p53 and p73 genes are both activated in response to DNA damage to induce either cell cycle arrest or apoptosis, depending on the strength and the quality of the damaging stimulus. p53/p73 transcriptional activity must be tightly regulated to ensure that the appropriate biological response is achieved and to allow the cell to reenter into the cell cycle after the damage has been repaired. In addition to multiple transcriptionally active (TA) isoforms, dominant negative (DN) variants, that lack the amino-terminal transactivation domain and function as trans-repressors of p53, p63 and p73, are expressed from a second internal promoter (P2-p73Pr).Here we show that, in response to a non apoptotic DNA damage induced by low doses of doxorubicin, p53 binds in vivo, as detected by a p53-speci®c chromatin immunoprecipitation assay, and activates the P2-p73 promoter. DN-p73a protein accumulates under the same conditions and exogenously expressed DN-p73a is able to counteract the p53-induced activation of the P2-p73Pr. These results suggest that DN-p73 may contribute to the autoregulatory loops responsible for the termination of p53/p73 responses in cells that do not undergo apoptosis. Accordingly, the activation of the P2-p73Pr is markedly enhanced in both p737/7 murine ®broblasts and in human cells in which p73 transcripts are selectively knocked-out by p73-speci®c small interfering RNAs.

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The Wnt pathway, which controls crucial steps of the development and differentiation programs, has been proposed to influence lipid storage and homeostasis. In this paper, using an unbiased strategy based on high-content genome-wide RNAi screens that monitored lipid distribution and amounts, we find that Wnt3a regulates cellular cholesterol. We show that Wnt3a stimulates the production of lipid droplets and that this stimulation strictly depends on endocytosed, LDL-derived cholesterol and on functional early and late endosomes. We also show that Wnt signaling itself controls cholesterol endocytosis and flux along the endosomal pathway, which in turn modulates cellular lipid homeostasis. These results underscore the importance of endosome functions for LD formation and reveal a previously unknown regulatory mechanism of the cellular programs controlling lipid storage and endosome transport under the control of Wnt signaling.

The NAD؉ -dependent histone deacetylase hSirT1 regulates cell survival and stress responses by inhibiting p53-, NF-B-, and E2F1-dependent transcription. Here we show that the hSirT1/PCAF interaction controls the E2F1/p73 apoptotic pathway. hSirT1 represses E2F1-dependent P1p73 promoter activity in untreated cells and inhibits its activation in response to DNA damage. hSirT1, PCAF, and E2F1 are corecruited in vivo on theP1p73 promoter. hSirT1 deacetylates PCAF in vitro and modulates PCAF acetylation in vivo. In cells exposed to apoptotic DNA damage, nuclear NAD ؉ levels decrease and inactivate hSirT1 without altering the hSirT1 interaction with PCAF and hSirT1 binding to the P1p73 promoter. hSirT1, the mammalian homologue of Sir2 (silent information regulator 2), is a NAD-dependent class III deacetylase (15, 33) that regulates cell survival, stress responses, and metabolism by inhibiting p53 (3, 18, 19, 28)-, E2F1 (1, 30)-, NF-B (31)-, and Forkhead (2)-dependent transcription. The role of hSirT1 in the regulation of mammalian cell survival in response to DNA damage is supported by several observations. hSirT1-deficient mice display increased levels of radiation-induced apoptosis and p53 hyperacetylation (4). hSirT1-dependent deacetylation attenuates the ability of p53 to trans-activate the cell cycle (p21) and apoptotic (bax) target genes (19,28). The constitutive expression of the tumor suppressor hypermethylated in cancer 1 (Hic1) represses hSirT1 transcription, thereby allowing the accumulation of acetylated p53 species and the enhancement of p53-mediated growth arrest and apoptosis in response to DNA damage (3). hSirT1 deacetylase function also modulates p53-independent pathways involved in the DNA damage response. The targeted disruption of the SirT1 gene in p53-deficient cells strongly sensitizes cells to radiation-, cisplatin-, and etoposide-induced cell death (20). hSirT1 deacetylates the DNA damage repair protein Ku70, and deacetylated Ku70 prevents Bax translocation to mitochondria to initiate apoptosis (5). hSirT1 maintains the Nijmegen breakage syndrome protein (NBS1) hypoacetylated and susceptible to be phosphorylated by ATM in response to DNA damage (32). Finally, etoposide treatment results in the E2F1-dependent induction of hSirT1 expression, and the abrogation of hSirT1 expression sensitizes cells to E2F1-dependent apoptosis (30).The E2F family of transcription factors has critical roles in the control of cell proliferation and apoptosis (7). E2F transcriptional activity is tightly regulated during the cell cycle through the association with pRb or the related pocket proteins p107 and p130, leading, in quiescent cells, to the recruitment of transcriptional corepressors, including histone deacetylases (HDACs), methyltransferases, and polycomb group proteins, onto the promoters of proliferation-associated E2F target genes (7). As cells progress into the cell cycle, cyclin-dependent kinases phosphorylate pRb, releasing free E2F and allowing it to interact with transcriptional coactivators, and direc...

Optimal activation of Rel / NF‐κB transcription factors in T lymphocytes requires a CD28‐delivered co‐stimulatory signal in addition to TCR engagement. Although, Rel / NF‐κB transcription factors are critical regulators of many T cell functions, the mechanisms and molecules, which link the surface receptors to their activation, are poorly characterized. Using Jurkat T cells stimulated with superantigen presented on B7‐positive APC, we showed that CD28‐ and TCR‐stimulated NF‐κB‐dependent transcription is associated to the activation of IκB kinase β (IKKβ) and, to a lesser extent, of IκB kinase α (IKKα). A dominant negative mutant of the MAP3 kinase MEKK1, a kinase known to regulate the JNK pathway and to activate NF‐κB‐dependent transcription in many cell types, strongly inhibits CD28‐ and TCR‐induced IKK activity, whereas the dominant negative mutants of the NF‐κB‐inducing kinase (NIK) did not exert any significant effects. In addition, TCR / CD28 stimulation results in the recruitment and autophosphorylation of endogenous MEKK1, whereas endogenous NIK was not detectably activated. Our data identify MEKK1 as a critical step in coupling signals initiated by TCR and CD28 to the downstream pathways which lead to both AP‐1 and NF‐κB activation in T lymphocytes.

Most cells acquire cholesterol by endocytosis of circulating low‐density lipoproteins (LDLs). After cholesteryl ester de‐esterification in endosomes, free cholesterol is redistributed to intracellular membranes via unclear mechanisms. Our previous work suggested that the unconventional phospholipid lysobisphosphatidic acid (LBPA) may play a role in modulating the cholesterol flux through endosomes. In this study, we used the Prestwick library of FDA‐approved compounds in a high‐content, image‐based screen of the endosomal lipids, lysobisphosphatidic acid and LDL‐derived cholesterol. We report that thioperamide maleate, an inverse agonist of the histamine H3 receptor HRH3, increases highly selectively the levels of lysobisphosphatidic acid, without affecting any endosomal protein or function that we tested. Our data also show that thioperamide significantly reduces the endosome cholesterol overload in fibroblasts from patients with the cholesterol storage disorder Niemann–Pick type C (NPC), as well as in liver of Npc1−/− mice. We conclude that LBPA controls endosomal cholesterol mobilization and export to cellular destinations, perhaps by fluidifying or buffering cholesterol in endosomal membranes, and that thioperamide has repurposing potential for the treatment of NPC.

How trafficking pathways and organelle abundance adapt in response to metabolic and physiological changes is still mysterious, although a few transcriptional regulators of organellar biogenesis have been identified in recent years. We previously found that the Wnt signaling directly controls lipid droplet formation, linking the cell storage capacity to the established functions of Wnt in development and differentiation. In the present paper, we report that Wnt-induced lipid droplet biogenesis does not depend on the canonical TCF/LEF transcription factors. Instead, we find that TFAP2 family members mediate the pro-lipid droplet signal induced by Wnt3a, leading to the notion that the TFAP2 transcription factor may function as a ‘master’ regulator of lipid droplet biogenesis.

In NPC, the traffic of LDL-derived cholesterol from lysosomes to the ER is defective, which impairs not only cholesterol esterification, but also the feedback transcriptional regulation of cholesterol metabolism via the SREBP pathway (2-4). The disease is caused by loss-of-function mutations in either one of the two late endosome/lysosome proteins NPC1 (5), a multispanning protein of the limiting membrane, and NPC2 (6), a globular protein present in the lumen. Solid evidence shows that both proteins bind cholesterol (7, 8), and structural and mutagenesis data suggest that cholesterol is transferred from NPC2 to NPC1, thereby facilitating export from the organelle (9-12). However, the precise mechanism of export remains unclear. Similarly, it is not clear how cholesterol is transferred from late endosome/ lysosomes to reach the regulatory pool in the ER. Several direct routes have been proposed (13), but recent findings indicate that the plasma membrane may be the primary destination of LDL-derived cholesterol before reaching the ER (14). This view is consistent with the earlier notion that cholesterol rapidly equilibrates between the plasma membrane and the regulatory ER pool (15). There is no approved treatment against NPC, with the exception of miglustat, which delays but does not arrest the progression of the disease (16). Over the last decade, cyclodextrins are emerging as a possible therapeutic strategy. These cyclic oligosaccharides clear cholesterol storage and reestablish the feedback regulation in cultured cells and NPC mice (17-21). They also improve neurological symptoms and survival in NPC animal models (22, 23) and were Abstract In specialized cell types, lysosome-related organelles support regulated secretory pathways, whereas in nonspecialized cells, lysosomes can undergo fusion with the plasma membrane in response to a transient rise in cytosolic calcium. Recent evidence also indicates that lysosome secretion can be controlled transcriptionally and promote clearance in lysosome storage diseases. In addition, evidence is also accumulating that low concentrations of cyclodextrins reduce the cholesterol-storage phenotype in cells and animals with the cholesterol storage disease Niemann-Pick type C, via an unknown mechanism. Here, we report that cyclodextrin triggers the secretion of the endo/lysosomal content in nonspecialized cells and that this mechanism is responsible for the decreased cholesterol overload in Niemann-Pick type C cells. We also find that the secretion of the endo/lysosome content occurs via a mechanism dependent on the endosomal calcium channel mucolipin-1, as well as FYCO1, the AP1 adaptor, and its partner Gadkin. We conclude that endo-lysosomes in nonspecialized cells can acquire secretory functions elicited by cyclodextrin and that this pathway is responsible for the decrease in cholesterol storage in Niemann-Pick C cells.-Vacca, F.