Florence W. L. Tsui

Notochordal cells protect nucleus pulposus cells from degradation and apoptosis: implications for the mechanisms of intervertebral disc degeneration

IntroductionThe relative resistance of non-chondrodystrophic (NCD) canines to degenerative disc disease (DDD) may be due to a combination of anabolic and anti-catabolic factors secreted by notochordal cells within the intervertebral disc (IVD) nucleus pulposus (NP). Factors known to induce DDD include interleukin-1 beta (IL-1ß) and/or Fas-Ligand (Fas-L). Therefore we evaluated the ability of notochordal cell conditioned medium (NCCM) to protect NP cells from IL-1ß and IL-1ß +FasL-mediated cell death and degeneration.MethodsWe cultured bovine NP cells with IL-1ß or IL-1ß+FasL under hypoxic serum-free conditions (3.5% O2) and treated the cells with either serum-free NCCM or basal medium (Advanced DMEM/F-12). We used flow cytometry to evaluate cell death and real-time (RT-)PCR to determine the gene expression of aggrecan, collagen 2, and link protein, mediators of matrix degradation ADAMTS-4 and MMP3, the matrix protection molecule TIMP1, the cluster of differentiation (CD)44 receptor, the inflammatory cytokine IL-6 and Ank. We then determined the expression of specific apoptotic pathways in bovine NP cells by characterizing the expression of activated caspases-3, -8 and -9 in the presence of IL-1ß+FasL when cultured with NCCM, conditioned medium obtained using bovine NP cells (BCCM), and basal medium all supplemented with 2% FBS.ResultsNCCM inhibits bovine NP cell death and apoptosis via suppression of activated caspase-9 and caspase-3/7. Furthermore, NCCM protects NP cells from the degradative effects of IL-1ß and IL-1ß+Fas-L by up-regulating the expression of anabolic/matrix protective genes (aggrecan, collagen type 2, CD44, link protein and TIMP-1) and down-regulating matrix degrading genes such as MMP-3. Expression of ADAMTS-4, which encodes a protein for aggrecan remodeling, is increased. NCCM also protects against IL-1+FasL-mediated down-regulation of Ank expression. Furthermore, NP cells treated with NCCM in the presence of IL-1ß+Fas-L down-regulate the expression of IL-6 by almost 50%. BCCM does not mediate cell death/apoptosis in target bovine NP cells.ConclusionsNotochordal cell-secreted factors suppress NP cell death by inhibition of activated caspase-9 and -3/7 activity and by up-regulating genes contributing anabolic activity and matrix protection of the IVD NP. Harnessing the restorative powers of the notochordal cell could lead to novel cellular and molecular strategies in the treatment of DDD.

Association of an ERAP1 ERAP2 haplotype with familial ankylosing spondylitis

Objectives To assess whether there is excess transmission of alleles from the ERAP1 ERAP2 locus in families with ankylosing spondylitis (AS). Methods 199 multiplex families with AS with four non-synonymous single nucleotide polymorphisms (SNPs), three in the endoplasmic reticulum aminopeptidase 1 (ERAP1) gene (rs27044, rs10050860 and rs30187) and one in the endoplasmic reticulum aminopeptidase 2 (ERAP2) gene (rs2549782), were genotyped and family-based association analyses were performed. Results Family-based association testing (FBAT –e; empirical variance option) analysis showed that ERAP1 rs30187[T] was associated with AS (additive model: p=0.02; dominant model: p=0.007). Haplotype permutation tests (HBAT-p) showed that a haplotype in the ERAP1 and ERAP2 locus (rs27044[G] rs30187[T] rs2549782[T]) was significantly associated with AS (two-sided p value by permutation test 0.009 for additive and 0.008 for dominant model, respectively). Conclusion This study shows that one ERAP1 SNP and a haplotype in the ERAP1 and ERAP2 locus are associated with familial AS.

Molecular Basis of the Motheaten Phenotype

Mice homozygous for the autosomal recessive motheaten (me) or the allelic viable motheaten (mev) mutations manifest a unique immunological disease associated with severe immunodeficiency and autoimmunity. Over the past few years, our group has used the motheaten mouse as a model system for elucidating the genetic and cellular events that contribute to expression of normal hematopoietic and immune cell function. To this end, we have sought to identify the gene responsible for the motheaten phenotype. In our initial studies, our general approach involved the use of subtractive hybridization to identify genes that were differentially expressed in the mutant versus control mice and which might thus provide clues as to the primary gene defect. Using this approach, we showed that genes encoding stefin A cysteine proteinase inhibitors are markedly overexpressed in bone marrow cells of me and mev mice compared to bone marrow cells of normal congenic animals. However, the motheaten mutation has been mapped to mouse choromosome 6 while the stefin A gene cluster was localized to mouse chromosome 16. Stefin gene therefore does not represent the primary gene defect. Our second strategy aimed at identifying the primary gene defect underlying the motheaten phenotype was prompted by the recent localization of a protein tyrosine phosphatase gene to human chromosome 12p12-p13, a region containing a large segment of homology with the region on mouse chromosome 6 where the motheaten locus has been mapped. We have shown that abnormal Hcph transcripts are expressed in me and mev bone marrow cells and that the generation of these altered transcripts is due to RNA splicing defects caused by single basepair changes in the Hcph genes of the mutant mice. These mutant mice thus provide a valuable model system for elucidating the biological roles of HCP in vivo and defining the mechanism whereby defective function of a hematopoietic cell phosphatase leads to expression of the motheaten phenotype of severe immunodeficiency and systemic autoimmunity.

The genetic basis of ankylosing spondylitis: new insights into disease pathogenesis

Ankylosing spondylitis (AS) is a complex disease involving multiple risk factors, both genetic and environmental. AS patients are predominantly young men, and the disease is characterized by inflammation and ankylosis, mainly at the cartilage–bone interface and enthesis. HLA-B27 has been known to be the major AS-susceptibility gene for more than 40 years. Despite advances made in the past few years, progress in the search for non-human leukocyte antigen susceptibility genes has been hampered by the heterogeneity of the disease. Compared to other complex diseases, such as inflammatory bowel disease (IBD), fewer susceptibility loci have been identified in AS. Furthermore, non-major histocompatibility-complex susceptibility loci discovered, such as ERAP1 and IL23R, are likely contributors to joint inflammation. Identification and confirmation of functional variants remains a significant challenge of investigations involving genome-wide association studies (GWAS). It remains unclear why none of the AS-susceptibility genes identified in GWAS appear to be directly involved in the ankylosing process. Numerous reviews have recently been published on the genetics of AS. Therefore, aside from a brief summary of what AS GWAS has successfully achieved thus far, this review will focus on directions that could address unanswered questions raised by GWAS.

Epitope studies indicate that histidyl-tRNA synthetase is a stimulating antigen in idiopathic myositis.

The most frequently found myositis‐specific antibody, the anti‐Jo‐1 antibody (anti‐HRS), binds to histidyl‐tRNA synthetase (HRS). Although this antibody reacts with HRS, it is unclear whether HRS is the stimulating antigen or is merely a protein that cross‐reacts with a yet undefined antigen. Because antibody directed against an unrelated antigen would not be expected to cross‐react with HRS at multiple sites, we mapped the epitopes on HRS to resolve this issue. We found by Western blot analyses that immunoglobulins G (IgG) from 18 of 19 anti‐HRS positive patient sera react with amino acids 2‐44 and 286‐509 of HRS. Patient IgG specific for these two epitopes were found not to inhibit HRS enzyme activity. Instead, the inhibitory property of anti‐HRS was found to be associated with antibodies that do not react to HRS in immunoblots, indicating the presence of other epitopes. In addition, antibodies that react in immunoblots were found to represent only a small fraction of total anti‐HRS antibody. Our finding that patient IgG recognized at least three distinct epitopes on HRS strongly suggests that the immunological response at some point in the disease is directed against HRS and not against a cross‐reactive anti‐gen.—Martin, A., Shulman, M. J., Tsui, F. W. L. Epitope studies indicate that histidyl‐tRNA synthetase is a stimulating antigen in idiopathic myositis. FASEB J. 9, 1226‐1233 (1995)

Differential activation of human and guinea pig complement by pentameric and hexameric IgM

Human and mouse IgM can be polymerized as a hexamer in addition to a pentamer. Our previous work with mouse IgM measured activation of guinea pig complement by highly enriched preparations of hexamer and pentamer and showed that hexamer is >100‐fold more active than pentamer. In this report pentamer and hexamer were compared for their capacity to activate complement in a homogeneic system, i.e. chimeric mouse V/human Cμ IgM pentamer and hexamer were assayed separately for their capacity to activate human (and guinea pig) complement. In both the homogeneic and the xenogeneic systems hexamer was more active than pentamer, but the magnitude of the difference between hexamer and pentamer depended on the complement source. Whereas chimeric hexamer activated guinea pig complement >100‐fold more efficiently than did chimeric pentamer, this hexamer was only 4–13‐fold more active than pentamer when assayed with human complement. Similarly, mouse hexamer, which was >100‐fold more active than mouse pentamer with guinea pig complement, was only ∼2‐fold more active than mouse pentamer with human complement. Mouse hexameric and pentameric IgM were each ∼20‐fold more active with human complement than were the corresponding chimeric isoforms of IgM.

Serum levels of novel noggin and sclerostin-immune complexes are elevated in ankylosing spondylitis

Background Unravelling the basis of joint inflammation and ankylosis represents a major challenge in ankylosing spondylitis (AS) research. As noggin (NOG) and sclerostin (SOST) have recently been associated with the disease process in mouse and human studies, respectively, we explored the immune responses to these two molecules in AS. Methods Immune complexes (IC) composed of IgG autoantibodies to NOG and SOST were detected by immunoprecipitation and Western blot analyses. Epitope-specific IgG were measured using peptide-binding ELISA. Serum samples were obtained from healthy controls and patients with AS, mechanical back pain (MBP) and inflammatory bowel disease (IBD) with or without concomitant AS. Results NOG and SOST-IgG IC were present in NOG-treated and untreated ank/ank (progressive ankylosis), but not in wild-type mice. Higher than normal levels of NOG and SOST-IgG IC are present in AS sera (p<0.001). We showed a SOST peptide (SOST-S146, with homology to a bacterial glycotransferase peptide) binds to a NOG peptide (NOG-N54), which contains a N-glycosylation site. AS patients have higher levels of IgG recognising the NOG-N54 and SOST-S146 peptides compared to the levels in normal controls, IBD and MBP patients (one way analysis of variance p<0.0001). Conclusions This is the first report showing IgG autoantibodies to NOG and SOST in normal individuals, and higher levels of NOG and/or SOST-IgG IC probably contribute to neo-ossification in AS patients. These novel findings hold the promise of earlier diagnosis, better management of AS with comorbidities and new therapeutic approaches to modulate ankylosis in AS.

From gene expression to serum proteins: biomarker discovery in Ankylosing Spondylitis

Objectives: Studying post-infliximab gene expression changes could provide insights into the pathogenesis of ankylosing spondylitis (AS). Methods: Gene expression changes were screened by microarray on peripheral blood RNA of 16 AS patients at baseline and 2 weeks post-infliximab, and selected results were confirmed by quantitative real-time (qRT)–PCR. Corresponding serum-soluble LIGHT (sLIGHT) was estimated by ELISA and the fold change in sLIGHT was correlated to the fold change in erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and the Bath AS disease activity index. Results: Post-infliximab, 69% of the patients (11/16) achieved an ASAS20 response. Six candidate genes were differentially expressed by microarray; four of which were validated by qRT–PCR. sLIGHT showed the most significant difference. There was good correlation of baseline sLIGHT with CRP (R  =  0.60; p = 0.01) and ESR (R  =  0.51; p = 0.04). The fold change in sLIGHT correlated with change in both CRP (R  =  0.71, p = 0.002) and ESR (R  =  0.77, p<0.001). Conclusion: LIGHT is significantly downregulated by infliximab. sLIGHT correlated well with changes in inflammatory markers.

Modulation of the ERG K+ current by the tyrosine phosphatase, SHP-1.

We reported previously (Cayabyab, F. S., and Schlichter, L. C. (2002) J. Biol. Chem.277, 13673–13681) a functional interaction between the ERG-1 K+ channel and Src tyrosine kinase, which increased the current. We now show that the tyrosine phosphatase, SHP-1, which is present in microglia, is increased after brain damage, and is activated by colony-stimulating factor-1, associates with ERG-1 and regulates the current. Patch clamp recordings from the MLS-9 microglia cells were made with pipette solutions containing a recombinant SHP-1 protein: wild type (SHP-1 wild type (wt)), catalytically active (SHP-1 S6), or the substrate-trapping mutant (SHP-1 Cys → Ser). SHP-1 wt and SHP-1 S6 proteins decreased the current, an effect that was reversed by the phosphatase inhibitor, pervanadate, whereas SHP-1 Cys → Ser increased the current. Moreover, transient transfection with cDNA for SHP-1 wt or SHP-1 S6 decreased the ERG current without decreasing the protein level. Tyrosine phosphorylation of ERG-1 was decreased by transfection with SHP-1 wt and increased by SHP-1 Cys → Ser. The decrease in current by active SHP-1 was partly attributed to changes in the voltage dependence of activation and steady-state conductance, whereas inactivation kinetics and voltage dependence were not affected. Our results show that ERG-1 is a SHP-1 substrate constituting the first report that an ion current is regulated by SHP-1.

Role of the Intronic Elements in the Endogenous Immunoglobulin Heavy Chain Locus EITHER THE MATRIX ATTACHMENT REGIONS OR THE CORE ENHANCER IS SUFFICIENT TO MAINTAIN EXPRESSION

High level expression in mice of transgenes derived from the immunoglobulin heavy chain (IgH) locus requires both the core enhancer (Eμ) and the matrix attachment regions (MARs) that flank Eμ. The need for both elements implies that they each perform a different function in transcription. While it is generally assumed that expression of the endogenous IgH locus has similar requirements, it has been difficult to assess the role of these elements in expression of the endogenous heavy chain gene, because B cell development and IgH expression are strongly interdependent and also because the locus contains other redundant activating elements. We have previously described a gene-targeting approach in hybridoma cells that overcomes the redundancy problem to yield a stable cell line in which expression of the IgH locus depends strongly on elements in the MAR-Eμ-MAR segment. Using this system, we have found that expression of the endogenous μ gene persists at substantially (∼50%) normal levels in recombinants which retain either the MARs or Eμ. That is, despite the dissimilar biochemical activities of these two elements, either one is sufficient to maintain high level expression of the endogenous locus. These findings suggest new models for how the enhancer and MARs might collaborate in the initiation or maintenance of transcription.