to IgG antibodies (immunoglobulin G). Seeling  microRNAs regulate osteoclastogenesis in RA microRNAs (miRNAs) are small, single-stranded, noncoding RNAs, consisting of 20-25 nucleotides, which regulate gene expression at post-transcriptional level. miRNAs bind to the 3'-Untranslated Region (3'-UTR) of their target messenger RNA (mRNA) via partial complementarity and they inhibit its translation or cause its degradation. Therefore, miRNAs inhibit the expression of their target gene at posttranscriptional level. miRNAs are encoded either by separate miRNA genes or by sequences located within the introns of other genes. Multiple miRNAs may regulate the expression of one target gene and each miRNA may regulate the expression of multiple target genes 77 . The expression of 90% of proteincoding genes is regulated by miRNAs. As a result, miRNAs participate in several biological processes, such as cell proliferation, cell differentiation, apoptosis and immune cell lineage commitment. Besides, altered expression of miRNAs is involved in the development of various diseases. During the last years, the involvement of miRNAs in the pathogenesis of RA has drawn notable attention and has been studied broadly 78 84 . miR-223 is also upregulated in the joints of a mouse model of RA, while inhibition of miR-223 ameliorated joint inflammation, attenuated osteoclastogenesis and reduced bone erosions in the RA mouse model 83 .
Another miRNA upregulated in RA is miR-146, which is overexpressed in synovial membrane macrophages and T cells from patients with RA, as well as in peripheral blood monocytes and T cells 85,86 . However, unlike miR-155 and miR-223, miR-146 inhibits osteoclastogenesis, since its target is TNF Receptor-Associated Factor 6 (TRAF6) which participates in RANKL signaling and in osteoclastogenesis 87 . The expression of miR-146 is induced by NF-κΒ, while its targets TRAF6 and IRAK1 (IL-1 receptor-associated kinase 1) participate in innate immunity, cytokine production and osteoclastogenesis. Thus, it seems that the role of miR-146 in RA involves control of cytokine signaling and osteoclastogenesis through a negative feedback regulation loop 87,88 . Nakasa et al. investigated the potential of utilizing miR-146 for the prevention of bone erosions in RA. They administrated miR-146 in a mouse model of RA, resulting in a reduction of osteoclast infiltration and prevention of bone destruction without amelioration of joint inflammation 87 .
miR-124 is another miRNA which was recently studied for its potential of being used in the treatment of RA. One of miR-124 targets is NFATc1, which participates in RANKL signaling and osteoclastogenesis. Administration of miR-124 in a rat model of RA resulted in amelioration of arthritis, downregulation of NFATc1 and RANKL expression, inhibition of osteoclastogenesis, reduction of osteoclast numbers and attenuation of bone destruction 89 .
Therefore, alterations in miRNA expression contribute in increased osteoclastogenesis and development of bone erosions in RA joints. Besides, certain miRNAs that inhibit osteoclast differentiation could be used in order to attenuate bone destruction in RA. However, more preclinical studies in this field are needed, in order to address delivery and safety issues, before miRNAs are tested in human patients with RA.

Osteoblast differentiation and function are impaired in RA
Development of bone erosions in RA is attributed not only to enhanced osteoclast differentiation and activation but also to impaired osteoblast differentiation and function. Inhibition of differentiation and function of osteoblasts in RA leads to impaired bone formation and limited capacity of bone erosions to repair. Walsh et al. demonstrated that bone formation was reduced at bone surfaces adjacent to inflamed synovial membrane at sites of bone erosion in a mouse model of RA, compared to bone surfaces distal to the inflamed synovium. In addition, there was a paucity of mature osteoblasts at the sites of bone erosion, whereas there was an abundance of osteoblast progenitor cells. Thus, inflammation impairs osteoblast differentiation and bone formation, leading to failure of bone erosions to repair in RA 90 . Besides, resolution of inflammation in the same mouse model of RA led to an increase in the number of mature osteoblasts populating the sites of bone erosion, resulting in increased bone formation and erosion repair 91 .
The pathogenetic mechanisms leading to impaired osteoblast differentiation and function and reduced capacity of the erosions to repair will be addressed in the following sections.

Cytokines inhibit osteoblast differentiation and function in RA
TNF, a key factor in RA pathogenesis, is a potent inhibitor of osteoblast differentiation 92,93 . Ιt promotes degradation of transcription factor Runx2, which plays central role in osteoblast differentiation 94 . Furthermore, TNF inhibits the production of several bone matrix components by osteoblasts, such as type I collagen and osteocalcin, as well as the production of alkaline phosphatase, which participates in bone matrix mineralization. Thirdly, TNF induces the apoptosis of osteoblasts 95 . Besides, TNF-transgenic mice, a mouse model of RA which overexpress TNF, are characterised by impaired osteoblast differentiation and activity, leading to reduced bone formation and lack of repair in bone erosions 96 .
IL-1, another cytokine participating in RA pathogenesis, inhibits osteoblast proliferation and expression of bone matrix components, resulting in reduced bone formation 97 . Furthermore, IL-1 inhibits chemotactic migration of osteoblasts and impairs their recruitment to sites of bone erosions in RA 98 .
IL-6 also takes part in RA pathogenesis and has been recently demonstrated to mediate the inhibition of osteoblast differentiation by TNF. Furthermore, IL-6 inhibits bone morphogenetic protein (BMP)-2/7-mediated osteoblast differentiation 99 .

The canonical Wnt signaling pathway is inhibited in RA
The canonical Wnt signaling pathway plays a key role in osteoblast differentiation and activity. It is activated by soluble Wnt ligands, which bind to the Frizzled protein receptors and the lipoprotein receptor-related protein (LRP)-5/6 co-receptors. Activation of the canonical Wnt pathway leads to inhibition of Glycogen Synthase Kinase (GSK)-3β and translocation of transcription factor β-catenin to nucleus, where it induces expression of genes controlling osteoblast differentiation. The canonical Wnt pathway is inhibited by soluble antagonists, including secreted Frizzled-Related Proteins (SFRP), Dickkopf (Dkk) proteins and Sclerostin (SOST). SFRPs bind to Wnt ligands and prevent their interaction with the Frizzled-LRP5/6 complex, while Dkk proteins and sclerostin bind to LRP5/6 co-receptors and inhibit Wnt pathway activation 100 .
Several studies have investigated the role of Wnt antagonists in osteoblast inhibition and development of bone erosions in RA. Diarra et al. (2007) demonstrated that RA synovial tissue fibroblasts express increased levels of Dickkopf-1 (Dkk-1) and TNF induces Dkk-1 expression. In addition, serum levels of Dkk-1 are increased in RA patients in comparison with healthy population and are correlated with disease activity. Besides, inhibition of Dkk-1 with an anti-Dkk-1 antibody in a mouse model of RA enhanced osteoblast differentiation and function, increased bone formation and prevented development of bone erosions, while it did not influence joint inflammation. Therefore, increased expression of the Wnt signaling antagonist Dkk-1 in RA contributes to development of bone erosions via inhibition of osteoblast differentiation and function 101 . Other studies confirmed increased expression of Dkk-1 by synovial fibroblasts in RA 102 and increased serum levels of Dkk-1 in RA patients [103][104][105] . Dkk-1 peripheral blood levels correlate with disease activity and are associated with increased risk of developing bone erosions. Treatment with TNF inhibitors and an IL-1 receptor antagonist reduces Dkk-1 levels in RA patients [103][104][105] . Besides, three single nucleotide polymorphisms (SNPs) of the Dkk-1 gene were associated with bone destruction in a cohort of RA patients, while one Dkk-1 SNP was associated with serum levels of Dkk-1 106 . Furthermore, Dkk-1 enhances TNFmediated expression of sclerostin by osteoblasts 107 , while, unlike TNF, IL-6 suppresses Dkk-1 expression by synovial fibroblasts 108 .
Besides Dkk-1, the expression of other Wnt signaling antagonists is upregulated in arthritic joints as well. In a mouse model of RA, in which osteoblast differentiation was impaired at sites of bone erosion, synovial membrane expressed increased levels of Dkk-1, Dkk-3, SFRP-1, SFRP-2 and SFRP-4 in comparison with nonarthritic mice 90 . In the same mouse model of RA, resolution of inflammation resulted in downregulation of the expression of SFRP-1 and SFRP-2 by synovial membrane and upregulation of the Wnt ligand Wnt10b, leading to an increase in osteoblast differentiation, bone formation and repair of erosions 91 .
Sclerostin is another Wnt signaling antagonist and polymorphisms (SNPs) of its gene have been associated with bone destruction of the joints in patients with RA 106 . In a mouse model of RA (TNF transgenic mice), inhibition of sclerostin with a monoclonal antibody stopped the progression of bone erosions, as well as periarticular and systemic bone loss. Moreover, combination treatment with anti-sclerostin and anti-TNF antibodies led to an increase of periarticular osteoblast numbers and regression of bone erosions 109 . On the other hand, a small study with 40 RA patients did not find significantly different levels of serum sclerostin between RA patients and healthy controls. It also did not find significant correlation between serum sclerostin level and progression of bone destruction in the joints 110 . In addition, inhibition of sclerostin in mice with collagen-induced arthritis (mouse model of RA) was not able to prevent the development of bone erosions or repair them, although it prevented or reversed systemic bone loss 111 . Furthermore, a recent study by Wehmeyer et al. found that sclerostin inhibition in TNF transgenic mice accelerates inflammatory arthritis and join destruction. In another mouse model of RA (K/BxN serumtransfer induced arthritis model), which is independent of TNF signaling, sclerostin inhibition ameliorated disease severity. Wehmeyer et al. concluded that sclerostin may play a protective role in TNF-mediated inflammation 112 . Therefore, more studies are needed on the role of sclerostin in RA pathogenesis and development of bone erosions.
Besides, alterations in the expression of microRNAs (miRNAs) by inflamed synovial tissue influence bone formation in RA and potentially participate in the development of bone erosions. Recent study on a mouse model of RA identified 22 miRNAs whose expression in synovial tissue differed between arthritic and nonarthritic mice and several of which targeted Wnt and BMP signaling pathway. miR-221-3p, which was overexpressed in inflamed synovial tissue, targets Dkk-2, which facilitates osteoblast differentiation and bone matrix mineralization. Moreover, TNF induced the expression of miR-221-3p by fibroblast-like synoviocytes (FLS) derived from RA patients, as well as the secretion of miR-221-3p by FLS, packaged in extracellular vesicles called exosomes. Therefore, it appears that inflammation in RA synovium upregulates the expression of miR-221-3p by FLS. FLS secrete exosomes containing miR-221-3p, which are taken up by osteoblasts at erosion sites. As a result, Dkk-2 expression in osteoblasts is downregulated, leading to inhibition of osteoblast differentiation and defective bone formation in RA erosions 113 .
Thus, increasing number of preclinical and clinical studies indicate a pathogenetic role for the canonical Wnt signaling pathway in RA. It seems that downregulation of this pathway leads to impaired osteoblast differentiation and activity and consequently, to reduced capacity of bone erosions to repair. Preclinical studies show that these findings could have implications in RA treatment, although more studies are required in this direction.

Bone morphogenetic protein (BMP) signaling in RA
Bone Morphogenetic Protein (BMP) signaling and particularly BMP-2, -4, -5, -6 and -7 play an important role in osteoblast differentiation and induce bone formation 114 . Expression of BMP-4 and BMP-5 is significantly decreased in synovial tissue of RA patients compared to that of healthy controls 115 and may be partially responsible for reduced bone formation at sites of bone erosion. In addition, expression of BMP-4, BMP-6 and BMP-7 was found to be downregulated in the joints of a mouse model of RA, whereas expression of BMP receptors type IA and II was upregulated 116 . On the other hand, serum levels of BMP-7 were higher in RA patients in comparison with healthy controls 117 .
BMP-3 is a BMP signaling antagonist and its involvement in the erosive process in RA was recently studied on two murine models of RA. Matzelle et al. (2016) found that BMP-3 was highly expressed by osteoblasts at the sites of bone erosion and also TNF induced BMP-3 expression. Thus they proposed that upregulated BMP-3 inhibits osteoblast differentiation and function in RA and contributes to the development of bone erosions 118 .
Therefore, dysregulated BMP signaling seems to participate in the inhibition of osteoblast activity and defective bone formation at sites of bone erosions in RA, although further studies are needed in this direction.

Therapeutic implications
Bone erosions progress in time, cause joint damage and impair functional capacity of patients 3,4 . Thus, inhibition of erosive process and repair of bone erosions is an important therapeutic target in RA. Conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), such as methotrexate, sulfasalazine and chloroquine, have demonstrated limited results, since they achieve limited repair of bone erosions in a small proportion (7-10%) of RA patients 119,120 . Thus, attention has been drawn to biological agents (biological DMARDs), whose introduction in RA therapeutics has permitted the achievement of clinical remission or low disease activity. During the last years, it has been investigated whether control of inflammation with biological agents in RA can lead to the repair of bone erosions.
Finzel et al. studied the effect of TNF inhibitors (TNFi) on bone erosions; they used high resolution micro-computed tomography (μCT) in order to assess width and depth of bone erosions in RA patients treated with TNFi versus methotrexate. Bone erosions in patients receiving TNFi showed partial repair and decrease in depth after 1 year of treatment, in contrast to bone erosions in patients receiving methotrexate which deteriorated. Repair was noted only in erosions with evidence of bone apposition (sclerosis). Moreover, deeper erosions were more prone to repair, implying that repair begins from the endosteal lining and the bone marrow 121 . Other studies have also shown that TNFi halt the progression of bone erosions and may achieve partial repair of them [122][123][124] . Finzel et al. (2013) also investigated the effect of blockade of IL-6 receptor (IL-6R) with a monoclonal antibody on bone erosions in RA patients. IL-6R blockade induced partial repair in erosions exhibiting sclerosis; repair was more prominent in larger erosions 125 . Other studies also showed that blockade of IL-6R stops the progression of bone erosions in RA 126,127 . Furthermore, abatacept, a biological agent that inhibits T cell co-stimulation, has been shown to slow the progression of bone erosions in RA patients 128 . Besides biological agents, targeted synthetic DMARDs, such as JAK inhibitors, have been recently added in the armamentarium of RA therapies. Janus kinases (JAK) participate in pathways transmitting signals of multiple cytokine receptors which take part in RA pathogenesis 129,130 . Tofacitinib inhibits JAK1 and JAK3 and has been shown to arrest the progression of bone erosions in RA patients 129,131 . At the same time, Baricitinib, blocks the JAK1 and JAK2 kinases. Baricitinib reversibly inhibits JAK1 and JAK2 while it is less selective for the JAK3 kinase. In general, JAK inhibitors, act through a signal transduction pathway involving STAT proteins, which conclusively regulates down-stream gene expression in immunological cells 132 . Recently, baricitinib was shown to reduce the progression of bone erosions and structural joint damage in patients with RA 133 .
Therefore, biological and synthetic DMARDs control inflammation in RA, achieve clinical remission or low disease activity, halt the erosive process and lead to partial repair of bone erosions. However, these drugs cannot induce full repair and regression of the erosions. A reason for this failure seems to be residual joint inflammation, in spite of clinical remission. In a mouse model of RA mentioned above, inflammation inhibited osteoblast differentiation and function in the sites of bone erosions 90  Besides, bone erosions which progressed in RA patients despite treatment with TNFi were characterized by presence of inflammation (osteitis) in MRI 122 . Thus, administration of osteoanabolics alongside DMARD treatment was considered in order to achieve full erosion repair. However, although coadministration of parathormone (PTH) and TNFi in a mouse model of RA led to full regression of bone erosions 96 , coadministration of PTH analogue teriparatide and TNFi in RA patients failed to achieve erosion repair 135,136 . Apart from teriparatide, denosumab is another antiosteoporotic drug which was studied for its effect on bone erosions in RA. Denosumab is a monoclonal antibody targeting RANKL, with anticatabolic and antiosteoclastogenic effects. Several studies have shown that denosumab halts the erosive process in RA [137][138][139] . Moreover, a recent study found that denosumab led to partial repair of bone erosions in RA patients, since it reduced their width, depth and volume. Moreover denosumab increased bone mineral density in the margin of the erosions 140 .
Lately, inhibition of pathogenetic mechanisms participating in the erosive process has been studied in preclinical studies as a novel therapeutic strategy in RA treatment. Blockade of TLR4 with a monoclonal antibody in a mouse model of RA reduced osteoclast infiltration of the joints 68 , while blockade of TLR5 inhibited osteoclast differentiation and reduced bone erosions in the same RA mouse model 58 . Moreover, enhancement of the sialylation of IgG immune complexes with the sialic acid precursor N-acetylmannosamine (ManNAc) in a mouse model of RA led to a significant reduction of osteoclast infiltration in the joints and prevented the formation of bone erosions 76 . Besides, inhibition of miR-223 in a RA mouse model attenuated osteoclastogenesis and reduced bone erosions 83 , while administration of miR-146 reduced osteoclast infiltration and prevented bone destruction 87 . Similarly, administration of miR-124 downregulated NFATc1 and RANKL expression, inhibited osteoclastogenesis, reduced osteoclast numbers and attenuated bone destruction 89 . However, these are preclinical studies in animal models of RA. Clinical studies are needed in order to determine whether these strategies are effective in the treatment of RA and in the prevention of bone erosions.

Gene therapy in RA
Besides, new innovative therapeutic approaches have emerged for the treatment of RA, including gene therapy and cell based therapy. Several gene targets have been studied for RA gene therapy. In a double-blinded, placebo-controlled, phase 1 study, an adeno-associated virus vector containing a TNF antagonist gene was injected intra-articularly in patients with RA. It was found to be safe and well tolerated, but was not shown to be superior to placebo after 12 weeks of follow-up 141 . In another clinical study, two patients with RA received ex vivo gene therapy with the gene of IL-1 receptor antagonist (IL-1Ra). Autologous synovial fibroblasts were transduced with a retrovirus carrying IL-1Ra and then were injected in the arthritic joints of the patients. After 4 weeks, pain and swelling were reduced in arthritic joints, while increased expression of IL-1Ra and reduced expression of IL-1 and matrix-metalloproteinase-3 were measured in the synovium 142 . In a more recent study on an animal model of RA, the IL-1Ra gene was transfected in mesenchymal stem cells (MSCs) which were then encapsulated in alginatepoly-L-lysine (APA) microcapsules. The APA microcapsules containing the IL-1Ra gene modified MSCs were shown to attenuate the arthritis in the rat model of RA 143 . IL-10 is an anti-inflammatory cytokine which has also been studied in RA gene therapy. Nanoparticles containing IL-10 encoding plasmid DNA were injected in a rat model of RA, resulting in a significant reduction of systemic and joint expression of pro-inflammatory cytokines (TNF, IL-1 and IL-6), as well as in attenuation of inflammation and joint damage 144 .
Apart from cytokines, calcium channels have also been studied as gene targets in RA. CD4 T cells of patients with RA express increased levels of calcium release-activated calcium (CRAC) channels, resulting in upregulation of Ca 2+ influx and expression of pro-inflammatory cytokines 145 . Systemic gene silencing of CRACM3 in a RA mouse model, using lentiviral vectors containing short hairpin RNA targeting this specific gene, resulted in significant decrease of cytokine expression (including TNF, IL-2, IL-6, Il-17), a marked attenuation of arthritis severity, as well as a significant reduction on inflammatory infiltration, pannus formation, cartilage destruction and bone erosions 146 . Therefore, gene therapy in RA has shown its effectiveness in several preclinical and some clinical studies and it is a promising approach in RA treatment. However, additional studies are needed in order to attain more information concerning pharmacokinetics and safety issues, before RA gene therapy reaches clinical practice 147 .

Cell based therapy in RA
Another novel field in RA therapeutics is cell based therapy; mesenchymal stem cells and dendritic cells have been studied during the last years for their potential in the treatment of RA. Thus, the systemic administration of mesenchymal stem cells (MSCs) in RA patients and in animal models of RA has been shown to have anti-inflammatory and immunomodulatory effects. Indeed, MSC transplantation has been shown to reduce the number of GM-CSF-expressing CD4 T cells 148 , as well as the number of T follicular helper cells 149 and CD4 Th1 and Th17 cells 150 , which play central role in RA pathogenesis. Besides, MSC administration leads to an increase in the number of regulatory T cells in draining lymph nodes 148 . Therefore, systemic administration of MSCs in animal models of RA attenuates the severity of arthritis, since it reduces synovitis, formation of pannus and destruction of cartilage and bone 148,150 . Besides, MSCs attenuate local bone destruction, periarticular and systemic bone loss in animal RA models, by inhibiting RANKL-induced osteoclastogenesis 151 . Two clinical trials have studied the administration of MSCs in RA patients who have not responded to standard therapy. The first one enrolled 172 patients with RA who received human umbilical cord MSCs (UC-MSCS) plus DMARDs or DMARDs alone. No serious adverse events were observed and UC-MSCs reduced TNF and IL-6 serum levels, increased the population of regulatory T cells in peripheral blood and induced a significant remission of RA 152 . Recently, a multicentre, randomized, single-blinded (double-blinded for efficacy), placebo-controlled, phase Ib/IIa clinical trial showed the safety of allogenic adipose-derived MSCs in 53 patients with active refractory RA. In addition, it demonstrated a trend for clinical efficacy for the MSCs 153 .
Tolerogenic dendritic cells have also been studied as a novel therapeutic approach for RA. Dendritic cells (DCs) play key role in immune system since they present antigens to T cells. Tolerogenic DCs present antigens without adequate costimulation, resulting in T cell anergy, clone deletion, induction of regulatory T cells and ultimately, antigen tolerance. Ren et al. incubated immature DCs with tacrolimus in order to generate tolerogenic DCs and then injected them to a mouse model of RA. Tolerogenic DCs attenuated the development of arthritis and altered the population of CD4 T cells, promoting Th1 and suppressing Th17 differentiation 154 . In another pleclinical study, Zhang et al. injected IL-10-and TGF-betaexpressing DCs to a RA mouse model and demonstrated that these cells ameliorated arthritis, reduced cartilage and bone erosions, inhibited Th17 differentiation and induced generation of regulatory T cells 155 . Besides, Park et al. found that tolerogenic semimature DCs ameliorate arthritis in a mouse model of RA, by enhancing regulatory T cell population and suppressing Th1/Th17 immunity 156 . Furthermore, a phase 1 clinical trial generated tolerogenic DCs by treating DCs with an NF-κΒ inhibitor and exposing them to four citrullinated peptide antigens. The DCs were then administered intradermally to 18 RA patients who were HLA-DRB1 and ACPA positive. The immunotherapy was well tolerated, increased regulatory T cell population and reduced disease activity 157 . Recently, another phase 1 clinical trial assessed the safety of intraarticular autologous tolerogenic DCs in patients with RA and inflammatory arthritis. The tolerogenic DCs were incubated with autologous synovial fluid as a source of autoantigens and then were injected arthroscopically to the patients. Intraarticular tolerogenic DC therapy was demonstrated to be safe, although no clinical or immunomodulatory effects in peripheral blood were detected 158 .
Therefore, mesenchymal stem cells and tolerogenic dendritic cells have demonstrated promising results in preclinical and in preliminary clinical studies. They have immunomodulatory effects and their administration in early stages of RA could achieve remission. However, larger clinical studies are needed in order to provide more evidence concerning their efficacy and safety.

Conclusion
Bone erosions develop early in the course of RA and are predictive of a worse prognosis. They deteriorate gradually and cause joint damage, resulting in impaired functional capacity and disability. Lately, a considerable number of studies have increased our understanding of the pathogenetic mechanisms participating in the development of bone erosions in RA. Osteoclasts are the responsible cells and multiple factors have been identified to stimulate their differentiation and function. RANKL and other cytokines have been known for a long time to enhance osteoclastogenesis, but the role of other pathways has also been revealed recently. Thus, dysregulation of innate immune mechanisms in RA -including both activation of the ITAM signaling pathway and increased expression of Toll-like receptors in osteoclast precursor cells -results in increased osteoclastogenesis. In addition, anti-citrullinated protein antibodies bind to citrullinated protein epitopes on the cellular membrane of osteoclast precursors and stimulate their differentiation, while autoantibody immune complexes bind to receptors on the same cells and also enhance their differentiation. Furthermore, dysregulation of microRNA expression by osteoclasts and cells of the synovial membrane contributes to increased osteoclastogenesis in RA. Besides to excessive osteoclastogenesis, impaired osteoblast differentiation and function also plays part in bone erosion formation in RA. Inflamed synovial membrane produces increased levels of cytokines and antagonists of the canonical Wnt signaling pathway, which inhibit osteoblast differentiation and function. In addition, lower production of bone morphogenetic proteins impairs osteoblastogenesis.
Increased understanding of the mechanisms contributing to the development of bone erosions in RA has created new therapeutic targets. Currently existing diseasemodifying antirheumatic drugs (DMARDs) -either synthetic or biological -control inflammation and disease activity, alleviate symptoms, stop the development of bone erosions and may even partially repair some of them. However, DMARDs never fully repair existing bone erosions. Targeting pathogenetic mechanisms taking part in the erosive process may add to the therapeutic effect of DMARDs and achieve prevention and full repair of bone erosions in RA. There are a small number of preclinical studies in animal models of RA indicating the efficacy of blocking TLR4 and TLR5 in preventing the erosive process, as well as the efficacy of enhancing sialylation of immune complexes, inhibiting miRNAs that facilitate osteoclastogenesis or administrating miRNAs that reduce osteoclastogenesis. Furthermore, novel therapeutic approaches have emerged, including gene therapy and cell based therapy. Nevertheless, more preclinical and clinical studies are needed in order to confirm whether these therapeutic strategies are effective, safe and prevent disease progression and bone erosions. Besides, considering that low bone mineral density at the cortical bone has been associated with bone erosions in patients with RA, an interesting approach would be to treat with agents that not only address inflammation but also improve cortical bone 159


extfieldsofstudy: Medicine Chemistry

text:
Collapsin Response Mediator Protein-2 Ameliorates Sevoflurane-Mediated Neurocyte Injury by Targeting PI3K-mTOR-S6K Pathway

Background Collapsin response mediator protein-2 (CRMP-2) is the first member of the CRMP family that has been identified in primary neuronal cells; it was originally found and identified in the regulation of microtubule dimerization into microtubules. Material/Methods In the present study, we aimed to investigate the roles and mechanisms of CRMP-2 in sevoflurane-induced neurocyte injury. Cell viability, proliferation, and apoptosis were measured by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Colorimetry was performed to measure the activity of caspase-3. Western blot and quantitative real-time reverse transcription assays were used to evaluate the related mRNAs and proteins expression. Results We found that CRMP-2 reversed the inhibitory effect of sevoflurane on the viability of nerve cells. Moreover, CRMP-2 accelerated the proliferation and suppressed the apoptosis of sevoflurane-induced nerve cells. CRMP-2 modulated the expression levels of apoptosis-associated protein in sevoflurane-induced nerve cells. Furthermore, it was demonstrated that CRMP-2 impacted the PI3K-mTOR-S6K pathway. Conclusions CRMP2 ameliorated sevoflurane-mediated neurocyte injury by targeting the PI3K-mTOR-S6K pathway. Thus, CRMP2 might be an effective target for sevoflurane-induced neurocyte injury therapies.

Background
Sevoflurane (SEV) was first discovered by Terrell and was synthesized by Regan in 1968 [1,2]. It is well known that SEV is commonly used in pediatric anesthesia [3]. However, at present, the cytoprotective effect or toxicity effect of SEV on pediatric patients remains controversial. A previous investigation demonstrated that SEV ameliorated the cognitive functions of 4-to 5-month-old rats [4]. It has also been reported that SEV affected the spatial reference memory and spatial working memory of P7 rats [5]. Another study found that repeated exposure to volatile anesthetics, such as isoflurane and SEV, enhanced the GABA A receptor activity and increased glutamate levels during subsequent neurodevelopmental stages, suggesting that it is a compensatory response [6,7]. However, the accurate pathogenesis of SEV-induced neurocyte injury is still unclear.
The PI3K/mTOR/S6K pathway, a crucial signaling pathway, combines inputs from at least 5 major intracellular and extracellular cues, involving amino acids, oxygen, energy status, stress, and growth factors, which further modulates main progressions such as lipid and protein synthesis and autophagy [8]. It has been demonstrated previously that the PI3K/mTOR pathway participated in the progression of anticancer, chemosensitization, and radiosensitization [9]. Additionally, the PI3K/mTOR pathway has been proven to be critical for hepatocyte proliferation and the apoptosis of breast cancer cells [10,11]. However, there has been limited investigations regarding the effects of the PI3K/mTOR/S6K pathway on the nerve cells induced by SEV.
Collapsin response mediator proteins (CRMPs) represent a novel family of microtubule-associated proteins (MAPs), which consist of 5 highly conserved phosphorylated proteins (CRMP1-5) [12]. CRMP-2 is the first member of the CRMPs family that has been identified in primary neuronal cells, and it was originally found and identified in the regulation of microtubule dimerization into microtubules [13]. Recently, CRMP-2 expression has also been found in non-neuronal cells such as leukocytes [14], fibroblasts [15], and neuroblastoma [16]. In addition, it has been suggested that CRMP-2 could modulate neuronal viability through the PI3K/mTOR/S6K pathway [17]. However, the exact roles and mechanisms of CRMP-2 in the protection of SEV-induced neurocyte injury have not been studied yet.
In the current research, we analyzed the correlation between CRMP-2 and the neurocytes injury induced by SEV. Furthermore, it was also fascinating to explore the exact roles and mechanisms of CRMP-2 and PI3K/mTOR/S6K pathway in the protection of SEV-induced nerve cells.

Animals
Pregnant Sprague-Dawley (SD) rats (2 months old, pregnancy cycle 18 days, weight 180-200 g) were kept in cages at 22±1°C. The rats had free access to food and water. The light/dark cycle of the animal house was 12 hours/12 hours, and the light time was 8 am to 8 pm. The animals used for experimental research had been approved by the ethics committee of the Hanzhong Central Hospital (Shaanxi, China), and the associated permit number was 170621.

Acquisition of hippocampal neuron cells
The following extraction and cultivation methods have been described in detail in previous articles [18,19]. The pregnant SD rats were anesthetized by ether, and the fetus removed. The fetuses were sterilized in culture dishes which contained 75% ethanol. The heads were cut using an animal breaker (Ugo Basile, #7950; Guangzhou, Guangdong, China). Afterwards, the hippocampus was removed and digested with 0.125% trypsin (Beyotime, Shanghai, China) for 10 min. The supernatant was discarded, and then Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) was added followed by gently shaking. The obtained nerve cells were then maintained in a 5% CO 2 atmosphere at 37°C. The morphology of the cells was observed under the TMM-220 inverted microscope (TX; Shanghai, China).

Cell viability analysis
Cell Counting Kit-8 (CCK-8; Beyotime, Shanghai, China) assay was performed to measure cell viability. About 6×10 4 cells/mL of nerve cells in the logarithmic phase were seeded into 96-well plates and maintained in an incubator (37°C, 5% CO 2 ) for 12 hours. After that, cells were treated as previously described. Cells were then maintained in the incubator (37°C, 5% CO 2 ) for 6 hours, 12 hours, and 24 hours, respectively. Then 10 μL of CCK8 reagent was added to each well. Cells were then maintained with 5% CO 2 at 37°C for 3 hours. Microplate reader (Bio-Rad Laboratories, Inc., USA) was used to read the absorbance at 450 nm. Cell viability was evaluated by the percentage of cell survival compared with the control.

Flow cytometry (FCM)
Cultured nerve cells were treated with 0.25% trypsin (Beyotime). The supernatant was removed, and the nerve cells were suspended in the incubation buffer at a density of 1×10 6 cells/mL. Nerve cells were then maintained with Annexin V-FITC and propidium iodide (PI) (XiLongScientific) in the dark at room temperature for 15 min. Finally, FACSCalibur (BD Biosciences, San Diego, CA, USA) was performed to measure cell proliferation and apoptosis.

Statistical analysis
IBM SPSS statistics 20 software was used in the statistical analysis of this study. Results of our study are shown as mean ±SD of at least 3 independent experiments. The experimental data was analyzed by one-way analysis of variance (ANOVA), Kruskal-Wallis, and Tukey's test. The statistical significance was defined as P<0.05.

Authentication of the rat primary-cultured hippocampal neurons
In the present study, the hippocampus was separated from SD fetal rats and the primary nerve cells were cultured with DMEM medium containing 10% FBS. After incubating for 48 hours, the neurons were observed by inverted microscope. As shown by the arrowhead (Figure 1A), 2 to 3 protuberances could be observed in each nerve cell, and the length of protuberances was longer than before. Moreover, a fraction of glial cells with flat polygonal shape were observed ( Figure 1A). Nevertheless, after incubating for 6 days, as shown by the arrowhead, the morphology  of nerve cells was more typical. The protuberances of nerve cells linked into a dense network and the cell bodies were fuller. Visible halo could be noted around the nerve cells ( Figure 1B). The nerve cells were harvested and used for the later studies.
The cell viability of SEV-induced nerve cells SEV was enhanced by CRMP-2 In order to confirm the accurate concentration of SEV that was used to establish the neurocyte injury model, we evaluated the cell viability of nerve cells treated with different concentration of SEV mixed gas. According to the CCK-8 data, it was noted that cell viability of nerve cells was evidently reduced when the concentration of SEV mixed gas was over 3.0% (Figure 2A). Hence, we selected the 3.0% SEV mixed gas as the concentration of SEV that was used to establish the neurocyte injury model in our study. Furthermore, the CRMP-2 overexpression plasmid, together with its empty vector, was used in the current investigation. qRT-PCR and western blot results indicated that compared with other groups, the expression level of CRMP-2 in nerve cells transfected with CRMP-2 was significantly upregulated ( Figure  2B, 2C). To further explore whether CRMP-2 impacted the cell growth of nerve cells, the cell viability of nerve cells treated with CRMP-2 together with its empty vector and 3.0% SEV mixed gas

4986
was measured. As the CCK-8 data showed, compared with other groups, SEV treatment inhibited cell viability of nerve cells. However, in the groups that were transfected with CRMP-2, the cell viability of SEV-induced nerve cells was enhanced ( Figure 3). These results suggested that CRMP-2 possessed the ability to accelerate the cell viability of nerve cells induced by SEV.

CRMP-2 accelerated the proliferation of SEV-induced nerve cells
CRMP-2 reversed the ability of SEV to inhibit the viability of neural cells according to CCK-8 data, we thereby conjectured that CRMP-2 might also affect the cell proliferation of nerve cells suffered from SEV treatment. Hence, flow cytometry (FCM) was carried out to assess the proliferation capacity of nerve cells from each treatment group. The FCM results revealed that the proliferation number of nerve cells was significantly reduced by SEV treatment. However, the proliferation number of nerve cells were distinctly increased in CRMP-2+SEV group (Figure 4). These results suggested that SEV reduced the proliferation capacity of nerve cells, while CRMP-2 could evidently promote the cell proliferation of SEV-induced nerve cells. Thus, it was determined that CRMP-2 accelerated the proliferation of nerve cells induced by SEV.

CRMP-2 suppressed the SEV-mediated apoptosis of nerve cells
Additionally, we also evaluated the apoptosis ability of nerve cells treated with CRMP-2 and SEV. Based on the FCM data, the percentage of apoptosis nerve cells in the SEV group was 24.57%, which was markedly higher than the control group (6.90%). Nevertheless, when the cells were co-processed by CRMP-2 and SEV, the apoptosis rate of SEV-induced nerve cells was decreased from 28.44% to 17.21% ( Figure 5). According to the FCM results, we noted that the percentage of apoptosis nerve cells in the CRMP-2+SEV group was distinctly lower than that of the SEV+NC group, which suggested that CRMP-2 could lessen the apoptosis capacity of SEV-induced nerve cells.

CRMP-2 modulated the expression levels of apoptosisassociated protein in SEV-induced nerve cells
In order to further investigate the molecular mechanism of CRMP-2 inhibition of SEV-induced neuronal apoptosis, the apoptosis-related factors were assessed. We first detected the activity of caspase-3 in nerve cells from all of the treatment groups. It was revealed that SEV obviously strengthened the caspase-3 activity in nerve cells, while CRMP-2 could distinctly reduce  the caspase-3 activity in SEV-induced nerve cells ( Figure 6A). Moreover, the mRNA and protein levels of apoptosis-associated proteins, involving caspase-3, B-cell lymphoma 2-associated x protein (Bax), and B-cell lymphoma 2 (Bcl-2) in nerve cells were measured in the current investigation. On the basis of qRT-PCR data, we found that the expression levels of caspase-3 and Bax in nerve cells treated with SEV were obviously upregulated. However, in the groups that were transfected with CRMP-2, decreases in the expression levels of caspase-3 and Bax in SEV-induced nerve cells were evidently observed. Moreover, it was also revealed that SEV treatment markedly lessened the Bcl-2 expression in nerve cells, while CRMP-2 transfection distinctly upregulated the Bcl-2 expression in SEV-induced nerve cells ( Figure 6B). At the same time, western blot data also showed similar trends of caspase-3, Bax, and Bcl-2 expression in nerve cells from the treatment groups ( Figure 6C). Based on these findings it was confirmed that CRMP-2 suppressed the apoptosis of SEV-induced nerve cells through modulating the expression levels of caspase-3, Bax, and Bcl-2.

CRMP-2 impacted the PI3K-mTOR-S6K pathway
On the basis of cell proliferation and apoptosis results, we demonstrated that CRMP-2 promoted the proliferation capacity and suppressed the apoptosis of nerve cells induced by SEV. In order to further investigate the accurate mechanisms of CRMP-2 on the protection of nerve cells against SEV, we detected the related signaling pathway. Western blot results suggested that the expression levels of synapsin-I, p-mTOR, p-S6K, and p-S6 were markedly downregulated by treating with SEV. Nevertheless, when cells were treated with SEV and CRMP-2 together, evident increases in synapsin-I, p-mTOR, p-S6K, and p-S6 expression were observed, relative to the NC+SEV group (Figure 7). However, no significant difference was observed in the expression levels of mTOR, S6K, and S6 in nerve cells from all of the treatment groups. Thus, it was affirmed that CRMP-2 could enhance the phosphorylation of mTOR, S6K, and S6 in nerve cells induced by SEV, which indicated that CRMP-2 could impact the PI3K-mTOR-S6K pathway in nerve cells treated with SEV.

Discussion
The hippocampus is the limbic system of the brain, and it is related functions of the central nervous system such as learning and memory [2122]. Meanwhile, hippocampal tissue is composed of a large number of neural stem cells, with structure of neat, relatively independent cells. Therefore, the model of cultured hippocampal neurons in vitro is considered an ideal experimental model. The hippocampus of rodents, especially newborn mice (24 hours), is easy to locate and removed and thus is often used [23][24][25]. Hence, we extracted hippocampal neurons cells from neonatal rats as a model.
Recent studies have shown that exposure to clinically relevant doses of narcotic drugs, such as isoflurane and SEV, can cause nerve structural disorder in rats, change hippocampal synapses to reduce the density of dendritic spines in prefrontal cortex of rats, decrease the expression of related proteins involved in the development of connections and axons, causing cortical axons needle disorder [26][27][28]. SEV anesthesia has been Synapsin-I p-mTOR mTOR # ## * ** ** *** * proven to led to more death of hippocampal neurons [29]. In addition, SEV has been used as an inducer to successfully construct a model of nerve cell injury, which makes cell proliferation decrease and apoptosis increase [30]. Hence, in the present study, the hippocampal neurons separated from 18-day SD fetal rats were chosen to establish the SEV-induced neurocyte injury model. Similarly, we found that SEV could markedly inhibit the proliferation of hippocampal neurons and promote apoptosis.
Because CRMP-2 has been suggested to possess multiple functions in the modulation of hippocampal neurons growth, we thereby selected CRMP-2 as the object of our study on SEV-induced neurocyte injury [31]. After transfecting with CRMP-2 and its empty vector, the data indicated that CRMP-2 could reduce the viability of SEV-suppressed nerve cells. Additionally, some researchers have found that CRMP-2 involves the development of the nervous system, as well suppresses apoptosis of various cancer cells [16,[32][33][34]. Hence, we suspected that CRMP-2 also plays a role in the apoptosis 4989 of nerve cells. Our results showed that CRMP-2 obviously suppressed the apoptosis of SEV-induced nerve cells. Furthermore, the related apoptosis factors were also investigated in our study. According to the experimental data, we found that CRMP-2 distinctly downregulated the expression levels of caspase-3 and Bax, while enhancing the Bcl-2 expression in SEV-induced nerve cells. These results suggested that CRMP-2 might suppress the apoptosis of SEV-induced nerve cells by modulating the expression levels of caspase-3, Bax, and Bcl-2.
Previous investigations have confirmed that the PI3K-mTOR-S6K pathway plays a crucial role in the proliferation and apoptosis of tumor cells [35]. Furthermore, recent studies have confirmed that CRMP-2 regulates neuronal growth via controlling the PI3K-mTOR-S6K pathway [17]. Thus, the roles and mechanisms of the PI3K-mTOR-S6K pathway in the proliferation and apoptosis of SEV-induced nerve cells affected by CRMP-2 were explored in our research. Based on the western blot data, it was noted that CRMP-2 significantly upregulated the expression level of synapsin-I and enhanced the phosphorylation of mTOR, S6K, and S6 in nerve cells induced by SEV. However, no significant difference was observed in the expression levels of mTOR, S6K, and S6 in nerve cells. Hence, we could affirm that CRMP-2 might modulate the proliferation and apoptosis of SEV-induced nerve cells via the regulation of the PI3K-mTOR-S6K pathway.
Altogether, our study suggested that CRMP-2 alleviated neurocyte injury induced by SEV through the modulation of the PI3K-mTOR-S6K pathway. And the study results provide a new thread for understanding the pathogenesis of neurocyte injury and afford a fascinating approach for the therapy of neurocyte injury.

Conclusions
Collectively, our current work highlights that CRMP-2 alleviated neurocyte injury induced by SEV through the modulation of the PI3K-mTOR-S6K pathway. These findings have a momentous impact on the understanding of the mechanisms of CRMP-2 in rat nerve cells. It indicates that CRMP-2 might be an effective target for the therapy of neurocyte injury.


extfieldsofstudy: Biology Medicine

text:
Demonstration of translation elongation factor 3 activity from a non-fungal species, Phytophthora infestans

In most eukaryotic organisms, translation elongation requires two highly conserved elongation factors eEF1A and eEF2. Fungal systems are unique in requiring a third factor, the eukaryotic Elongation Factor 3 (eEF3). For decades, eEF3, a ribosome-dependent ATPase, was considered “fungal-specific”, however, recent bioinformatics analysis indicates it may be more widely distributed among other unicellular eukaryotes. In order to determine whether divergent eEF3-like proteins from other eukaryotic organisms can provide the essential functions of eEF3 in budding yeast, the eEF3-like proteins from Schizosaccharomyes pombe and an oomycete, Phytophthora infestans, were cloned and expressed in Saccharomyces cerevisiae. Plasmid shuffling experiments showed that both S. pombe and P. infestans eEF3 can support the growth of S. cerevisiae in the absence of endogenous budding yeast eEF3. Consistent with its ability to provide the essential functions of eEF3, P. infestans eEF3 possessed ribosome-dependent ATPase activity. Yeast cells expressing P. infestans eEF3 displayed reduced protein synthesis due to defects in translation elongation/termination. Identification of eEF3 in divergent species will advance understanding of its function and the ribosome specific determinants that lead to its requirement as well as contribute to the identification of functional domains of eEF3 for potential drug discovery.

Introduction
Translation is a highly conserved process during which proteins are synthesized from messenger RNA. This process is divided into the four phases of initiation, elongation, termination and ribosome recycling, each of which requires a specialized set of soluble protein factors (reviewed in [1]). During the initiation phase of translation, eukaryotic initiation factors facilitate the binding of an 80S ribosome at the start codon of the mRNA. This is followed by a repetitive cycle of aminoacyl-tRNA (aa-tRNA) delivery, peptide bond formation and ribosomal translocation during the elongation phase. In most eukaryotic organisms, translation elongation is catalyzed by two soluble elongation factors (eEFs), eEF1A and eEF2. eEF1A, the functional homolog of bacterial EF-Tu, is a G-protein that binds and recruits aa-tRNAs to the A-site of the ribosome. When a codon-anticodon match occurs, the ribosome stimulates eEF1A-mediated GTP hydrolysis resulting in the release of inactive GDP-bound eEF1A from the ribosome and accommodation of the aa-tRNA into the A-site. Following peptide bond formation, eEF2, the homolog of the bacterial GTPase EF-G, catalyzes the translocation of the peptidyl-tRNA from the A-site to the P-site of the ribosome thereby positioning the next codon for decoding. When a stop codon is encountered by the ribosome, release factors release the polypeptide from the ribosome and the ribosomal subunits are recycled for another round of protein synthesis. Fungal translation elongation has long been considered unique among eukaryotes in its requirement for a third elongation factor, eEF3. eEF3 is a ribosome-dependent ATPase required for in vitro translation elongation assays when using ribosomes purified from yeast [2]. In contrast, Saccharomyces cerevisiae eEF1A and eEF2 alone catalyze translation elongation with rat liver ribosomes [3]. Therefore, it is likely a ribosome-specific determinant that establishes the need for eEF3. The reason why ribosomes from yeast require eEF3 and the function of eEF3 itself are not well understood. In one study, eEF3 facilitated the release of de-acetylated tRNA from the E-site of the ribosome [4]. eEF3 also stimulates eEF1A-mediated binding of cognate aa-tRNA to the A-site [5,6] potentially through a direct interaction with eEF1A. [7,8]. in vivo experiments confirm the important role of eEF3 in yeast protein synthesis. eEF3 is encoded by an essential gene in S. cerevisiae and strains harboring either temperature sensitive or mutant forms of eEF3 display protein synthesis and translation elongation defects [7][8][9][10]. eEF3 orthologues from other yeasts including Candida albicans, another ascomycete and Cryptococcus neoformans, a basidiomycete, can complement the loss of eEF3 in S. cerevisiae suggesting that the function of eEF3 is likely conserved among fungi [11,12].
Structural studies of eEF3 identified five domains: an amino-terminal HEAT repeat followed by a four-helix bundle domain and two ATP-binding cassette (ABC) domains, the second of which is interrupted by a chromodomain insertion [13]. A cryo-EM reconstruction of the eEF3-ATP-post-translocation 80S ribosome complex demonstrated that eEF3 binds the ribosome near the E-site in agreement with its proposed function in E-site tRNA release [13]. The chromodomain insertion is proposed to interact with the ribosome and stabilize the ribosomal L1 stalk in an open conformation which may facilitate tRNA release. Mutations in this domain, however, show significant reduction in ATPase activity without affecting overall ribosome binding [10].
Recent bioinformatic analysis identified potential eEF3 orthologues in multiple non-fungal, lower eukaryotic species [14,15]. These eEF3-like protein sequences are as similar to S. cerevisiae eEF3 as the functionally complementary C. neoformans eEF3, suggesting that these putative eEF3s are likely to maintain at least a subset of eEF3 functions. In this study, we present the first direct evidence suggesting that functional eEF3 orthologues exist outside the fungal kingdom. We have expressed the eEF3 orthologue from Schizosaccharomyes pombe and the oomycete Phytopthora infestans, the causative agent of late potato blight, in S. cerevisiae and showed that either can provide the essential functions of eEF3. in vitro studies demonstrated that P. infestans eEF3, like S. cerevisiae eEF3, possesses ribosome-stimulated ATPase activity.

Plasmid construction
S. cerevisiae eEF3: The sequence encoding an N-terminal 6x-His tagged S. cerevisiae eEF3 was cloned as a BamHI fragment from plasmid pTKB1142 into plasmid pTKB328 (CEN LEU2) where it is expressed from the S. cerevisiae TEF5 promoter (pTKB1263).
Schizzosaccharomyces pombe eEF3: The S. pombe tef3 + gene was amplified by PCR from fission yeast genomic DNA prepared using the Epicentre Masterpure Yeast DNA Purification Kit. The PCR was performed using the following primers: Forward Primer-5' ATTGTTTG ATCAATGCATCATCATCATCATCATTCTGCAAAGAGTGA-GAATAA-3', Reverse Primer-5'CTATTAGGGCCCTTACAGGTCACTGACCTC-3'. The PCR product encoding N-terminal 6x-His tagged S. pombe eEF3 was cloned into the BamHI and ApaI restriction sites of the pTKB328 vector and is expressed from the S. cerevisiae TEF5 promoter (TKB1269).
P. infestans eEF3: The sequence encoding P. infestans eEF3 (NCBI Reference Sequence XP_002906761.1) was synthesized as two codon-optimized gblock gene fragments with an Nterminal 6x-His tag (Integrated DNA Technologies). These two gene fragments were cloned into plasmid pTKB328 using Gibson assembly (New England Biolabs) such that N-terminal 6x-His tagged P. infestans eEF3 is expressed from the S. cerevisiae TEF5 promoter (pTKB1264). The Sac1-XhoI fragment of pTKB1264 containing the TEF5 promoter and P. infestans eEF3 was cloned into pTKB372 (2μ LEU2) creating pTKB1268 Strains and growth conditions S. cerevisiae strains used in this study are listed in Table 1. Yeast cells were grown in either yeast extract-peptone-dextrose (YEPD; 1% Bacto-yeast extract, 2% Bacto-tryptone, 2% dextrose) or defined synthetic complete medium (C) lacking the indicated amino acid and supplemented with 2% dextrose as a carbon source. Growth curves were generated by diluting exponentially growing cultures to an A 600 of 0.1 and then measuring the A 600 at the indicated time points.
Heterologous eEF3 proteins were expressed as the only form of eEF3 by plasmid shuffling in TKY1617. Briefly, TKY1617 was individually transformed with the plasmids described above using a lithium acetate method. Transformants were counterselected on media containing 5-fluoroorotic acid to select cells that have lost the complementing S. cerevisiae eEF3 expressing plasmid.

Immunoblotting
Yeast cells in mid-log phase growth were lysed with glass beads in 100 mM Tris-HCl pH 8.0, 20% glycerol, 1 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride and cell debris was removed by centrifugation. Protein concentration in cell lysates was determined using the BioRad reagent according to the manufacturer's instructions. Twenty-five micrograms of total protein were separated on a 7.8% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was stained with Ponceau S and immunoblotted with either a monoclonal anti-His antibody (BD Biosciences) or a monoclonal anti-PGK antibody (Novex). Quantitation of immunoblots was performed using ImageQuant software (GE Healthcare).

Antibiotic sensitivity assays
Yeast strains in exponential growth were diluted to an A 600 of 0.6 and 200 μl was spread on YEPD plates. Sterile discs were placed on the plates and 10 μL of antibiotic at the indicated concentration was pipetted onto the disc. Plates were incubated at 30˚C for 2d at which time the diameter of the zone of growth inhibition surrounding the discs was measured. Antibiotic concentrations were 1 mM cycloheximide, 25 mM hygromycin and 800 mg/mL paromomycin.

Translation assays
For in vivo [ 35 S] methionine incorporation assays, yeast strains were grown in liquid culture in C-Met at 30˚C to mid-log phase and assayed as previously described [16]. All time points were analyzed in triplicate. Polyribosome extracts were harvested and run on a 7-47% sucrose density gradient as previously described [17]. The areas under the 80S and polyribosome peaks were determined using Image J (National Institutes of Health).

eEF3 activity is not unique to fungi
While originally designated as a fungal-specific elongation factor, recent bioinformatic analysis identified eEF3-like proteins in several lower eukaryotic species [14,15]. The most significant regions of conservation between these proteins and S. cerevisiae eEF3 are found within the two ABC-type ATPase domains. Therefore, it is important to demonstrate that these proteins are not simply related ATPases but that they can provide the essential functions of eEF3. To address this question, the genes encoding eEF3 from an evolutionarily distant ascomycete, S. pombe, and the putative form of eEF3 from the oomycete, P. infestans, were cloned into a low copy S. cerevisiae expression vector with an N-terminal 6x-His tag (materials and methods). S. cerevisiae eEF3 is approximately 62% identical to S. pombe eEF3 and 45% identical to P. infestans eEF3. S. pombe eEF3 was included in the analysis to broaden the understanding of the evolution of eEF3. Both eEF3 proteins are expressed from the strong TEF5 promoter and the P. infestans eEF3 gene sequence was codon optimized for expression in budding yeast. TheYEF3 gene encoding S. cerevisiae eEF3 was also cloned into the same expression vector as a control. The eEF3 expression plasmids were individually transformed into a S. cerevisiae strain in which the only copy of YEF3 is untagged and present on a URA3 plasmid. The ability of S. pombe or P. infestans eEF3 to function as the only form of the protein was assessed by growth on 5-FOA containing media which only allows growth of cells that have lost the URA3 plasmid. Both S. pombe and P. infestans eEF3 could support growth of a yef3Δ strain (Fig 1A). While expression of S. pombe eEF3 could support growth at a level similar to S. cerevisiae eEF3, the strain expressing P. infestans eEF3 grew at a reduced rate (Fig 1A and 1B). The average doubling time was 137±4 min for the S.c. eEF3 expressing strain, 135±2 min for the S.p. eEF3 expressing strain and 162±9 min for the P.i. eEF3 (CEN) expressing strain. The ability of P. infestans eEF3 to complement the loss of YEF3 in S. cerevisiae suggests that it can perform the essential functions of eEF3 and is a bona fide eEF3 homologue. The reduced growth rate of cells expressing P. infestans eEF3 may arise from either reduced protein expression or reduced activity in a heterologous system. Cell lysates were collected and immunoblotted with an anti-His antibody to detect eEF3 expression levels in strains whose growth was supported solely by either S. cerevisiae eEF3, S. pombe eEF3 or P. infestans eEF3 (Fig 1C). The steady state levels of both S. pombe eEF3 and P. infestans eEF3 were comparable but lower than that of the native S. cerevisiae eEF3 protein. These data suggest that there is a threshold level of eEF3 activity required for wild-type growth. To determine if increasing the expression of P. infestans eEF3 would rescue the slow growth phenotype, 6x-His tagged P. infestans eEF3 expressed from the same promoter was cloned into a high copy expression plasmid and its expression level and ability to support growth of a yef3Δ strain were determined as described above. Over expression of P. infestans eEF3 (average of 2.2-fold over S. cerevisiae eEF3) could rescue the growth of yef3Δ cells to wild-type (S. cerevisiae eEF3) levels with an average doubling time of 138±14 min. (Fig 1B and 1C).

P. infestans eEF3 shows ribosome-stimulated ATPase activity
A key characteristic of S. cerevisiae eEF3 is its ribosome-stimulated ATPase activity. To determine if P. infestans eEF3 demonstrated similar activity, P. infestans eEF3 was expressed and purified from budding yeast and its activity was compared to S. cerevisiae eEF3. S. cerevisiae eEF3 has a low basal ATPase activity that was stimulated 3.2-fold at 100 nM eEF3 by the inclusion of ribosomes as reported previously (Fig 2, [10, 21, 22]). The activity of P. infestans eEF3 in the absence of ribosomes was very low compared to S. cerevisiae eEF3 (6.2-fold lower) at the Fig 1. S. pombe (S.p.) and P. infestans (P.i.) eEF3 complement the loss of S. cerevisiae (S.c.) eEF3. (A) Yeast strains expressing eEF3 from the indicated species as the only form of eEF3 were streaked onto YEPD medium and incubated at 30˚C for 2 d. CEN-low copy number plasmid/low expression. 2μ -high copy number same concentration. The inclusion of S. cerevisiae ribosomes could stimulate P. infestans eEF3 activity (3.9-fold) which is similar to the observed ribosome stimulation of S. cerevisiae eEF3 activity. While the ribosome-stimulated P. infestans eEF3 activity was concentration dependent, its activity was still 5.2-fold lower than S. cerevisiae eEF3.

Cells expressing P. infestans eEF3 exhibit translation defects
The slow growth of P. infestans eEF3 cells compared to S. pombe expressing cells suggests that the function of P. infestans eEF3 is also compromised in the heterologous system. To determine whether the slow growth was due to a defect in translation, total protein synthesis was measured by [ 35 S] methionine incorporation in exponentially growing cells expressing the indicated forms of eEF3. Strains expressing either S. cerevisiae or S. pombe eEF3 had similar rates of protein synthesis over the course of the experiment while a strain with low P. infestans eEF3 expression showed a 23-31% reduction in total protein synthesis (Fig 3). This defect was rescued to near wild-type (S. cerevisiae and S. pombe) levels by increasing the amount of P. infestans eEF3 protein with a high copy expression vector. plasmid/high expression. (B) Growth curves were generated from A 600 measurements of exponentially growing cultures over the indicated time period. Error bars represent standard error. (C) Whole cell extracts were prepared from the indicated strains, separated by SDS-PAGE, and stained with Ponceau S (bottom panel). The membrane was then cut and immunoblotted with either an anti-His antibody to detect eEF3 or anti-Pgk1 antibody as a loading control. The control lane is an extract from a yeast strain (TKY1617) that does not express epitope tagged eEF3.
https://doi.org/10.1371/journal.pone.0190524.g001 To further probe the nature of the translation defect in the low P. infestans eEF3 expression strain, sensitivity to three antibiotics known to inhibit translation elongation was measured. The low P. infestans eEF3 expression strain showed an increase in sensitivity to paromomycin, hygromycin and cycloheximide compared to wild-type cells in an assay measuring the zone of growth inhibition (Fig 4A). Increased expression of P. infestans eEF3 could either fully (hygromycin) or partially rescue (cycloheximide and paromomycin) the level of sensitivity. In these assays, S. pombe eEF3 also demonstrated a small increase in sensitivity to all three elongation inhibitors suggesting that its reduced expression level compared to S. cerevisiae eEF3 does result in a mild translation defect that does not significantly impact growth rates or total protein synthesis. To examine more specifically which steps of protein synthesis were affected in the low P. infestans eEF3 strain, polyribosome profiles were analyzed for each of the indicated strains. The polyribosome profiles of both S. cerevisiae eEF3 and S. pombe eEF3 expressing strains were similar in the absence of cycloheximide while the low P. infestans eEF3 expressing strain showed an increase in the number of polyribosomes and a concomitant decrease in the 80S ribosome peak (Fig 4B). The polyribosome profile observed in a strain expressing high levels of P. infestans eEF3, however, did not significantly differ from that seen in a strain expressing low levels of P. infestans eEF3. These results are consistent with a defect in translation elongation/termination in P. infestans eEF3 expressing strains.

Bioinformatic analysis shows divergence in the chromodomain of P. infestans eEF3
To help identify the regions of eEF3 important for its activity, a domain-specific comparison of sequence identity was performed for all three eEF3 proteins ( Fig 5A). As expected, the ABC1 and ABC2 domains involved in ATP binding have a high level of identity in both S. pombe eEF3 and P. infestans eEF3 when compared to S. cerevisiae eEF3. The four-helix bundle domain, whose function is currently unknown, has the lowest level of conservation across all three proteins. Outside of this domain, P. infestans eEF3 shows significantly less sequence conservation in the HEAT repeat domain and the chromodomain region, both of which have been shown to contact the ribosome in cryo-EM reconstructions [13]. The largest difference between S. pombe and P. infestans eEF3 in comparison to S. cerevisiae eEF3 is found in the chromodomain region. S. pombe eEF3 is 79.1% identical to S. cerevisiae eEF3 in this region while P. infestans eEF3 is only 36.1% identical. This region disrupts the second ABC domain and has been shown to be important for ribosome-dependent ATPase activity [10]. A sequence alignment of the chromodomain of all three eEF3s is shown in Fig 5B. Notably, P. infestans eEF3 is lacking 13-amino acids compared to both S. cerevisiae eEF3 and S. pombe eEF3. These data suggest that the chromodomain may be a species-specific determinant of ATPase activity either independently or through its role in ribosome binding.

Discussion
The reason fungal ribosomes require eEF3 and how mammalian ribosomes have evolved to synthesize proteins without it have been long-standing questions in the field of protein synthesis. In this report, we present the first evidence that eEF3 activity exists outside the fungal kingdom. P. infestans is a filamentous eukaryotic microorganism of the oomycetes class. Oomycetes were originally grouped with fungi based on their phenotypic characteristics and are still often referred to as fungi despite genetic evidence to the contrary. Sequencing efforts have in fact shown that oomycetes are phylogenetically distinct from fungi and are instead classified with brown algae and diatoms [26,27]. Phylogenetic comparison of another translation elongation factor, eEF1A, from P. infestans with other taxonomic groups supports the hypothesis that oomycetes evolved independently of fungi [28] and in a phylogenetic tree constructed using eEF3 sequences from a variety of unicellular eukaryotes, P. infestans eEF3 is located in the same clade as green algae and brown algae/diatoms, separate from fungi ( Fig  5C). Therefore, P. infestans eEF3 is a non-fungal eEF3 which can perform the essential function of the yeast protein in vivo.
Despite its ability to complement the loss of S. cerevisiae eEF3, P. infestans eEF3 exhibits lower activity in the S. cerevisiae system. S. pombe eEF3 and P. infestans eEF3 are expressed at a , and P. infestans (P.i.) eEF3 were aligned using Clustal Omega and the percentage identity to S. cerevisiae eEF3 is shown [23,24]. The amino acids comprising each domain in S. cerevisiae are indicated. (B) Alignment of the chromodomain of S. cerevisiae, S. pombe, and P. infestans eEF3 was performed using Clustal Omega and shading based on identity was done using Jalview [25]. Dark gray represents identity in all three species and light gray represents identity in two species. (C) Maximum likelihood tree with 500 bootstrap replicates created using phylogeny.fr. https://doi.org/10.1371/journal.pone.0190524.g005 Translation elongation factor 3 activity in Phytophthora infestans similar level, both lower than the S. cerevisiae eEF3, but only the expression of P. infestans eEF3 is associated with an effect on growth rate. Furthermore, increasing the expression of P. infestans eEF3 using a high copy expression plasmid rescues this effect on growth. These observations correlate with what was observed in vitro. P. infestans eEF3 had a lower ATPase activity in the absence and presence of ribosomes. However, P. infestans eEF3 ATPase activity displayed a similar fold stimulation of activity upon the addition of ribosomes as S. cerevisiae eEF3. One explanation for these results is that it may not be an inability to interact with S. cerevisiae ribosomes but an overall low ATPase activity which causes the growth defect observed in vivo. Similar effects on growth rate were observed with C. neoformans eEF3 complementation. However, in this study, purified C. neoformans eEF3 activity showed weak stimulation by addition of S. cerevisiae ribosomes and an altered eEF3-ribosome interaction was suspected as the cause of the low activity [11].
The slow growth phenotype observed in P. infestans eEF3 is likely due to defects in protein synthesis. Cells with low P. infestans eEF3 expression showed a defect in [ 35 S] methionine incorporation that was rescued to near wild-type levels by increasing its levels. However, increased expression of P. infestans eEF3 did not complement the polyribosome profile defect seen in cells expressing this form of eEF3. Previous experiments have shown that altered translation elongation can exist even when total protein synthesis is apparently normal [29] and may be due to the different aspects of translation being examined in each assay. These results suggest that there is still a minor elongation defect when P. infestans eEF3 is overexpressed which is consistent with the mild antibiotic sensitivities observed in this strain.
Bioinformatic analysis of existing genome databases has identified eEF3 orthologues in at least 18 different non-fungal species, including multiple green algae [14]. Since molecular genetic techniques have not been developed for many of these organisms, complementation of the loss of S. cerevisiae eEF3 may be the most expedient way to assay eEF3-like functions for these proteins, however, this technique has its limitations. For example, as noted above, heterologous eEF3 proteins are often expressed at lower levels than endogenous S. cerevisiae eEF3 from the same expression construct even when codon-optimized. We have performed similar complementation experiments using a codon-optimized construct to express Chlamydomonas reinhardtii eEF3 and were unable to make any conclusion on functionality because expression levels were very low even when expressed from a high copy plasmid (unpublished observations). In S. cerevisiae, it has been observed that point mutations in key Walker ATP binding motifs and the chromodomain affect the expression of eEF3 resulting in little or no protein (unpublished observations, [10]). While an effect on RNA stability cannot be excluded, these data suggest that eEF3 expression levels may be regulated by either proper folding or protein activity. In addition, lack of complementation in this system does not preclude the possibility that the heterologous eEF3 proteins retain eEF3-like function in its native organism as negative results may result from ribosomal or other divergence within the protein synthesis machinery.
The identification of eEF3 in non-fungal species will facilitate the utilization of phylogenetics to gain a greater mechanistic understanding of the role of eEF3 in translation and will provide important information on the evolution of protein synthesis. For example, the analysis of the conservation of domains among the three eEF3 proteins analyzed here, indicates that the chromodomain insertion in ABC2 maybe an important species-specific functional domain. Previous studies suggesting that this domain is important for ATPase activity [10] and the observations detailed here point to possible unique aspects of ribosome association and ATPase activation of eEF3 proteins.
Since its initial discovery as an essential, fungal-specific translation elongation factor, eEF3 has been put forth as a potential therapeutic target for the treatment of fungal infections. This work identifies a factor with eEF3 activity in another important class of pathogens. Oomycetes represent a significant threat to global food security through effects on both agriculture and fish farming [30]. If eEF3 is shown to play an essential role in oomycetes, as it does in yeast, then targeting eEF3 activity in these organisms may prove to be an effective mechanism to control these pathogens.