RESUMO
Nonvesicular transport of cholesterol plays an essential role in the distribution and regulation of cholesterol within cells, but it has been difficult to identify the key intracellular cholesterol transporters. The steroidogenic acute regulatory-related lipid-transfer (START) family of proteins is involved in several pathways of nonvesicular trafficking of sterols. Among them, STARD4 has been shown to increase intracellular cholesteryl ester formation and is controlled at the transcriptional level by sterol levels in cells. We found that STARD4 is very efficient in transporting sterol between membranes in vitro. Cholesterol levels are increased in STARD4-silenced cells, while sterol transport to the endocytic recycling compartment (ERC) and to the endoplasmic reticulum (ER) are enhanced upon STARD4 overexpression. STARD4 silencing attenuates cholesterol-mediated regulation of SREBP-2 activation, while its overexpression amplifies sterol sensing by SCAP/SREBP-2. To analyze STARD4's mode of action, we compared sterol transport mediated by STARD4 with that of a simple sterol carrier, methyl-ß-cyclodextrin (MCD), when STARD4 and MCD were overexpressed or injected into cells. Interestingly, STARD4 and cytosolic MCD act similarly by increasing the rate of transfer of sterol to the ERC and to the ER. Our results suggest that cholesterol transport mediated by STARD4 is an important component of the cholesterol homeostasis regulatory machinery.
Assuntos
Colesterol/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Motivos de Aminoácidos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Ésteres do Colesterol/biossíntese , Retículo Endoplasmático/metabolismo , Ergosterol/análogos & derivados , Ergosterol/metabolismo , Esterificação , Recuperação de Fluorescência Após Fotodegradação , Corantes Fluorescentes/metabolismo , Técnicas de Silenciamento de Genes , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinética , Lipossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Estrutura Terciária de Proteína , Interferência de RNA , Esterol O-Aciltransferase/antagonistas & inibidores , Esterol O-Aciltransferase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Imagem com Lapso de Tempo , Transferrina/metabolismo , Vesículas Transportadoras/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
Cell migration is an essential step in cancer invasion and metastasis. A number of orchestrated cellular events involving tyrosine kinases and signaling receptors enable cancer cells to dislodge from primary tumors and colonize elsewhere in the body. For example, activation of the Src and Abl kinases can mediate events that promote tumor cell migration. Also, activation of the Robo1 receptor can induce tumor cell migration. However, while the importance of Src, Abl, and Robo1 in cell migration have been demonstrated, molecular mechanisms by which they collectively influence cell migration have not been clearly elucidated. In addition, little is known about mechanisms that control Robo1 expression. We report here that Src activates Abl to stabilize Robo1 in order to promote cell migration. Inhibition of Abl kinase activity by siRNA or kinase blockers decreased Robo1 protein levels and suppressed the migration of transformed cells. We also provide evidence that Robo1 utilizes Cdc42 and Rac1 GTPases to induce cell migration. In addition, inhibition of Robo1 signaling can suppress transformed cell migration in the face of robust Src and Abl kinase activity. Therefore, inhibitors of Src, Abl, Robo1 and small GTPases may target a coordinated pathway required for tumor cell migration.