Type I collagen is the most abundant protein in human body. Fibrosis is characterized by excessive synthesis of type I collagen in parenchymal organs. It is a leading cause of morbidity and mortality worldwide, about 45% of all natural deaths are attributable to some fibroproliferative disease. There is no cure for fibrosis. To find specific antifibrotic therapy targeting type I collagen, critical molecular interactions regulating its synthesis must be elucidated. Type I and type III collagen mRNAs have a unique sequence element at the 5' end, the 5' stem-loop. This stem-loop is not found in any other mRNA. We cloned LARP6 as the protein which binds collagen 5' stem-loop with high affinity and specificity. Mutation of the 5' stem-loop or knock down of LARP6 greatly diminishes collagen expression. Mice with mutation of the 5' stem-loop are resistant to development of liver fibrosis. LARP6 associates collagen mRNAs with filaments composed of nonmuscle myosin; disruption of these filaments abolishes synthesis of type I collagen. Thus, LARP6 dependent collagen synthesis is the specific mechanism of high collagen expression seen in fibrosis. We developed fluorescence polarization (FP) method to screen for drugs that can inhibit binding of LARP6 to 5' stem-loop RNA. FP is high when LARP6 is bound, but decreases to low levels when the binding is competed out. Thus, by measuring decrease in FP it is possible to identify chemical compounds that can dissociate LARP6 from the 5' stem-loop. The method is simple, fast and suitable for high throughput screening.

The heavy metal mercury elicits a genetically restricted, anti-nucleolar autoantibody response that targets fibrillarin, a 34-kDa protein component of many small nucleolar ribonucleoprotein particles. The mechanisms by which a toxin such as mercury elicits an autoantibody response that predominantly targets a single intracellular protein autoantigen remain uncertain, but may be prefaced by mercury gaining access to the intracellular environment. Mercury-induced cell death was associated with loss of fibrillarin antigenicity and modification of the molecular properties of fibrillarin as revealed by aberrant migration under nonreducing conditions in SDS-PAGE. Addition of mercury to isolated nuclei also resulted in aberrant migration of fibrillarin, but not other nuclear autoantigens. The sensitivity of the HgCl2-induced modification of fibrillarin to 2-ME, iodoacetamide, and hydrogen peroxide suggested interaction of mercury with the two cysteines in the fibrillarin sequence. This was confirmed by mutation of the cysteines to alanines, which abolished the aberrant migration of fibrillarin in the presence of HgCl2. The modification of the molecular structure of fibrillarin by mercury reduced immunoprecipitation by anti-fibrillarin autoantibodies, pointing to unmodified fibrillarin as the B cell Ag and implicating mercury-modified fibrillarin as the source of T cell antigenicity. These observations demonstrate for the first time that an environmental toxin can alter the physicochemical properties of an autoantigen and may help to explain the antigenic specificity of mercury-induced murine autoimmunity.

In the past few years, a role for apoptotic processes in the development of autoimmune diseases has been suggested. An increasing number of cellular proteins, which are modified during apoptosis, has been described, and many of these proteins have been identified as autoantigens. We have studied the effects of apoptosis on the La protein in more detail and for the first time demonstrate that this autoantigen is rapidly dephosphorylated after the induction of apoptosis. Dephosphorylation of the La protein was observed after induction of apoptosis by several initiators and in various cell types. Furthermore, we demonstrate that at least a subset of the La protein is proteolytically cleaved in vivo, generating a 45 kDa fragment. Dephosphorylation as well as cleavage of La is inhibited by ZnSO4 as well as by several tetrapeptide caspase inhibitors, indicating that these processes require the activation of caspases. Dephosphorylation of La is inhibited by low concentrations of okadaic acid, suggesting that a PP2A-like phosphatase is involved. Generation of the 45 kDa fragment is consistent with proteolytic cleavage at amino acids 371 and/or 374. The possible significance of the apoptotic changes in the La protein for autoantibody production is discussed.

The effects of recombinant mouse DNase on a well established murine model of spontaneous SLE have been evaluated. Daily intraperitoneal injections of DNase were given to female NZB/NZW F1 mice during the period of disease development from 4 to 7 months of age or at the height of disease activity from the age of 7 months for 3 weeks. This treatment was compared with the injections of diluent and with an immunosuppressive dose of dexamethasone. The effects of treatment were evaluated using the immunological parameters of disease activity (antinucleoprotein antibody, immune complexes, serum immunoglobulins, anti-cardiolipin antibodies), protein-uria, serum creatinine and renal histopathology (light microscopy, immunofluorescence and electron microscopy). The dose of dexamethasone used (1 mg/kg per day from the age of 4 months) was sufficient to suppress the development of lupus entirely. Treatment with DNase starting at the age of 4 months postponed the development of the disease by about 1 months and extended the period from the onset of disease to death by about 30%. Mice treated for 3 weeks during the most active phase of the disease at 7 months of age showed more dramatic effects. Proteinuria and serum creatinine were significantly reduced and renal histopathology was strikingly less severe than in the control group. Immune complexes involving DNA-containing antigens are believed to play a crucial role in the pathogenesis of SLE. DNA-nucleoprotein, even in immune complexes, can be destroyed by DNase. This enzyme therefore provides a rational way to interfere with the disease process. The results reported here encourage a trial of recombinant human DNase in human SLE and lupus nephritis.

Podocyte-derived vascular endothelial growth factor (VEGF) is upregulated in diabetes and may contribute to albuminuria. Although believed to act upon the glomerular endothelium, VEGF may have pronounced effects on the podocyte itself. The functionality of this VEGF autocrine loop was investigated in conditionally immortalized mouse podocytes. Exogenous VEGF(164) increased the production of alpha3(IV) collagen, an integral component of the glomerular basement membrane (GBM); this effect was completely prevented by SU5416, a pan-VEGF receptor inhibitor. The VEGF inhibitor also partially prevented the stimulation of alpha3(IV) collagen by transforming growth factor (TGF)-beta1, establishing a novel role for endogenous VEGF. However, VEGF did not influence the production of another novel chain of collagen IV, alpha5(IV) collagen, and SU5416 failed to reverse the known inhibitory effect of TGF-beta1 on alpha5(IV) collagen production. Cultured mouse podocytes possess at least the VEGFR-1 receptor, confirmed by RT-PCR, immunoblotting, and immunocytochemistry. By these techniques, however, VEGFR-2 is absent. VEGF signaling proceeds via autophosphorylation of VEGFR-1 and activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Thus, podocyte-derived VEGF operates in an autocrine loop, likely through VEGFR-1 and PI3K, to stimulate alpha3(IV) collagen production. The TGF-beta1-stimulated endogenous VEGF may have significant implications for podocyte dysfunction in diabetic glomerulopathy, manifesting as GBM thickening and altered macromolecular permeability.

The increase in autoantibodies with age of both experimental animals and humans has been thought to reflect a shift in the antibody repertoire from foreign to self antigens. In mice, before immunization, the age-associated increase in antibodies reactive with a prototypic autoantigen, bromelain-treated autologous erythrocytes (BrMRBC), reflected a 3-fold increase in serum IgM and the number of IgM-secreting spleen cells in old compared with young mice. However, the percentage of the IgM-secreting spleen cell repertoire reactive with BrMRBC in old mice was actually approximately 50% that in young mice. In contrast, after immunization with sheep erythrocytes (SRBC), old mice showed a 5-fold increase in the percentage of IgM-secreting cells reactive with BrMRBC while young mice showed no significant increase. The converse is true for the percentage of IgM-secreting spleen cells in old mice specific for SBRC, which is 10% the number generated by young mice. The increased autoantibody response of old mice is not, however, linked to their poor response to the nominal antigen. Thus, immunization with phosphorylcholine (PC) conjugated keyhole limpet hemocyanin, an antigen that induces a comparable anti-PC response in old and young mice, also induced more autoantibody forming cells in old than young mice. The increased autoantibody response of old mice after immunization can be accounted for by both an increased number of Ig-secreting spleen cells as well as an increased percentage of the expressed repertoire of IgM-secreting spleen cells that react with autoantigens.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND: The podocyte is bathed in an angiotensin II (AngII)-rich ultrafiltrate, but the impact of AngII on podocyte pathobiology is not well known. Because podocytes play a direct role in the glomerular basement membrane (GBM) thickening of diabetes, the alpha3(IV) collagen chain was examined. Podocyte expression of alpha3(IV) collagen may involve the transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF) systems. METHODS: Cultured mouse podocytes were treated with various doses of AngII for selected periods of time, with or without inhibitors of TGF-beta and VEGF signalling, SB-431542 and SU5416, respectively. TGF-beta1 and VEGF were assayed by enzyme-linked immunosorbent assay (ELISA); alpha3(IV) collagen, TGF-beta type II receptor and phospho-Smad2 were assayed by immunoblotting. RESULTS: AngII >or=10(-10) M was found to stimulate the production of alpha3(IV) collagen significantly in as short a time as 3 h. The expression of alpha3(IV) collagen was influenced by the TGF-beta system, but AngII did not increase the podocyte's production of TGF-beta1 ligand; rather, it increased the expression of the TGF-beta type II receptor and activated the TGF-beta signalling system through Smad2. Despite the TGF-beta receptor upregulation, synergy between AngII and TGF-beta1 to boost alpha3(IV) collagen production was not observed. However, blockade of TGF-beta signalling with SB-431542 prevented AngII from stimulating alpha3(IV) collagen production. Podocyte expression of alpha3(IV) collagen was also increased by the autocrine activity of VEGF. Podocytes were stimulated to secrete VEGF by 10(-10) M or higher AngII after 48 h. Blockade of the endogenous VEGF activity by SU5416 prevented AngII-stimulated alpha3(IV) collagen production. CONCLUSIONS: AngII stimulates the podocyte to produce alpha3(IV) collagen protein via mechanisms involving TGF-beta and VEGF signalling. Alterations in alpha3(IV) collagen production may contribute to GBM thickening and perhaps proteinuria in diabetes.

BACKGROUND: Inhaled isocyanate binds with cytokeratin (CK) of the epithelial cells, which could induce immune responses. OBJECTIVE: To elucidate the possible existence of an isocyanate-induced, asthma-associated autoantigen from the bronchial epithelial cells, which may be associated with toluene diisocyanate (TDI)-induced asthma development. METHODS: We cultured bronchial epithelial cells with incubation of TDI-human serum albumin (HSA) conjugate. Gene expression profiles of cultured epithelial cells were analyzed using a microarray technique. CK19 protein expression within the epithelial cells was confirmed by IgG immunoblot using monoclonal antibody to CK19. Serum IgG to CK19 and specific IgG and IgE antibodies to TDI-HSA conjugate were detected by enzyme-linked immunosorbent assay in 68 TDI asthma patients (group 1) and compared with 40 allergic asthma patients (group 2) and 80 unexposed healthy controls (group 3). RESULTS: After TDI exposure, increased expression of CK19 and CK14 genes from the culture bronchial epithelial cells was noted using microarray analysis. IgG immunoblot analysis confirmed increased expression of CK19 after the TDI exposure. The levels of serum IgG to CK19 were significantly higher in the TDI asthma group than in groups 2 and 3 (P=.008). The prevalence of IgG to CK19 was significantly higher in group 1 (38.2%) than group 2 (22.5%) or group 3 (1.3%)(P=.008). Significant associations were noted between IgG to CK19 and specific IgG to TDI-HSA conjugate and transglutaminase (P=.02) but not with specific IgE to TDI-HSA conjugate. CONCLUSION: We suggest that TDI exposure can augment CK19 expression from the bronchial epithelial cell, which may involve immune responses as an autoantigen to induce airway inflammation in TDI-induced asthma.

Islet-cell antigen 512 (IA-2) and phogrin (IA-2beta) are atypical members of the receptor protein tyrosine phosphatase (PTP) family that are characterized by a lack of activity against conventional PTP substrates. The physiological role(s) of these proteins remain poorly defined, although recent studies indicate that IA-2 may be involved in granule trafficking and exocytosis. To further understand their function, we have embarked upon developing low-molecular-mass inhibitors of IA-2 and IA-2beta. Previously, we have shown that a general PTP inhibitor, 2-(oxalylamino)benzoic acid (OBA), can be developed into highly selective and potent inhibitors of PTP1B. However, since wild-type IA-2 and IA-2beta lack conventional PTP activity, a novel strategy was designed whereby catalytically active species were generated by 'back-mutating' key non-consensus catalytic region residues to those of PTP1B. These mutants were then used as tools with which to test the potency and selectivity of OBA and a variety of its derivatives. Catalytically competent IA-2 and IA-2beta species were generated by 'back-mutation' of only three key residues (equivalent to Tyr(46), Asp(181) and Ala(217) using the human PTP1B numbering) to those of PTP1B. Importantly, enzyme kinetic analyses indicated that the overall fold of both mutant and wild-type IA-2 and IA-2beta was similar to that of classic PTPs. In particular, one derivative of OBA, namely 7-(1,1-dioxo-1 H -benzo[ d ]isothiazol-3-yloxymethyl)-2-(oxalylamino)-4,7-dihydro-5 H -thieno[2,3- c ]pyran-3 -carboxylic acid ('Compound 6 ' shown in the main paper), which inhibited IA-2beta((S762Y/Y898P/D933A))(IA-2beta in which Ser(762) has been mutated to tyrosine, Tyr(898) to proline, and Asp(933) to alanine) with a K (i) value of approximately 8 microM, appeared ideal for future lead optimization. Thus molecular modelling of this classical, competitive inhibitor in the catalytic site of wild-type IA-2beta identified two residues (Ser(762) and Asp(933)) that offer the possibility for unique interaction with an appropriately modified 'Compound 6 '. Such a compound has the potential to be a highly selective and potent active-site inhibitor of wild-type IA-2beta.

It is generally believed that autoimmune processes are initiated when tolerance to self-proteins is broken. Primary biliary cirrhosis (PBC) is an autoimmune liver disease of unknown etiology. Autoimmune attack in PBC is predominantly organ-specific, despite the presence of mitochondrial autoantigens, the major targets of autoimmunity in PBC, in all nucleated cells. Although the events that provoke initial activation remain unknown, the hypothesis of molecular mimicry implies that foreign pathogens with homology to self-protein or modified self-protein can break tolerance. Several reports have suggested the association of autoimmune diseases with drugs, chemicals, and other environmental factors. Specifically, many xenobiotics are metabolized in the liver. Liver autoantigens exposed to these chemicals could be modified and become immunogenic. We propose that exposure to the environmental xenobiotics is one of the initiating factors that leads to the loss of tolerance to self-proteins in genetically susceptible hosts, resulting in development of PBC.