Cloning and characterization of a lymphoid-specific, inducible human protein tyrosine phosphatase, Lyp.
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Protein tyrosine phosphatases act in conjunction with protein kinases to regulate the tyrosine phosphorylation events that control cell activation and differentiation. We have isolated a previously undescribed human phosphatase, Lyp, that encodes an intracellular 105-kD protein containing a single tyrosine phosphatase catalytic domain. The noncatalytic domain contains four proline-rich potential SH3 domain binding sites and an NXXY motif that, if phosphorylated, may be recognized by phosphotyrosine binding (PTB) domains. Comparison of the Lyp amino acid sequence with other known proteins shows 70% identity with the murine phosphatase PEP. The human Lyp gene was localized to chromosome 1p13 by fluorescence in situ hybridization analysis. We also identified an alternative spliced form of Lyp RNA, Lyp2. This isoform encodes a smaller 85-kD protein with an alternative C-terminus. The lyp phosphatases are predominantly expressed in lymphoid tissues and cells, with Lyp1 being highly expressed in thymocytes and both mature B and T cells. Increased Lyp1 expression can be induced by activation of resting peripheral T lymphocytes with phytohemagglutinin or anti-CD3. Lyp1 was found to be constitutively associated with the proto-oncogene c-Cbl in thymocytes and T cells. Overexpression of lyp1 reduces Cbl tyrosine phosphorylation, suggesting that it may be a substrate of the phosphatase. Thus, Lyp may play a role in regulating the function of Cbl and its associated protein kinases. (+info)
The identification of a novel T cell activation state controlled by a diabetogenic gene.
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The cyclin-dependent kinase inhibitor p27(kip) regulates the cell cycle at the G(1)-S phase restriction point. S phase entry and cell cycle commitment in peripheral T cells requires p27(kip) degradation, normally initiated by the receipt of costimulatory signals such as those provided by B7.1 or IL-2. We have previously reported that T cells from BioBreeding (BB)-diabetes-prone (DP) rats exhibit decreased costimulatory requirements for activation and cell cycle entry. In the present study, we find that peripheral T cell subsets from BB-DP rats demonstrate activation-like characteristics, including significantly reduced levels of p27(kip) as well as increased levels of proliferating cell nuclear Ag (PCNA). Since our previous studies have established that expression of extracellular activation markers are relatively low in unmanipulated peripheral BB-DP T cells; this p27(low) PCNA(high) phenotype represents a novel activation state. Analyses of T cell subsets from congenic rats demonstrate that this phenotype segregates with the lyp diabetogenic locus and that the p27(low) PCNA(high) phenotype is T cell specific. This p27(low) PCNA(high) phenotype is not seen in medullary thymocytes, but appears abruptly in the recent thymic emigrant population, suggesting that the lyp locus does not act directly on cell cycle regulators but rather alters the interaction between T cells and the peripheral environment. These results provide a biochemical basis for costimulation-independent activation and suggest a mechanism whereby a diabetes susceptibility gene contributes to disease development. (+info)
Evidence for the extrathymic origin of intestinal TCRgammadelta(+) T cells in normal rats and for an impairment of this differentiation pathway in BB rats.
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The BB rat lyp mutation, one of its diabetes susceptibility genes, is responsible for a 5-fold decrease in the number of peripheral TCRalphabeta(+) T cells. In this study we show that TCRgammadelta(+) T cells are virtually undetectable among splenic T cells and intestinal intraepithelial T lymphocytes (IEL) of BB rats, while they account for 3 and 30% of these two T cell populations, respectively, in normal animals. It has been shown that murine IEL expressing TCRgammadelta develop extrathymically. We determined whether this is the case in rats. Athymic radiation chimeras reconstituted with normal hemopoietic precursors were devoid of donor-derived TCRalphabeta(+) T cells and TCRgammadelta(+) splenocytes but contained a normal number of TCRgammadelta(+) IEL, suggesting that in unmanipulated rats some of the TCRgammadelta(+) IEL may have an extrathymic origin. This was further supported by the observation that RAG1 transcripts are present in IEL of unmanipulated animals. No T cells developed in chimeras reconstituted with BB hemopoietic precursors, demonstrating that the BB rat lyp mutation inhibits both intrathymic and extrathymic development of TCRgammadelta(+) T cells. (+info)
Lymphopenia in the BB rat model of type 1 diabetes is due to a mutation in a novel immune-associated nucleotide (Ian)-related gene.
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The BB (BioBreeding) rat is one of the best models of spontaneous autoimmune diabetes and is used to study non-MHC loci contributing to Type 1 diabetes. Type 1 diabetes in the diabetes-prone BB (BBDP) rat is polygenic, dependent upon mutations at several loci. Iddm1, on chromosome 4, is responsible for a lymphopenia (lyp) phenotype and is essential to diabetes. In this study, we report the positional cloning of the Iddm1/lyp locus. We show that lymphopenia is due to a frameshift deletion in a novel member (Ian5) of the Immune-Associated Nucleotide (IAN)-related gene family, resulting in truncation of a significant portion of the protein. This mutation was absent in 37 other inbred rat strains that are nonlymphopenic and nondiabetic. The IAN gene family, lying within a tight cluster on rat chromosome 4, mouse chromosome 6, and human chromosome 7, is poorly characterized. Some members of the family have been shown to be expressed in mature T cells and switched on during thymic T-cell development, suggesting that Ian5 may be a key factor in T-cell development. The lymphopenia mutation may thus be useful not only to elucidate Type 1 diabetes, but also in the function of the Ian gene family as a whole. (+info)
Characterization of a myeloid tyrosine phosphatase, Lyp, and its role in the Bcr-Abl signal transduction pathway.
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The Bcr-Abl protein-tyrosine kinase is implicated in the development of chronic myeloid leukemia. The potential role of protein-tyrosine phosphatase in the regulation of Bcr-Abl signaling was explored. First, expression patterns of tyrosine phosphatases in leukemic cell lines were investigated using degenerate primers for reverse transcription-PCR followed by cloning and sequencing of the cDNA. Distinct patterns of distribution of phosphatase were found in erythroid and myeloid leukemic cell lines. Whereas some phosphatases were ubiquitously expressed, others were limited to specific cell types. Surprisingly, a previously cloned "lymphocyte-specific" phosphatase, Lyp, was frequently detected in a number of myeloid cell lines as well as normal granulocytes and monocytes. Lyp was localized to the cytosol, and overexpression of Lyp caused reduction in the phosphorylation levels of multiple proteins in KCL22 chronic myeloid leukemia blast cells including Cbl, Bcr-Abl, Erk1/2, and CrkL. Co-expression of Lyp and Bcr-Abl in Cos-7 cells resulted in decreased levels of Bcr-Abl, Grb2, and Myc. Overexpression of Lyp markedly suppressed anchorage-independent clonal growth of KCL22 cells. Taken together, the data suggest that Lyp may play an antagonistic role in signaling by the Bcr-Abl fusion protein. (+info)
Involvement of eotaxin, eosinophils, and pancreatic predisposition in development of type 1 diabetes mellitus in the BioBreeding rat.
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Allergy and autoimmunity are both examples of deregulated immunity characterized by inflammation and injury of targeted tissues that have until recently been considered disparate disease processes. However, recent findings have implicated mast cells, in coordination with granulocytes and other immune effector cells, in the pathology of these two disorders. The BioBreeding (BB) DRlyp/lyp rat develops an autoimmune insulin-dependent diabetes similar to human type 1 diabetes mellitus (T1DM), whereas the BBDR+/+ rat does not. To better understand immune processes during development of T1DM, gene expression profiling at day (d) 40 (before insulitis) and d65 (before disease onset) was conducted on pancreatic lymph nodes of DRlyp/lyp, DR+/+, and Wistar-Furth (WF) rats. The eosinophil-recruiting chemokine, eotaxin, and the high-affinity IgE receptor (FcepsilonRI) were up-regulated >5-fold in d65 DRlyp/lyp vs d65 DR+/+ pancreatic lymph nodes by microarray (p < 0.05) and quantitative RT-PCR studies (p < 0.05). DR+/+, WF, and d40 DRlyp/lyp animals possessed normal pancreatic histology; however, d65 DRlyp/lyp animals possessed eosinophilic insulitis. Therefore, immunohistochemistry for pancreatic eotaxin expression was conducted, revealing positive staining of d65 DRlyp/lyp islets. Islets of d65 DR+/+ rats also stained positively, consistent with underlying diabetic predisposition in the BB lineage, whereas WF islets did not. Other differentially expressed transcripts included those associated with eosinophils, mast cells, and lymphocytes. These data support an important role for these inflammatory mediators in BB rat T1DM and suggest that the lymphopenia due to the Ian5/(lyp) mutation may result in a deregulation of cells involved in insulitis and beta cell destruction. (+info)
The non-major histocompatibility complex quantitative trait locus Cia10 contains a major arthritis gene and regulates disease severity, pannus formation, and joint damage.
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OBJECTIVE: To construct rats congenic for the chromosome 2 arthritis-regulatory quantitative trait locus Cia10, originally identified in a (DA x ACI)F(2) intercross rat strain that had been assessed for collagen-induced arthritis (CIA), and to determine the effect of this congenic interval on arthritis severity, joint histologic structure, and cytokine transcription in rats with pristane-induced arthritis (PIA). METHODS: A 52.6-MB interval derived from the ACI (CIA- and PIA-resistant) strain and containing the Cia10 interval was introgressed into the DA (arthritis-susceptible) background through genotype-guided congenic breeding. Homozygous male and female DA.ACI(Cia10) congenic rats were studied for their susceptibility to and severity of PIA, and were compared with same-sex DA rats. Histologic analyses were done on hind paws collected on day 32 following the pristane injection. Levels of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) messenger RNA (mRNA) were measured with real-time polymerase chain reaction on synovial tissues from day-32 ankles. RESULTS: Both male and female DA.ACI(Cia10) congenic rats developed a significantly milder form of arthritis, with a 95% and 92% reduction in the arthritis severity index compared with DA male and female controls, respectively (males P < or = 0.001 and females P = 0.003). DA.ACI(Cia10) congenic rat synovial tissue was more likely to preserve its normal histologic architecture, including minimal to no cartilage and bone erosions, synovial hyperplasia, and pannus formation, and reduced numbers of vessels (angiogenesis), when compared with DA synovial tissue. There was a 2.7- and 2.4-fold reduction in the amount of IL-1beta and TNFalpha mRNA, respectively, in the synovial tissue of DA.ACI(Cia10) congenic rats compared with DA rats. Sequencing analyses of complementary DNA for the Cia10-predicted candidate gene Ptpn8, the rat homolog of the rheumatoid arthritis (RA)-susceptibility gene PTPN22, revealed no polymorphisms between the DA and ACI strains. CONCLUSION: This study determined that Cia10 harbors a major autoimmune arthritis-regulatory gene. This gene regulates clinical disease severity, histologic damage, and the levels of at least two central proinflammatory cytokines. We are in the process of narrowing down the critical region for positional cloning of the Cia10 gene. The identification of this gene will provide novel targets or pathways for focused candidate-gene studies in RA. (+info)
Association of a functional single-nucleotide polymorphism of PTPN22, encoding lymphoid protein phosphatase, with rheumatoid arthritis and systemic lupus erythematosus.
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OBJECTIVE: To assess the possible association between the PTPN22 gene 1858C-->T polymorphism and the predisposition and clinical expression of 2 systemic autoimmune diseases, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). METHODS: Our study population consisted of 826 RA patients, 338 SLE patients, and 1,036 healthy subjects. All subjects were of Spanish Caucasian origin. Genotyping of the PTPN22 gene 1858C-->T polymorphism was performed by real-time polymerase chain reaction technology, using the TaqMan 5'-allele discrimination assay. RESULTS: The overall distribution of genotypes in the RA patients was significantly different from that in the controls (P = 0.005, by chi-square test with 2 x 3 contingency tables). We observed a statistically significant difference in the distribution of the PTPN22 1858T allele between healthy subjects (7.4%), and RA patients (10.4%) (P = 0.001, odds ratio [OR] 1.45 [95% confidence interval (95% CI) 1.15-1.83]). In addition, PTPN22 1858 C/T and T/T genotypes were present at a significantly higher frequency in SLE patients than in controls (P = 0.02, OR 1.55 [95% CI 1.05-2.29]). Differences were also observed when allele frequencies were compared, with the PTPN22 1858T allele being present at a higher frequency among SLE patients (P = 0.03, OR 1.45 [95% CI 1.01-2.09]). CONCLUSION: These results suggest that the PTPN22 1858T allele may confer differential susceptibility to RA and SLE in the Spanish population. (+info)