Motheaten and viable motheaten mice have mutations in the haematopoietic cell phosphatase gene.

Mice with the recessive motheaten (me) or the allelic viable motheaten (mev) mutations express a severe autoimmune and immunodeficiency syndrome. We have shown that the basic defect in these mice involves lesions in the gene which encodes haematopoietic cell phosphatase (HCP). These mice thus provide excellent models for investigating the roles of phosphatases in haematopoiesis and the nature of the genetic and cellular events linking impaired haematopoiesis to severe immunodeficiency and expression of systemic autoimmunity.

B and T cells are not required for the viable motheaten phenotype.

Hematopoietic cell phosphatase (HCP), encoded by the hcph gene, (also called PTP1C, SHP, SH-PTP1, and PTPN6) is deficient in motheaten (me/me), and the allelic viable motheaten (me(v)/me(v)) mice. Since HCP is expressed in many cell types and protein phosphorylation is a major mechanism of regulating protein function, it is not surprising that the motheaten phenotype is pleiotropic. It is commonly thought that immune system involvement causes this disease. If so, the motheaten disease ought to be alleviated when the recombination activation gene-1 (RAG-1) is disrupted because there will be no V(D)J rearrangement and thus impaired development of B and T cells. We bred homozygous, double-mutant me(v)/me(v) x RAG 1 -/- mice and found that, in fact, inflamed paws, and splenomegaly with elevated myelopoiesis. Thus, except for autoantibodies, the motheaten phenotype does not depend on the presence of B and T cells. This observation cautions the use of motheaten mice as a model of autoimmune disease.

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.

Amplification of the gene for histidyl-tRNA synthetase in histidinol-resistant Chinese hamster ovary cells.

Histidinol-resistant (HisOHR) mutants with up to a 30-fold increase in histidyl-tRNA synthetase activity have been isolated by stepwise adaptation of wild-type Chinese hamster ovary (CHO) cells to increasing amounts of histidinol in the medium. Immunoprecipitation of [35S]methionine-labeled cell lysates with antibodies to histidyl-tRNA synthetase showed increased synthesis of the enzyme in histidinol-resistant cells. The histidinol-resistant cell lines had an increase in translatable polyadenylated mRNA for histidyl-tRNA synthetase. A cDNA for CHO histidyl-tRNA synthetase has been cloned, using these histidyl-tRNA synthetase-overproducing mutants as the source of mRNA. Southern blot analysis of wild-type and histidinol-resistant cells with this cDNA showed that the histidyl-tRNA synthetase DNA bands were amplified in the resistant cells. These HisOHR cells owed their resistance to histidinol to amplification of the gene for histidyl-tRNA synthetase.

SHP-1 variant proteins are absent in motheaten mice despite presence of splice variant transcripts with open reading frames.

Motheaten mice have a mutation that causes abnormal splicing of the SHP-1 gene producing transcripts that are out of frame. Thus, motheaten mice cannot produce normal SHP-1 protein. However, we have found that the SHP-1 locus in normal mice is expressed as multiple in-frame splice variant transcripts. We report here that the motheaten SHP-1 gene is likewise expressed in multiple spliced forms, two of which are in frame. One of these two variants, which is also present in normal mice, lacks the exon containing the motheaten mutation and is therefore expected to encode an active phosphatase with only one of the two SH2 domains of SHP-1. We showed that both of these variants produce phosphatases with a higher specific activity than SHP-1, suggesting that motheatein mice are not SHP-1 null. The possibility that motheaten mice produce disregulated phosphatases offered a simple explanation for the puzzling observation that substrates of SHP-1 are hypo-phosphorylated in motheaten mice. We tested this by measuring for SHP-1 protein and activity in motheaten macrophages. However, we did not detect specific activity, and found that one of these variant proteins was unstable. These findings likewise suggest that little or no SHP-1 variant proteins exist in normal cells.

Structural analysis of the 5' region of the chromosomal gene for hamster histidyl-tRNA synthetase.

The chromosomal gene (HRS) coding for hamster histidyl-tRNA synthetase, like many other housekeeping genes, lacks many of the features associated with promoters of RNA-polymerase-II-transcribed genes. HRS transcripts have multiple start points. Using RNase protection analysis, we also identified a 300-bp exon located only 36 bp away from the 5'-most start point of the HRS transcript. This exon hybridizes to a 3.5-kb transcript which transcribes from a different strand of DNA in the 5' region of the HRS gene. This divergent 3.5-kb transcript also has multiple transcription start points. The identity and function of this 3.5-kb transcript is not known.

Transcriptional analyses of the gene region that encodes human histidyl-tRNA synthetase: identification of a novel bidirectional regulatory element.

A recombinant phage clone containing the 5' end of the gene HRS encoding human histidyl-tRNA synthetase (HRS) has been isolated. Primer extension analyses indicated that there are two types of HRS transcripts. The longer transcripts were initiated from a single transcription start point (tsp) located approximately 455 bp upstream and the shorter transcripts were initiated from multiple tsp located approximately 38 to 82 bp upstream from the HRS ATG start codon. Functionally, we have identified two regions (+1 to -122; -185 to -502), each of which when placed 5' of a promoterless cat construct can initiate transcription in both orientations after transfection into HeLa cells. A pair of imperfect inverted repeats (IIR) was located within the region +1 to -122. Using mobility shift assays, we have identified a nuclear factor that binds specifically to each half of the IIR. However, this pair of IIR (-73 to -110) was not sufficient for bidirectional transcription activity. At least one copy of a 27-bp oligodeoxyribonucleotide (oligo), which spans -94 to -120, was required in order to facilitate bidirectional transcription activity. From mobility shift assays using HeLa cell nuclear extracts and this 27-bp oligo, we have identified two DNA-protein complexes, both of which are presumably required to initiate bidirectional transcription.

Anti-Jo-1 autoantibodies and the immunopathogenesis of autoimmune myositis.

Polymyositis and dermatomyositis are inflammatory myopathies characterized by proximal muscle weakness and myopathic electromyographic and histological findings. While the causes of myositis are not known, the close association of these disorders with a spectrum of autoantibodies suggests an etiologic and/or pathogenetic role for autoimmune processes. Of particular interest in this regard are antibodies directed against histidyl as well as other tRNA synthetases which are almost uniquely associated with myositis and may define a distinct subset of patients. Recently we isolated the histidyl tRNA synthetase gene which encodes the autoantigen representing the most frequent target of the myositis autoimmune response. The isolation and expression of this gene has allowed us to investigate both the autoreactive epitopes on histidyl-tRNA synthetase and the extent to which these correlate with functional epitopes on the molecule. As described here, the results of these studies as well as other recent data pertaining to the immunopathogenesis of myositis, provide a framework for delineating the mechanisms which render synthetases and other translation-related proteins autoantigenic in myositis, and allow one to examine the significance of such autoimmune responses in the etiology and pathogenesis of inflammatory myopathy.

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.

Isolation, structure and expression of mammalian genes for histidyl-tRNA synthetase.

A full length cDNA clone that codes for human histidyl-tRNA synthetase (HRS) and cDNA clones that span the full length transcript of hamster HRS have been isolated. The full length human HRS cDNA was expressed after transfection into Cos 1 cells and a CHO ts mutant defective in the gene for HRS. The complete nucleotide sequence of the hamster and human gene were obtained and extensive homologies were observed in three regions on comparing these sequences between themselves and with the sequence of HRS derived from yeast. These results provide unequivocal evidence that we have indeed cloned the hamster and human gene for HRS. Three overlapping phage recombinants containing the complete hamster chromosomal gene for HRS have also been isolated. The genomic HRS is divided into 13 exons. The precise locations of each of the 5' and 3' exon-intron boundaries were defined by sequencing the appropriate regions of the cloned genomic DNA and aligning them with the sequence of HRS cDNAs. These studies provide the basis for future structural and functional analysis of the gene for HRS. In particular, it will be of interest to examine if different exons of HRS correlate to different domains of the HRS polypeptide.

Targeted removal of the mu switch region from mouse hybridoma cells. A test of its role in gene expression in the endogenous IgH locus.

The switch regions adjoining the DNA encoding the Ig heavy chain constant regions have been implicated in gene expression as well as isotype switching, in that transgenic mice express switch-containing transgenes at a level 100- to 1000-fold higher than the corresponding switch-deleted transgenes. To test whether the switch region of the natural IgH locus is also required for high level expression we have used homologous recombination to generate targeted recombinant hybridoma cell lines that lack the switch region sequences from the major intron of the mu gene. The expression pattern of these switch knock-out cell lines was compared with that of the parental cell line as well as to that of control recombinants using both steady-state mRNA level and nuclear run-on activity to assess heavy chain gene expression. In striking contrast with the results reported for transgenic animals, we have found only a minimal effect, if any, of deleting the switch element from the natural chromosomal location.

Cellular localization of the target structures recognized by the anti-Jo-1 antibody: immunofluorescence studies on cultured human myoblasts.

Antibodies to Jo-1 (alpha Jo-1) are most characteristically detected in patients with the idiopathic inflammatory muscle disease polymyositis (PM). The Jo-1 antigen has previously been identified as histidyl-tRNA synthetase (HRS). In order to clarify the cellular localization of the antigenic targets recognized by the alpha Jo-1 antibody, immunofluorescence (IF) studies were performed with cultured human myoblasts. Incubation with alpha Jo-1 positive sera demonstrated granular cytoplasmic as well as nuclear staining, but only the cytoplasmic fluorescence was specifically inhibited by preabsorbing the sera with recombinant histidyl-tRNA synthetase (rHRS). A polyclonal rabbit anti-rHRS sera demonstrated granular cytoplasmic IF which was also specifically inhibited by preincubation with rHRS protein. Alpha Jo-1 negative healthy control or patient sera demonstrated nonspecific low intensity staining. 35S methionine biosynthetically labelled myoblast cell extracts immunoprecipitated with alpha Jo-1 positive sera and analyzed by SDS-PAGE revealed a specific band of the same molecular weight as the rHRS antigen. Our studies demonstrate that alpha Jo-1 specifically binds to antigen in the cytoplasm of cultured myoblasts. Alpha Jo-1 has been shown to inhibit HRS activity in vitro. Given the importance of aminoacyl tRNA synthetases such as HRS to intracellular protein assembly, intracytoplasmic binding and enzyme inhibition in vivo may potentially contribute to the pathogenesis of autoimmune muscle damage in PM.

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 antigen.

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 (Emu) and the matrix attachment regions (MARs) that flank Emu. 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-Emu-MAR segment. Using this system, we have found that expression of the endogenous mu gene persists at substantially (approximately 50%) normal levels in recombinants which retain either the MARs or Emu. 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.

Murine SHP-1 splice variants with altered Src homology 2 (SH2) domains. Implications for the SH2-mediated intramolecular regulation of SHP-1.

SHP-1 is a protein-tyrosine phosphatase with two Src homology 2 (SH2) domains. These SH2 domains determine which proteins SHP-1 associates with, but they also autoregulate the activity of the catalytic domain. In this report, we find that the murine SHP-1 transcript is processed to yield a series of alternatively spliced in-frame transcripts, the majority of which exclude exons encoding one or the other SH2 domain. We have examined the corresponding protein isoforms in several ways. First, our measurements of V(max) and K(m) under different conditions indicate that the SH2 variants have elevated activity because of lessened autoregulation. Second, to ascertain whether regulation by the SH2 domains reflects intra- or intermolecular effects, we analyzed the state of SHP-1 by high performance liquid chromatography and sucrose density gradient centrifugation. Our results showed that SHP-1 is a monomer and, thus, is regulated in an intramolecular manner. Third, our analyses detected shape differences between SHP-1 and the active splice variant protein deleted of the amino-terminal SH2 domain; i.e. SHP-1 was globular and resistant to proteolytic digestion, while the splice variant protein was "rod-shaped" and more susceptible to proteolytic digestion.

Overexpression of the protein tyrosine phosphatase, nonreceptor type 6 (PTPN6), in human epithelial ovarian cancer.

Our current understanding of human ovarian tumorigenesis is limited by the lack of a discrete precursor lesion as well as a limited knowledge of the steps in tumor progression. Since the alterations in the regulation of the tyrosyl residues on various cellular proteins appear to be an important pathway in neoplastic transformation, it is possible that changes in the expression of the proteins that control tyrosine phosphorylation (i.e., tyrosine kinases and phosphatases) may play a role in ovarian cancer development. Protein tyrosine phosphatase, nonreceptor type 6 (PTPN6), contains two src homology 2 domains and is expressed primarily in hematopoietic and epithelial cells. Using Northern blot and immunoblotting analysis, we showed that both the PTPN6 transcripts and proteins were overexpressed two- to four-fold in 7 of the 8 ovarian epithelial carcinoma cell lines studied. In addition, we showed that there was also a two- to threefold increase in expression of the PTPN6 transcript in 10 of 11 (91%) invasive ovarian epithelial cancer tissues examined. These observations suggest that the PTPN6 gene is potentially of etiologic relevance to a majority of ovarian cancers.

Mouse NKR-P1B, a novel NK1.1 antigen with inhibitory function.

The mouse NK1.1 Ag originally defined as NK cell receptor (NKR)-P1C (CD161) mediates NK cell activation. Here, we show that another member of the mouse CD161 family, NKR-P1B, represents a novel NK1.1 Ag. In contrast to NKR-P1C, which functions as an activating receptor, NKR-P1B inhibits NK cell activation. Association of NKR-P1B with Src homology 2-containing protein tyrosine phosphatase-1 provides a molecular mechanism for this inhibition. The existence of these two NK1.1 Ags with opposite functions suggests a potential role for NKR-P1 molecules, such as those of the Ly-49 gene family, in regulating NK cell function.

Epitope mapping of the cloned human autoantigen, histidyl-tRNA synthetase. Analysis of the myositis-associated anti-Jo-1 autoimmune response.

Autoantibodies that bind aminoacyl-tRNA synthetases are strongly associated with the human inflammatory myopathies polymyositis and dermatomyositis, but their molecular origins and relationship to pathogenesis are not known. To address these issues, we wished to identify the autoantigenic epitopes which react with these autoantibodies and to this end, we previously isolated a full length cDNA clone encoding the target Ag recognized most frequently by myositis sera, histidyl-tRNA synthetase (HRS). In the present study, we have analyzed the HRS autoepitopes by two amino acid insertion linker mutagenesis of HRS proteins expressed in Cos 1 cells. A series of mutant HRS cDNA were constructed and the expressed proteins were tested for enzyme activity and for immune reactivity with a panel of sera with anti-Jo-1 antibodies. Immunoblotting and immunoprecipitation analyses revealed that anti-Jo-1 antibodies recognize multiple conformation-dependent and independent epitopes on HRS and that the autoepitopes vary among different myositis patients.

Molecular characterization and mapping of murine genes encoding three members of the stefin family of cysteine proteinase inhibitors.

Stefins or Type 1 cystatins belong to a large, evolutionarily conserved protein superfamily, the members of which inhibit the papain-like cysteine proteinases. We report here on the molecular cloning and chromosomal localization of three newly identified members of the murine stefin gene family. These genes, designated herein as mouse stefins 1, 2, and 3, were isolated on the basis of their relatively increased expression in moth-eaten viable compared to normal congenic mouse bone marrow cells. The open reading frames of the stefin cDNAs encode proteins of approximately 11.5 kDa that show between 50 and 92% identity to sequences of stefins isolated from various other species. Data from Southern analysis suggest that the murine stefin gene family encompasses at least 6 and possibly 10-20 members, all of which appear to be clustered in the genome. Analysis of interspecific backcross mice indicates that the genes encoding the three mouse stefins all map to mouse chromosome 16, a localization that is consistent with the recent assignment of the human stefin A gene to a region of conserved homology between human chromosome 3q and the proximal region of mouse chromosome 16.