G 13 protein, one of the heterotrimeric guanine nucleotide-binding proteins (G proteins), regulates diverse and complex cellular responses by transducing signals from the cell surface presumably involving more than one pathway. Yeast two-hybrid screening of a mouse brain cDNA library identified radixin, a member of the ERM family of three closely related proteins (ezrin, radixin, and moesin), as a protein that interacted with G␣ 13 . Interaction between radixin and G␣ 13 was confirmed by in vitro binding assay and by co-immunoprecipitation technique. Activated G␣ 13 induced conformational activation of radixin, as determined by binding of radixin to polymerized F-actin and by immunofluorescence in intact cells. Finally, two dominant negative mutants of radixin inhibited G␣ 13 -induced focus formation of Rat-1 fibroblasts but did not affect Ras-induced focus formation. Our results identifying a new signaling pathway for G␣ 13 indicate that ERM proteins can be activated by and serve as effectors of heterotrimeric G proteins.

Collagen-induced arthritis (CIA) and proteoglycan-induced arthritis (PGIA) are murine models for rheumatoid arthritis both in terms of their pathology and genetics. Using the F2 hybrids of the CIA-susceptible, but PGIA-resistant DBA/1 mice, and the CIA-resistant, but PGIA-susceptible BALB/c mice, our goals were to 1) identify both model-specific and shared loci that confer disease susceptibility, 2) determine whether any pathophysiological parameters could be used as markers that distinguish between nonarthritic and arthritic mice, and 3) analyze whether any immune subtraits showed colocalization with arthritis-related loci. To identify chromosomal loci, we performed a genome scan on 939 F2 hybrid mice. For pathophysiological analyses, we measured pro- and anti-inflammatory cytokines (IL-1, IL-6, TNF-α, IFN-γ, IL-4, IL-10, IL-12), Ag-specific T cell proliferation and IL-2 production, serum IgG1 and IgG2 levels of both auto- and heteroantibodies, and soluble CD44. In addition to multiple CIA- and PGIA-related loci identified in previous studies, we have identified nine new CIA- and eight new PGIA-linked loci. Comprehensive statistical analysis demonstrated that IL-2 production, T cell proliferation, and IFN-γ levels differed significantly between arthritic and nonarthritic animals in both CIA and PGIA populations. High levels of TNF-α, IFN-γ, IL-2, and Ab production were detected in F2 hybrids with CIA, whereas T cell proliferation, IL-2 and IFN-γ production, and a shift to IgG2a isotype were more characteristic of PGIA. Quantitative trait loci analysis demonstrated colocalization of numerous immune subtraits with arthritis-related traits. Quantitative trait loci on chromosomes 5, 10, 17, 18, and X were found to control arthritis in both models.

Astrocyte dysregulation correlates with the severity and the rate of human immunodeficiency virus (HIV)-associated dementia (HAD) progression, highlighting a pivotal role for astrocytes in HIV neuropathogenesis. Yet, astrocytes limit HIV, indicating that they posses an intrinsic molecular mechanism to restrict HIV replication. We previously established that this restriction can be partly overcome by priming astrocytes with gamma interferon (IFN-␥), which is elevated in the cerebral spinal fluid of HAD patients. We evaluated the mechanism of restrictive HIV replication in astrocytes and how IFN-␥ priming modulates this restriction. We demonstrate that the downstream effector of Wnt signaling, T-cell factor 4 (TCF-4), is part of a transcriptional complex that is immunoprecipitated with HIV TAR-containing region in untreated astrocytes but not in IFN-␥-treated cells. Blocking TCF-4 activity with a dominant-negative mutant enhanced HIV replication by threefold in both the astrocytoma cell line U87MG and primary fetal astrocytes. Using a TCF-4 reporter plasmid, we directly demonstrate that Wnt signaling is active in human astrocytes and is markedly reduced by IFN-␥ treatment. Collectively, these data implicate TCF-4 in repressing HIV replication and the ability of IFN-␥ to regulate this restriction by inhibiting TCF-4. Given that TCF-4 is the downstream effector of Wnt signaling, harnessing Wnt signaling as an intrinsic molecular mechanism to limit HIV replication may emerge as a powerful tool to regulate HIV replication within and outside of the brain.

c Molecular regulation of HIV transcription is a multifaceted process dictated in part by the abundance of cellular transcription factors that induce or repress HIV promoter activity. ␤-Catenin partners with members of the T cell factor (TCF)/LEF transcription factors to regulate gene expression. The interaction between ␤-catenin and TCF-4 is linked to inhibition of HIV replication in multiple cell types, including lymphocytes and astrocytes. Here, we evaluated the molecular mechanism by which ␤-catenin/ TCF-4 repress HIV replication. We identified for the first time multiple TCF-4 binding sites at ؊336, ؊143, ؉66, and ؉186 relative to the transcription initiation site on the HIV long terminal repeat (LTR). Two of the sites (؊143 and ؉66) were present in approximately 1/3 of 500 HIV-1 isolates examined. Although all four sites could bind to TCF-4, the strongest association occurred at ؊143. Deletion and/or mutation of ؊143, in conjunction with ␤-catenin or TCF-4 knockdown in cells stably expressing an LTR reporter construct, enhanced basal HIV promoter activity by 5-fold but had no effect on Tat-mediated transactivation of the HIV LTR. We also found that TCF-4, ␤-catenin, and the nuclear matrix binding protein SMAR1 tether at the ؊143-nucleotide (nt) site on the HIV LTR to inhibit HIV promoter activity. Collectively, these data indicate that TCF-4 and ␤-catenin at ؊143 associate with SMAR1, which likely pulls the HIV DNA segment into the nuclear matrix and away from transcriptional machinery, leading to repression of basal HIV LTR transcription. These studies point to novel avenues for regulation of HIV replication by manipulation of ␤-catenin signaling within cells.

Objective. To explore the effect of sex on clinical and immunologic traits in major histocompatibility complex-matched (H-2d) F 2 hybrid mice with proteoglycan (PG)-induced arthritis and to identify how the quantitative trait locus (QTL) on the X chromosome influences the onset QTL of another chromosome.Methods. (BALB/c ؋ DBA/2)F 2 hybrid mice were immunized with cartilage PG, and a genome-wide linkage analysis was performed using >200 simple sequence-length polymorphic markers. The major clinical traits (susceptibility, onset, and severity) were assessed, and PG-specific T and B cell responses, and the production of proinflammatory and antiinflammatory cytokines (tumor necrosis factor ␣, interleukin-1 [IL-1], IL-6, interferon-␥, IL-4, IL-10, and IL-12) were measured in 133 arthritic and 426 nonarthritic female and male F 2 hybrid mice. The major clinical and immunologic traits were linked to genetic loci, and potential linkages among these QTLs and the effect of sex were analyzed.Results. Thirteen QTLs reported in previous studies were confirmed. Binary traits (susceptibility to arthritis) and disease onset were female specific and were identified on chromosomes 3, 7, 10, 11, 13, and X. QTLs for disease severity were mostly male specific and were located on chromosomes 1, 4, 5, 8, 14, 15, and 19. In addition, we identified 4 new QTLs for the onset of arthritis on chromosomes 3, 4, and 11, and 1 new QTL for severity on chromosome 14; all showed a strong gender association. A locus on the X chromosome interacted with a QTL on chromosome 10, and these 2 loci together seemed to control disease incidence and onset. Most of the clinical traits (QTLs) shared common regions with the immunologic traits and frequently showed a locus-locus interaction.Conclusion. Numerous immunologic QTLs overlap with clinical QTLs, thus providing information about possible mechanisms underlying QTL function. Disease susceptibility and onset showed predominant linkage with the female sex, under the control of a QTL on the X chromosome, while the severity QTLs were more strongly linked to the male sex.

Objective Gender disparities in rheumatoid arthritis (RA) are well documented despite the lack of any known major RA susceptibility genes mapped to sex chromosomes. Murine chromosome 15 carries the sex-affected Pgia8 locus that mediates proteoglycan-induced arthritis (PGIA); homologous human loci are associated with RA. This study uses a Pgia8 congenic strain to identify genes/mechanisms implicated in gender disparities in arthritis. Methods Gene expression analysis was performed using RNA isolated from paws of congenic males and females with collagen antibody-induced arthritis (CAIA). Data were corroborated with RT-PCR and also studied in mice prior to disease onset. Ingenuity Pathways Analysis of the expression patterns and gene functions were used to discover locus-specific and sex-affected signature transcripts. Results We found that the Pgia8 locus regulates antibody-mediated inflammatory arthritis differently in males and females. In Pgia8 congenic males, arthritis severity was 30% less (p < 0.005) than in wild-type males, but the anti-inflammatory effect was similar in wild type and congenic females. Transcriptome analysis indicated that twelve genes within the locus were significantly deregulated in arthritic joints of congenic mice; expression of these genes was also gender specific. The genes that correlated the most with arthritis severity include collagen triple helix repeat containing-1 (Cthrc1), metalloproteinase Adamts12, R-spondyn (Rspo2) and Syndecan (Sdc2) (r=0.87–0.91). The level of Cthrc1 message also correlated with that of pro-inflammatory cytokines IL-1β and IL-6. Conclusion Gender-specific disparities in RA are linked to transcriptional regulation of genes involved in cartilage degradation (Adamts12) and canonical and non-canonical Wnt signaling (Cthrc1, Rspo2, Sdc2).

We found a spontaneous autosomal mutation in a mouse leading to neutrophil infiltration with ulceration in the upper dermis of homozygous offspring. These animals had increased neutrophil numbers, associated with normal lymphocyte count, in peripheral blood and bone marrow, suggesting a myeloproliferative disorder; however, granulocyte precursor proliferation in bone marrow was actually reduced (because circulating neutrophils were less susceptible to apoptosis). Neutrophil infiltration of the skin and other organs and high serum levels of immunoglobulins and autoantibodies, cytokines, and acute-phase proteins were additional abnormalities, all of which could be reduced by high-dose corticosteroid treatment or neutrophil depletion by antibodies. Use of genome-wide screening localized the mutation within an 0.4-Mbp region on mouse chromosome 6. We identified insertion of a B2 element in exon 6 of the Ptpn6 gene (protein tyrosine phosphatase, non-receptor type 6; also known as Shp-1). This insertion involves amino acid substitutions that significantly reduced the enzyme activity in mice homozygous for the mutation. Disease onset was delayed, and the clinical phenotype was milder than the phenotypes of other Ptpn6-mutants described in motheaten (me, mev) mice; we designated this new genotype as Ptpn6(meB2/meB2) and the phenotype as meB2. This new phenotype encompasses an autoinflammatory disease showing similarities to many aspects of the so-called neutrophilic dermatoses, a heterogeneous group of skin diseases with unknown etiology in humans.

BackgroundAngiotensin-converting enzyme (ACE) metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases, including asthma. Previously, a molecular mechanism underlying a 5-fold familial increase of blood ACE was discovered: Pro1199Leu substitution enhanced the cleavage-secretion process. Carriers of this mutation were Caucasians from Europe (mostly Dutch) or had European roots.Methodology/Principal FindingsWe have found a family of African-American descent whose affected members' blood ACE level was increased 13-fold over normal. In affected family members, codon TGG coding for Trp1197 was substituted in one allele by TGA (stop codon). As a result, half of ACE expressed in these individuals had a length of 1196 amino acids and lacked a transmembrane anchor. This ACE mutant is not trafficked to the cell membrane and is directly secreted out of cells; this mechanism apparently accounts for the high serum ACE level seen in affected individuals. A haplotype of the mutant ACE allele was determined based on 12 polymorphisms, which may help to identify other carriers of this mutation. Some but not all carriers of this mutation demonstrated airflow obstruction, and some but not all have hypertension.Conclusions/SignificanceWe have identified a novel Trp1197Stop mutation that results in dramatic elevation of serum ACE. Since blood ACE elevation is often taken as a marker of disease activity (sarcoidosis and Gaucher diseases), it is important for clinicians and medical scientists to be aware of alternative genetic causes of elevated blood ACE that are not apparently linked to disease.