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Maintenance of mitochondrial structure and function is critical for preventing podocyte apoptosis and eventual glomerulosclerosis in the kidney; however, the transcription factors that regulate mitochondrial function in podocyte injury remain to be identified. Here, we identified Kruppel-like factor 6 (KLF6), a zinc finger domain transcription factor, as an essential regulator of mitochondrial function in podocyte apoptosis. We observed that podocyte-specific deletion of Klf6 increased the susceptibility of a resistant mouse strain to adriamycin-induced (ADR-induced) focal segmental glomerulosclerosis (FSGS). KLF6 expression was induced early in response to ADR in mice and cultured human podocytes, and prevented mitochondrial dysfunction and activation of intrinsic apoptotic pathways in these podocytes. Promoter analysis and chromatin immunoprecipitation studies revealed that putative KLF6 transcriptional binding sites are present in the promoter of the mitochondrial cytochrome c oxidase assembly gene (SCO2), which is critical for preventing cytochrome c release and activation of the intrinsic apoptotic pathway. Additionally, KLF6 expression was reduced in podocytes from HIV-1 transgenic mice as well as in renal biopsies from patients with HIV-associated nephropathy (HIVAN) and FSGS. Together, these findings indicate that KLF6-dependent regulation of the cytochrome c oxidase assembly gene is critical for maintaining mitochondrial function and preventing podocyte apoptosis., Introduction The glomerulus is the main filtration barrier of the human body. Podocytes, which are terminally differentiated epithelial cells in the glomerulus, play a major role in the maintenance of [...]

We examined whether cardiolipin (CL) synthesis was required to support cholesterol (CH) production from palmitate in Hela cells. Knockdown of human cardiolipin synthase-1 (hCLS1) in Hela cells has been shown to reduce CL synthesis. Therefore Hela cells stably expressing shRNA for hCLS1 and mock control cells were incubated for 16 h with [[sup.14]C(U)]palmitate bound to albumin (1:1 molar ratio) in the absence or presence of serum. Knockdown of hCLS1 in Hela cells resulted in a reduction in [[sup.14]C(U)]palmitate incorporation into CL and CH. This reduction in [[sup.14]C(U)]palmitate incorporation into CH was most pronounced during incubation under serum-free conditions. The reduction in [[sup.14]C(U)]palmitate incorporation into CH was not due to alterations in total uptake of [[sup.14]C(U)]palmitate into cells or altered palmitate metabolism, since [[sup.14]C(U)]palmitate incorporation into phosphatidylcholine, the major [[sup.14]C(U)]palmitate-containing lipid, and its immediate precursor, 1,2-diacyl-sra-glycerol, were unaffected by hCLS1 knockdown. In addition, knockdown of hCLS1 did not affect CH pool size, indicating that CH catabolism was unaltered. Hydroxymethylglutaryl coenzyme A reductase enzyme activity and its mRNA expression were reduced by knockdown of hCLS1 and this was most pronounced in Hela cells cultured under serum-free conditions. These data indicate that CL synthesis is required to support human de novo CH biosynthesis under conditions of increased demand for CH. Key words: cardiolipin synthesis, cholesterol synthesis, human, hydroxymethylglutaryl coenzyme A reductase, palmitate, fatty acid, Hela cells, serum, metabolism, cardiolipin synthase, short hairpin RNA, phosphatidylcholine, phospholipids. Resume: Nous voulons verifier si la synthese de cardiolipine (CL) est essentielle a la production de cholesterol (CH) a partir du palmitate dans les cellules Hela. La suppression de l'activite de la cardiolipine synthase-1 humaine (hCLS1) dans les cellules Hela a pour effet de diminuer la synthese de CL. Des cellules Hela exprimant bien l'ARN court en epingle a cheveux (shRNA) pour l'hCLS1 et des simili-cellules de controle sont incubeees durant 16 h avec du [[sup.14]C(U)]palmitate lie a de l'albumine (rapport molaire 1 : 1) en l'absence ou en presence de serum. La suppression de la hCLS1 dans les cellules Hela suscite une diminution de l incorporation du [[sup.14]C(U)]palmitate dans la CL et le CH. La diminution de l incorporation du [[sup.14]C(U)]palmitate dans le CH est plus importante quand l'incubation se fait en l'absence de serum. Cette diminution n'est pas due a des modifications de l'apport total de [[sup.14]C(U)]palmitate dans les cellules ou a une modification du metabolisme du palmitate, car l'incorporation du [[sup.14]C(U)]palmitate dans la phosphatidylcholine, le lipide contenant le plus de [[sup.14]C(U)]palmitate, et dans le 1,2-diacyl-sra-glycerol, son precurseur, n'est pas affecteee par la suppression de l'activite; de la hCLS1. De plus, la suppression de l'activite; de la hCLS1 ne modifie pas la dimension du pool du CH, ce qui signifie que le catabolisme du CH n'est pas aftere. La suppression de l'activite; de la hCLS1 diminue l'activite; enzymatique de l hydroxyme;thylglutaryl coenzyme A re;ductase et de l expression de l ARNm et cette diminution est plus importante dans les cellules Hela incube;es en l'absence de serum. D'apres ces observations, la synthese de la CL est essentielle a la biosyntheese de novo du CH lorsque la demande de CH augmente. Mots-cles: synthese de cardiolipine, synthese du cholesterol, humain, hydroxymethylglutaryl coenzyme A reductase, palmitate, acide gras, cellules Hela, serum, meetabolisme, cardiolipine synthase, ARN court en eepingle a cheveux, phosphatidylcholine, phospholipide. [Traduit par la Redaction], Introduction Cardiolipin (CL) was the first polyglycerolphospholipid discovered and was first isolated from beef heart by Mary Pangborn (reviewed in Hostetler 1982). CL is both synthesized and localized exclusively within [...]

During chemotaxis, activation of the small guanosine triphosphatase Rac is spatially regulated to organize the extension of membrane protrusions in the direction of migration. In neutrophils, Rac activation is primarily mediated by DOCK2, an atypical guanine nucleotide exchange factor. Upon stimulation, we found that DOCK2 rapidly translocated to the plasma membrane in a phosphatidylinositol 3,4,5-trisphosphate--dependent manner. However, subsequent accumulation of DOCK2 at the leading edge required phospholipase D--mediated synthesis of phosphatidic acid, which stabilized DOCK2 there by means of interaction with a polybasic amino acid duster, resulting in increased local actin polymerization. When this interaction was blocked, neutrophils failed to form leading edges properly and exhibited defects in chemotaxis. Thus, intracellular DOCK2 dynamics are sequentially regulated by distinct phospholipids to localize Rac activation during neutrophil chemotaxis.

Presenilin (PS1/PS2) is a major component of [gamma]-secretase, the activity that mediates proteolysis of [beta]-amyloid precursor protein to generate [beta]-amyloid (A[beta]). Here we demonstrate that PS1, through its loop region, binds to phospholipase D1 (PLD1), thereby recruiting it to the Golgi/trans-Golgi network. Overexpression of wildtype PLD1 reduces A[beta] generation. Conversely, down-regulation of endogenous PLD1 by small hairpin RNA elevates A[beta] production. The A[beta]- lowering effect of PLD1 is independent of its ability to promote vesicular budding of [beta]-amyloid precursor protein. The data indicate that overexpression of PLD1 decreases, and downregulation of PLD1 increases, the catalytic activity, and the association of the subunits, of [gamma]-secretase. [beta]-amyloid precursor protein | [gamma]-secretase complex activity | negative regulator | protein interaction | trans-Golgi network

The signaling enzyme phospholipase D1 (PLD1) facilitates membrane vesicle trafficking. Here, we explore how PLD1 subcellular localization is regulated via Phox homology (PX) and pleckstrin homology (PH) domains and a PI4,5[P.sub.2]-binding site critical for its activation. PLD1 localized to perinuclear endosomes and Golgi in COS-7 cells, but on cellular stimulation, translocated to the plasma membrane in an activity-facilitated manner and then returned to the endosomes. The PI4,5[P.sub.2]-interacting site sufficed to mediate outward translocation and association with the plasma membrane. However, in the absence of PX and PH domains, PLD1 was unable to return efficiently to the endosomes. The PX and PH domains appear to facilitate internalization at different steps. The PH domain drives PLD1 entry into lipid rafts, which we show to be a step critical for internalization. In contrast, the PX domain appears to mediate binding to PI5P, a lipid newly recognized to accumulate in endocytosing vesicles. Finally, we show that the PH domain--dependent translocation step, but not the PX domain, is required for PLD1 to function in regulated exocytosis in PC12 cells. We propose that PLD1 localization and function involves regulated and continual cycling through a succession of subcellular sites, mediated by successive combinations of membrane association interactions.

The ADP ribosylation factor (ARF) GTP binding proteins are believed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. Here we show that ARF6 is specifically associated with dense-core secretory granules in neuroendocrine PC12 cells. Stimulation with a secretagogue triggers the recruitment of secretory granules to the cell periphery and the concomitant activation of ARF6 by the plasma membrane-associated guanine nucleotide exchange factor, ARF nucleotide binding site opener (ARNO). Expression of the constitutively inactive ARF6(T27N) mutant inhibits secretagogue-dependent exocytosis from PC12 cells. Using a mutant of ARF6 specifically impaired for PLD1 stimulation, we find that ARF6 is functionally linked to phospholipase D (PLD)1 in the exocytotic machinery. Finally, we show that ARNO, ARF6, and PLD1 colocalize at sites of exocytosis, and we demonstrate direct interaction between ARF6 and PLD1 in stimulated cells. Together, these results provide the first direct evidence that ARF6 plays a role in calcium-regulated exocytosis in neuroendocrine cells, and suggest that ARF6-stimulated PLD1 activation at the plasma membrane and consequent changes in membrane phospholipid composition are critical for formation of the exocytotic fusion pore.

Phosphatidylinositol 4-phosphate 5-kinase alpha, a downstream effector of the small G protein ARF6 in membrane ruffle formation, is discussed. When ARF6 is activated by stimulation from an agonist, it brings on recruitment of a diverse set of signaling molecules into active cytoskeletal and membrane rearrangement sites. These signaling molecules are interactive. Colocalization of ARF6 is seen in ruffling membranes formed where AlF(sub.4) and EGF stimulation exist. It is blocked by dominant-negative ARF6.