Background-Oxysterol binding protein (OSBP) has previously been implicated as a sterol sensor that regulates sphingomyelin synthesis and the activity of extracellular signal-regulated kinases (ERK). Methods and Results-We determined the effects of adenovirus-mediated hepatic overexpression of OSBP and its homologues ORP1L and ORP3 on mouse serum lipids. Whereas ORP1L and ORP3 had no effect on serum lipids, OSBP induced a marked increase of VLDL triglycerides (TG). Also, the liver tissue TG were elevated in the AdOSBP-injected mice, and their TG secretion rate was increased by 70%. The messenger RNAs for enzymes of fatty acid synthesis and their transcriptional regulator, SREBP-1c, as well as the Insig-1 mRNA, were upregulated two-fold in the OSBPexpressing livers. No change occurred in the messages of liver X receptor target genes ABCA1, ABCG5, and CYP7A1, and the Insig-2a mRNA was reduced. The phosphorylation of ERK was decreased in AdOSBP-infected liver and cultured hepatocytes. Importantly, silencing of OSBP in hepatocytes suppressed the induction of SREBP1-c by insulin and resulted in a reduction of TG synthesis. T he liver plays a central role in triglyceride (TG) and cholesterol homeostasis. Complex regulatory circuits within hepatocytes maintain the body lipid homeostasis under varying environmental conditions. Hepatic lipid syntheses and fluxes are controlled by transcription factors that respond to signals from a variety of lipidous ligands. The synthesis of cholesterol and fatty acids as well as the uptake of cholesterol and hepatic glucose use are controlled by sterol regulatory element binding proteins, SREBP. 1,2 A two-step proteolytic cleavage of SREBP precursors occurs within the Golgi complex and releases a basic helix-loop-helix-leucine zipper (bHLH-LZ) transcription factor denoted nuclear SREBP (nSREBP). The cleavage is controlled by the endoplasmic reticulum (ER) cholesterol content, which is sensed by SREBP cleavage activating protein (SCAP). SCAP, together with Insig proteins, retains SREBP within the ER when cholesterol is abundant but escorts it to the Golgi complex on cholesterol depletion. In addition to cholesterol, exogenously added oxysterol 25-hydroxycholesterol (25OH) is a potent inducer of SREBP activation, suggesting that also endogenous cellular oxysterols regulate the SREBP machinery. 2 Of the 3 SREBPs, SREBP-1c is particularly abundant in the liver where its expression is regulated by insulin and glucagon, and it plays a major role in controlling hepatic lipogenesis and glucose use. 1,3 SREBP-2, also expressed at relatively high levels in the liver, is responsible for control of cholesterol metabolism. The third family member, SREBP-1a, functions in both cholesterol and TG metabolism. In cultured cells, SREBP-1a is expressed at much higher levels than SREBP1c. 4 The cleavage of SREBP-1a and -2 precursors is regulated by cholesterol status, whereas the expression and maturation of SREBP-1c are primarily regulated by nutritional factors. SREBP-1c expression in liver, white adipos...
Caveolin-1 binds cholesterol and caveola formation involves caveolin-1 oligomerization and cholesterol association. The role of cholesterol in caveolae has so far been addressed by methods that compromise membrane integrity and abolish caveolar invaginations. To study the importance of sterol specificity for the structure and function of caveolae, we replaced cholesterol in mammalian cells with its immediate precursor desmosterol by inhibiting 24-dehydrocholesterol reductase. Desmosterol could substitute for cholesterol in maintaining cell growth, membrane integrity, and preserving caveolar invaginations. However, in desmosterol cells the affinity of caveolin-1 for sterol and the stability of caveolin oligomers were decreased. Moreover, caveolar invaginations became more heterogeneous in dimensions and in the number of caveolin-1 molecules per caveola. Despite the altered caveolar structure, caveolar ligand uptake was only moderately inhibited. We found that in desmosterol cells, Src kinase phosphorylated Cav1 at Tyr 14 more avidly than in cholesterol cells. Taken the role of Cav1 Tyr 14 phosphorylation in caveolar endocytosis, this may help to preserve caveolar uptake in desmosterol cells. We conclude that a sterol C24 double bond interferes with caveolin-sterol interaction and perturbs caveolar morphology but facilitates Cav1 Src phosphorylation and allows caveolar endocytosis. More generally, substitution of cholesterol by a structurally closely related sterol provides a method to selectively modify membrane protein-sterol affinity, structure and function of cholesterol-dependent domains without compromising membrane integrity.Caveolae are specialized plasma membrane (PM) 3 microdomains enriched in cholesterol and sphingolipids (1). These flask-shape invaginations contain caveolin-1 (Cav1) as their main structural protein component (2). Caveolae are relatively immobile structures but their internalization can be stimulated by a variety of cargo, including sphingolipids (3), integrins (4), and select viruses (5). Cav1 binds cholesterol (6) and cholesterol promotes the formation of Cav1 oligomers that are required for caveola formation (7,8). However, no detailed structural information on Cav1 membrane association is available (1). The role of cholesterol in caveolae has so far been addressed using treatments that sequester or remove cholesterol, thus compromising membrane integrity, causing the loss of invaginated caveolae and affecting many cellular processes (9, 10). Thus, functions assigned to caveolae/caveolin based on cholesterol depletion must be assessed critically (9).In this work, we studied the importance of the sterol structure for caveolae by a method circumventing the side effects of cholesterol removal or sequestration. Cells were treated with a specific inhibitor of 24-dehydrocholesterol reductase (DHCR24), the enzyme converting desmosterol to cholesterol. This resulted in the accumulation of desmosterol, which differs from cholesterol by an additional double bond between carbon atoms 24 and 25 ...
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