Endogenous versus exogenous fatty acid availability affects lysosomal acidity and MHC class II expression.

Although the immune system, inflammation, and cellular metabolism are linked to diseases associated with dyslipidemias, the mechanism(s) remain unclear. To determine whether there is a mechanistic link between lipid availability and inflammation/immune activation, we evaluated macrophage cell lines incubated under conditions of altered exogenous and endogenous lipid availability. Limiting exogenous lipids results in decreased lysosomal acidity and decreased lysosomal enzymatic activity. Both lysosomal parameters are restored with the addition of oleoyl-CoA, suggesting that fatty acids play a role in the regulation of lysosomal function. Cell surface expression of major histocompatibility complex (MHC)-encoded molecules is also decreased in the absence of exogenous lipids. Additionally, we observe decreased gamma-interferon stimulation of cell surface MHC class II. Using cerulenin to limit the endogenous synthesis of fatty acids results in decreased cell surface expression of MHC class II but does not appear to alter lysosomal acidity, suggesting that lysosomal acidity is dependent on exogenous, but not endogenous, fatty acid availability. Testing these conclusions in an in vivo mouse model, we observed statistically significant, diet-dependent differences in lysosomal acidity and MHC class II cell surface expression. Collectively, these data demonstrate a mechanistic link between lipid availability and early events in the immune response.

Paranemin and the organization of desmin filament networks.

De novo expression of vimentin, GFAP or peripherin leads to the assembly of an extended intermediate filament network in intermediate filament-free SW13/cl.2 cells. Desmin, in contrast, does not form extended filament networks in either SW13/cl.2 or intermediate filament-free mouse fibroblasts. Rather, desmin formed short thickened filamentous structures and prominent spot-like cytoplasmic aggregates that were composed of densely packed 9-11 nm diameter filaments. Analysis of stably transfected cell lines indicates that the inability of desmin to form extended networks is not due to a difference in the level of transgene expression. Nestin, paranemin and synemin are large intermediate filament proteins that coassemble with desmin in muscle cells. Although each of these large intermediate filament proteins colocalized with desmin when coexpressed in SW-13 cells, expression of paranemin, but not synemin or nestin, led to the formation of an extended desmin network. A similar rescue of desmin network organization was observed when desmin was coexpressed with vimentin, which coassembles with desmin, or with keratins, which formed a distinct filament network. These studies demonstrate that desmin filaments differ in their organizational properties from the other vimentin-like intermediate filament proteins and appear to depend upon coassembly with paranemin, at least when they are expressed in non-muscle cells, in order to form an extended filament network.

Vimentin-dependent utilization of LDL-cholesterol in human adrenal tumor cells is not associated with the level of expression of apoE, sterol carrier protein-2, or caveolin.

SW-13 adrenal tumor cells that lack detectable intermediate filaments (IF-free) exhibit an impaired capacity to esterify lipoprotein-derived cholesterol compared with cells that contain vimentin filaments. IF-free cells were found to synthesize and secrete significant amounts of apoE, while apoE secretion was nearly undetectable in cell lines that spontaneously express vimentin. However, stable transfectants that express a mouse vimentin cDNA exhibited elevated levels of cholesterol esterification and apoE secretion compared with untransfected IF-free cells, indicating that apoE secretion is not directly related to the capacity of these cells to esterify cholesterol. Some of the cell lines that differed in the level of apoE synthesis and secretion had similar levels of apoE mRNA, suggesting that the differences in expression involve a post-transcriptional mechanism. Treatment of these cells with forskolin and IBMX, 8br-cAMP, or TPA had no effect on apoE secretion. The level of sterol carrier protein-2 (SCP(2)) synthesis and the distribution of SCP(2) between membrane and soluble cellular fractions was not observably different in cells that contained or lacked vimentin. SW-13 cell lines contained little or no detectable caveolin-1 or caveolin-2. These studies demonstrate that the difference in the capacity of these adrenal tumor cells that contain or lack vimentin filaments to esterify low density lipoprotein-cholesterol is not obviously associated with the level of expression or distribution of apoE, SCP(2), or caveolins.

Vimentin and lipid metabolism.

Vimentin IFs form close associations with the lipid droplets that are characteristic of adipose and steroidogenic cells. There is good evidence that changes in vimentin expression or organization can alter the metabolism of specific lipid components in cultured preadipose and adrenal cell lines. However, the effect of vimentin on triglyceride stability that is observed in preadipose cells is not obviously reflected in adrenal cells or fibroblasts. Conversely, an effect of vimentin on the metabolism of lipoprotein-derived cholesterol observed in adrenal cells is not apparent in preadipose cells. While the complexity of the phenotypes observed in these cells might be associated with cell-type-specific differences in the metabolism of triglycerides or lipoprotein-derived cholesterol, these studies have not yet revealed a general role of vimentin in lipid metabolism that would indicate a common mechanism in all cell types. A key issue that needs to be addressed is whether the effect of vimentin IFs on the stability of triglycerides or the trafficking of GSLs and lysosomal cholesterol is due to a direct participation of vimentin IFs in some aspect of these processes, or perhaps reflects an indirect response of the lipid metabolism of these cells to an effect of vimentin on some other cellular process.

Tetracycline regulated expression of vimentin in fibroblasts derived from vimentin null mice.

Fibroblast cell lines were derived from vim-/- mice that express a mouse vimentin transgene in a tetracycline regulatable manner. Vimentin null mouse primary embryo fibroblasts were transformed with SV-40 early genes and vim- cell lines were isolated. A vim- cell line was then serially transfected with an expression plasmid encoding the tetracycline regulatable transactivator (tTA) and a mouse vimentin cDNA expression plasmid under the regulation of Escherichia coli tet operator and minimal CMV promoter sequences. Two stable cell lines were obtained that contained little or no vimentin in the presence of low concentrations of tetracycline but rapidly expressed abundant vimentin filaments after removal of tetracycline. The vimentin content of one cell line was similar to that of control vim+/+ fibroblasts. The level of transgene expression could be regulated by the concentration of tetracycline in a dose dependent fashion. Induction of vimentin expression in these cells did not observably affect cell growth, the distribution of microfilaments or microtubules, or the shape of the nucleus. Enucleation studies indicated that while disassembly of microfilaments significantly increased the sensitivity of the cells to enucleation, the presence or absence of vimentin had no detectable effect on the degree of enucleation with increasing sedimentation force. Monolayer wounding experiments demonstrated that vimentin expression did not alter the mobility of polarized cells at the edge of the wound. Experiments to more directly test the effect of vimentin expression on the capacity of these fibroblasts to survive mechanical trauma indicated that vimentin expression had no obvious effect on the survival of suspension cells subjected to nitrogen cavitation or the fraction of cells that survived the mechanical scraping of monolayer culture. These studies indicate that vimentin expression in a single population of cells does not have an obvious effect on cytoplasmic organization and provides a useful system to study the effects of IFs on the capacity of individual cells to resist mechanical injury.