Hormone signaling and fatty liver in females: analysis of estrogen receptor α mutant mice.

BACKGROUND: Treatment with estrogen in early menopausal women protects against development of hepatic steatosis and nonalcoholic fatty liver disease but estrogen has undesirable side effects, which negate its beneficial effects in premenopausal and postmenopausal women. Targeted therapies require better understanding of the target sites and mechanisms by which estrogen signaling exerts its protective effects in women. Estrogen receptor α (ERα) is thought to be the primary mediator for estrogen signaling to protect against hepatic steatosis. ERα has several mechanisms for signal transduction: (1) inducing gene transcription by direct binding to specific DNA sequences, (2) inducing tethered transcription with other DNA-binding factors, and (3) stimulating nongenomic action through membrane-associated ERα. However, it is still unclear which mechanisms mediate ERα-dependent protection against hepatic steatosis. METHODS: To understand the mechanisms of estrogen signaling for protection against hepatic steatosis in females, we analyzed the global ERα knockout mouse (αERKO), ERα DNA-binding domain mutant mouse (KIKO) and liver-specific ERα knockout mouse (LERKO) fed high-fat diets (HFD). The KIKO mouse disrupts the direct DNA-binding transcription activity but retains tethered transcription regulation and nongenomic action. Hepatic steatosis was evaluated by scoring the macrovesicular and microvesicular steatosis as well as serum alanine aminotransferase (ALT) levels. We analyzed serum testosterone to assess its correlation with hepatic steatosis. RESULTS: Liver fat accumulation was far greater in HFD-fed αERKO and KIKO females than in HFD-fed wild-type (WT) controls. Conversely, HFD-fed LERKO females did not accumulate excess liver fat. HFD-fed αERKO and KIKO females showed higher microvesicular steatosis and ALT levels than WT controls that correlated with increased serum testosterone levels. CONCLUSIONS: ERα-mediated direct transcription in non-hepatic tissues is essential for estrogen-mediated protection against hepatic steatosis in HFD-fed females. The balance between non-hepatic estrogen signaling and hepatic or non-hepatic testosterone action may control hepatic steatosis.

Influence of microbial stimulation on hypergammaglobulinemia and autoantibody production in pristane-induced lupus.

Pristane induces a lupus-like syndrome characterized by autoantibody production and glomerulonephritis in nonautoimmune strains of mice. Although it has been suggested that this syndrome results from nonspecific immune activation, there is little evidence so far that B cells are activated nonspecifically by pristane or that this promotes autoimmunity. In this study, we examined whether polyclonal hypergammaglobulinemia occurs in pristane-induced lupus, and its relationship to the production of anti-DNA, nRNP/Sm, and Su autoantibodies. In conventionally housed mice, there was a marked increase in total IgM and IgG3 2 weeks after i.p. pristane injection, followed by increased IgG1, IgG2a, and IgG2b levels. IgM levels were higher in pristane-treated specific pathogen-free (SPF) mice than in conventionally housed mice, whereas IgG and IgA levels were reduced. Pristane induced anti-nRNP/Sm and Su autoantibodies in SPF mice, but their onset was delayed and levels were lower than those in conventionally housed mice. There was no consistent relationship between total IgG1, 2a, and 2b hypergammaglobulinemia and production of anti-nRNP/Sm and Su autoantibodies. Moreover, the total Ig levels were similar in the anti-nRNP/Sm-positive and -negative groups. In contrast, production of IgM anti-ssDNA antibodies paralleled IgM hypergammaglobulinemia in some, but not all, mice. These studies indicate that pristane-induced lupus is associated with marked hypergammaglobulinemia, the magnitude of which is influenced by the microbial environment. However, anti-nRNP/Sm and Su autoantibody production is at least partly independent of polyclonal B cell activation. The data strongly suggest that pristane-induced lupus is not exclusively the consequence of nonspecific immune stimulation. They also point to the importance of microbial stimulation in the development of hypergammaglobulinemia in this inducible lupus model.

Human anti-nuclear ribonucleoprotein antigen autoimmune sera contain a novel subset of autoantibodies that stabilizes the molecular interaction of U1RNP-C protein with the Sm core proteins.

Anti-Sm Abs recognize Sm core proteins B'/B, D, E, F, and G, shared by U1, U2, U4-6, and U5 small nuclear ribonucleoproteins (snRNPs), while anti-nuclear ribonucleoprotein Ag (nRNP) Abs recognize the U1 RNP-specific 70K, A, and C proteins. However, although the autoimmune response to U1 snRNPs involves all components of the particle, not all are recognized equally. For example, all human anti-nRNP sera contain Abs against native U1-C, in contrast to their absence in MRL/lpr mice. In this study, autoantibody recognition of native U1 snRNPs was investigated by dissociating the particle into four components (U1-70K, U1-A, U1-C, and the Sm core particle) using 1 M MgCl2 or ribonuclease treatment. As expected, human anti-Sm and MRL/lpr sera immunoprecipitated only the Sm core proteins, and human anti-nRNP/Sm sera immunoprecipitated the Sm core proteins plus U1-C under both conditions. However, although human anti-nRNP sera immunoprecipitated U1-C when U1 snRNPs were dissociated before Ab binding, they unexpectedly immunoprecipitated the Sm core proteins when Abs were bound before dissociation. This apparent paradox was explained by the stabilizing effects of anti-nRNP sera on interactions of U1-C with the Sm core particle. All human anti-nRNP sera contained high levels of autoantibodies that prevent dissociation of U1-C from the U1 snRNP. These Abs were absent in MRL/lpr mice. Human autoimmune sera may prevent dissociation by recognizing the quaternary structure of the U1-C-Sm core protein complex or by altering its conformation. Stabilization of U1 snRNPs by autoantibodies could influence Ag processing and presentation, possibly with important effects on the development of autoimmunity to U1 snRNPs.

Autoantibodies to ribosomal P antigens with immune complex glomerulonephritis in SJL mice treated with pristane.

BALB/c ByJ mice develop a lupus-like syndrome characterized by anti-nRNP/Sm and Su autoantibodies and immune complex glomerulonephritis after a single i.p. pristane injection. In contrast, mercuric chloride induces anti-fibrillarin Abs only in SJL and other H-2s mice, and not in BALB/c (H-2d) mice. In the present study, the specificities of autoantibodies induced by pristane and HgCl2 were compared in SJL and BALB/c mice to examine whether these strains are "programmed" to make different sets of autoantibodies in response to nonspecific immune stimulation. Unexpectedly, the predominant autoantibodies induced by pristane in SJL mice were neither those characteristic of HgCl2-treated SJL mice nor those associated with pristane-induced disease in BALB/c mice but, rather, anti-ribosomal P, another lupus-related specificity. The autoantibodies were strongly reactive with the C-terminal 22 amino acids of the ribosomal P2 protein, indicating that they exhibited similar fine specificities to anti-P Abs in human SLE and MRL/Ipr mice. Like BALB/c mice, pristane-treated SJL mice developed severe glomerulonephritis characterized by proteinuria, mesangial proliferation, and glomerular immune complex deposits. This is the first evidence that the induction of a lupus-like syndrome by pristane is not restricted to BALB/c mice. The predominance of anti-P Abs in SJL mice contrasts sharply with the predominance of anti-nRNP/Sm and Su, in pristane-treated BALB/c mice, even though the renal lesions were similar in both strains. The data suggest that H-2s does not program mice to produce anti-fibrillarin Abs in response to nonspecific immune stimulation, arguing that autoantibody induction by pristane involves Ag-specific mechanisms.

Late development of anti-La/SS-B antibodies in a patient with Sjogren's syndrome and high titer anti-Ro/SS-A antibodies.

The frequent coexistence of anti-Ro and anti-La autoantibodies is well described, however, there is little evidence of sequential development of these two autoantibodies. We report a case of typical Sjogren's syndrome with high titer anti-Ro antibodies, who subsequently developed anti-La antibodies later in the course. This case suggests that the anti-La antibodies may actually follow the anti-Ro antibodies in some cases as hypothesized in the concept of linked set of autoantibodies, analogous to development of anti-Sm in certain anti-nRNP antibody positive SLE patients and animal models.

Distinctive immune response patterns of human and murine autoimmune sera to U1 small nuclear ribonucleoprotein C protein.

The Ul small nuclear ribonucleoprotein (snRNP), a complex of nine proteins with Ul RNA, is a frequent target of autoantibodies in human and murine systemic lupus erythematosus (SLE). Anti-Sm antibodies recognizing the B'/B, D, E, F, and G proteins of Ul snRNPs are highly specific for SLE, and are nearly always accompanied by anti-nRNP antibodies recognizing the Ul snRNP-specific 70K, A, and/or C proteins. Previous studies suggest that human anti-nRNP antibodies recognize primarily the U1-70K and Ul-A proteins, whereas recognition of Ul-C is less frequent. We report here that autoantibodies to U1-C are more common in human autoimmune sera than believed previously. Using a novel immunoprecipitation technique to detect autoantibodies to native Ul-C, 75/78 human sera with anti-nRNP/ Sm antibodies were anti-Ul-C (+). In striking contrast, only 1/65 anti-nRNP/Sm (+) MRL mouse sera of various Igh allotypes was positive. Two of ten anti-nRNP/Sm (+) sera from BALB/c mice with a lupus-like syndrome induced by pristane recognized Ul-C. Thus, lupus in MRL mice was characterized by a markedly lower frequency of anti-U1-C antibodies than seen in human SLE or pristane-induced lupus. The results may indicate different pathways of intermolecular-intrastructural diversification of autoantibody responses to the components of Ul snRNPs in human and murine lupus, possibly mediated by alterations in antigen processing induced by the autoantibodies themselves.

Initiation of autoimmunity to the p53 tumor suppressor protein by complexes of p53 and SV40 large T antigen.

Antinuclear antibodies (ANAs) reactive with a limited spectrum of nuclear antigens are characteristic of systemic lupus erythematosus (SLE) and other collagen vascular diseases, and are also associated with certain viral infections. The factors that initiate ANA production and determine ANA specificity are not well understood. In this study, high titer ANAs specific for the p53 tumor suppressor protein were induced in mice immunized with purified complexes of murine p53 and the Simian virus 40 large T antigen (SVT), but not in mice immunized with either protein separately. The autoantibodies to p53 in these mice were primarily of the IgG1 isotype, were not cross-reactive with SVT, and were produced at titers up to 1:25,000, without the appearance of other autoantibodies. The high levels of autoantibodies to p53 in mice immunized with p53/SVT complexes were transient, but low levels of the autoantibodies persisted. The latter may have been maintained by self antigen, since the anti-p53, but not the SVT, response in these mice could be boosted by immunizing with murine p53. Thus, once autoimmunity to p53 was established by immunizing with p53/SVT complexes, it could be maintained without a requirement for SVT. These data may be explained in at least two ways. First, altered antigen processing resulting from the formation of p53/SVT complexes might activate autoreactive T helper cells specific for cryptic epitopes of murine p53, driving anti-p53 autoantibody production. Alternatively, SVT-responsive T cells may provide intermolecular-intrastructural help to B cells specific for murine p53. In a second stage, these activated B cells might themselves process self p53, generating p53-responsive autoreactive T cells. The induction of autoantibodies during the course of an immune response directed against this naturally occurring complex of self and nonself antigens may be relevant to the generation of specific autoantibodies in viral infections, and may also have implications for understanding the pathogenesis of ANAs in SLE. In particular, our results imply that autoimmunity can be initiated by a "hit and run" mechanism in which the binding of a viral antigen to a self protein triggers an immune response that subsequently can be perpetuated by self antigen.

Phospholipase A2 engineering. X-ray structural and functional evidence for the interaction of lysine-56 with substrates.

Site-directed mutagenesis studies of bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli) showed that replacement of surface residue Lys-56 by a neutral or hydrophobic amino acid residue resulted in an unexpected and significant change in the function of the enzyme. The kcat for phosphatidylcholine micelles increases 3-4-fold for K56M, K56I, and K56F and ca. 2-fold for K56N and K56T but does not change for K56R. These results suggest that the side chain of residue 56 has significant influence on the activity of PLA2. In order to probe the structural basis for the enhanced activity, the crystal structures of wild-type and K56M PLA2 were determined by X-ray crystallography to a resolution of 1.8 A. The results suggest that the mutation has not only perturbed the conformation of the side chain of Met-56 locally but also caused conformational changes in the neighboring loop (residues 60-70), resulting in the formation of a hydrophobic pocket by residues Met-56, Tyr-52, and Tyr-69. Docking of a phosphatidylcholine inhibitor analogue into the active site of K56M, according to the structure of the complex of cobra venom PLA2-phosphatidylethanolamine inhibitor analogue [White, S.P., Scott, D. L., Otwinowski, Z., Gleb, M. H., & Sigler, P. (1990) Science 250, 1560-1563], showed that the choline moiety [N(CH3)3]+ is readily accommodated into the newly formed hydrophobic pocket with a high degree of surface complementarity. This suggests a possible interaction between residue 56 and the head group of the phospholipid, explaining the enhanced activities observed when the positively charged Lys-56 is substituted by apolar residues, viz., K56M, K56I, and K56F. Further support for this interpretation comes from the 5-fold enhancement in kcat for the mutant K56E with a negatively charged side chain, where there would be an attractive electrostatic interaction between the side chain of Glu-56 and the positively charged choline moiety. Our results also refute a recent report [Tomasselli, A. G., Hui, J., Fisher, J., Zürcher-Neely, H., Reardon, I.M., Oriaku, E., Kézdy, F.J., & Heinrikson, R.L. (1989) J. Biol. Chem. 264, 10041-10047] that substrate-level acylation of Lys-56 is an obligatory step in the catalysis by PLA2.