SummaryThis study investigates the molecular mechanisms underlying the induction of and protection from T cell activation-associated hepatic injury. When BALB/c mice were given a single intravenous injection of concanavalin A (Con A) (>10.3 rag/mouse), they developed acute hepatic injury as assessed by a striking increase in plasma transaminase levels within 24 h. Histopathologically, only the liver was injured while moderate infiltration of T cells and polymorphonuclear cells occurred in the portal areas and around the central veins. The induction of hepatic injury was dependent on the existence as well as the activation of T cells, as untreated BALB/c nu/nu mice or BALB/c mice pretreated with a T cell-specific immunosuppressive drug, FK506, failed to develop disease. Significant increases in the levels of various cytokines in the plasma were detected before an increase in plasma transaminase levels. Within 1 h after Con A injection, tumor necrosis factor (TNF) levels peaked, this being followed by production of two other inflammatory cytokines, interleukin 6 (IL-6) and IL-1. Passive immunization with anti-TNF but not with anti-IL-1 or anti-IL-6 antibody, conferred significant levels of protection. Moreover, administration of rlL-6 before Con A injection resulted in an IL-6 dose-dependent protection. A single administration of a given dose of rlL-6 completely inhibited the release of transaminases, whereas the same regimen induced only 40-50% inhibition of TNF production. More than 80% inhibition of TNF production required four consecutive rlL-6 injections. These results indicate that: (a) TNFs are critical cytokines for inducing T cell activation-associated (Con A-induced) hepatitis; (b) the induction of hepatitis is almost completely controlled by rlL-6; and (c) rlL-6 exerts its protective effect through multiple mechanisms including the reduction of TNF production.

Maintenance of cellular zinc homeostasis includes regulating the expression of cell membrane zinc transporters. Knowledge about the mechanisms underlying changes in mammalian zinc transporter mRNA abundance is poor. We demonstrated that when expressed in Chinese hamster ovary cells as N-terminal fusions to green fluorescent protein, two splice variants of ZnT5 adopt different subcellular locations (either in the Golgi apparatus or throughout the cell, including at the plasma membrane) indicating discrete roles in cellular zinc homeostasis. We demonstrated, using a ␤-galactosidase reporter gene, that both splice variants were expressed from a promoter region that was transcriptionally repressed by increased extracellular zinc (150 M compared with 3 M; ϳ40%) and by extracellular zinc depletion, using the chelator N,N,N,N-tetrakis(2-pyridylmethyl) ethylenediamine (ϳ20%). We mapped the zinc-responsive element to the region ؊154 to ؉50, relative to the predicted start of transcription, and showed that a consensus metal response element sequence (؊410 to ؊404) was not responsible for these effects. Changes in ZnT5 mRNA abundance in Caco-2 cells at different zinc concentrations were in parallel to the changes in promoter activity (ϳ40% reduction at 150 M zinc) but in the presence of actinomycin D, to prevent transcription, we observed a marked stabilization (1.7-2-fold accumulation over 24 h) of ZnT5 mRNA. We conclude that effects of zinc on ZnT5 transcription and mRNA stability act in opposition to balance mRNA abundance for cellular zinc homeostasis. To our knowledge, this is the first report that zinc affects the stability of a transcript with a direct role in cellular zinc homeostasis.

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Here, we describe a high-content microscopy-based screen that allowed us to systematically assess and rank proteins involved in Golgi-to-endoplasmic reticulum (ER) retrograde transport in mammalian cells. Using a cell line stably expressing a GFP-tagged Golgi enzyme, we used brefeldin A treatment to stimulate the production of Golgi-to-ER carriers and then quantitatively analysed populations of cells for changes in this trafficking event. Systematic RNA interference (RNAi)-based depletion of 58 Rab GTPase proteins and 12 Rab accessory proteins of the PRAF, YIPF and YIF protein families revealed that nine of these were strong regulators. In addition to demonstrating roles for Rab1a, Rab1b, Rab2a, and Rab6a or Rab6a′ in this transport step, we also identified Rab10 and Rab11a as playing a role and being physically present on a proportion of the Golgito-ER tubular intermediates. Combinatorial depletions of Rab proteins also revealed previously undescribed functional co-operation and physical co-occurrence between several Rab proteins. Our approach therefore provides a novel and robust strategy for a more complete investigation of the molecular components required to regulate Golgi-to-ER transport in mammalian cells.

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