RESUMEN
Miconazole is effective in treating inflammatory skin conditions and has well-established antifungal effects. To elucidate the underlying mechanisms mediating its additional beneficial effects, we assessed whether miconazole influences the inflammation induced by 27-hydroxycholesterol (27OHChol), an oxygenated cholesterol derivative with high proinflammatory activity, using THP-1 monocytic cells. Miconazole dose-dependently inhibited the expression of proinflammatory markers, including CCL2 and CCR5 ligands such as CCL3 and CCL4, and impaired the migration of monocytic cells and CCR5-positive T cells. In the presence of 27OHChol, miconazole decreased CD14 surface levels and considerably weakened the lipopolysaccharide response. Furthermore, miconazole blocked the release of soluble CD14 and impaired the transcription of the matrix metalloproteinase-9 gene and secretion of its active gene product. Additionally, it downregulated the expression of ORP3 and restored the endocytic function of THP-1 cells. Collectively, these findings indicate that miconazole regulates the 27OHChol-induced expression of proinflammatory molecules in monocytic cells, thereby suppressing inflammation in an oxysterol-rich milieu.
RESUMEN
Genome instability is an essential hallmark in tumor development, including colorectal cancer. We have recently identified the oxysterol binding protein-related protein 3 (ORP3), also known as oxysterol binding protein-like 3 (OSBPL3), as a novel ploidy-control gene, whose knock-out leads to aneuploidy induction and promotes tumor formation, indicating that ORP3 is a bona fide tumor suppressor protein. Here we analyzed expression of ORP3 in a cohort (n = 206) of colon cancer patients in relation to patient survival. We show that low ORP3 mRNA levels correlate with reduced survival of patients with advanced nodal metastasis (N2). While patient survival does not associate with grading when the whole cohort is evaluated, importantly, low ORP3 mRNA levels associate with worse survival of female patients with grade 3 colon cancer. Similarly, low ORP3 mRNA levels associate with worse survival of grade 3 colon cancer patients 70 years of age and younger while low ORP3 mRNA levels seem to be beneficial for colon cancer patients with a T2 tumor size. Together, the data show that ORP3 expression is downregulated during colon cancer progression, which correlates with reduced patient survival. Thus, ORP3 mRNA levels may be a prognostic marker for better stratification of colon cancer patients.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias del Colon/genética , Proteínas de Unión a Ácidos Grasos/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Regulación hacia Abajo , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Inestabilidad Genómica , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores SexualesRESUMEN
Oxysterol-binding protein (OSBP) and its related protein (ORP) constitute a conserved family of lipid transfer proteins (LTPs). ORPs have been implicated as intracellular lipid exchanger and sensor in recent years, which regulate the lipid homeostasis and signal pathway. OSBP-related protein 3 plays key role in controlling cell adhesion and migration and could be developed as the drug target for cancer therapy. Here, we report the crystal structures of human ORP3 ORD to 2.1 Å and ORD-PI4P complex to 3.2 Å. The binding assay in vitro confirms the ORP3 has the capability of PI4P binding. This study further verifies that the PI4P is the common ligand of all ORPs and ORPs should be the lipid exchanger in membrane contact sites(MCS).
Asunto(s)
Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Homología Estructural de ProteínaRESUMEN
Contact sites between the endoplasmic reticulum (ER) and plasma membrane (PM) regulate both non-vesicular lipid transfer as well as Ca2+ signaling with multiple interactions between the two pathways. Here I discuss recent findings that offer exciting insights into the role of store-operated Ca2+ entry (SOCE), Oxysterol-binding protein (OSBP)-related proteins ORP3, Arf5 and the Arf GEF IQSec1 in this crosstalk and how they regulate cell migration and focal adhesion disassembly.
Asunto(s)
Señalización del Calcio , Membrana Celular/metabolismo , Movimiento Celular , Retículo Endoplásmico/metabolismo , Lípidos/química , Animales , Adhesiones Focales/metabolismo , HumanosRESUMEN
Oxysterol-binding protein (OSBP)-related proteins (ORPs) mediate non-vesicular lipid transfer between intracellular membranes. Phosphoinositide (PI) gradients play important roles in the ability of OSBP and some ORPs to transfer cholesterol and phosphatidylserine between the endoplasmic reticulum (ER) and other organelle membranes. Here, we show that plasma membrane (PM) association of ORP3 (also known as OSBPL3), a poorly characterized ORP family member, is triggered by protein kinase C (PKC) activation, especially when combined with Ca2+ increases, and is determined by both PI(4,5)P2 and PI4P After activation, ORP3 efficiently extracts PI4P and to a lesser extent phosphatidic acid from the PM, and slightly increases PM cholesterol levels. Full activation of ORP3 resulted in decreased PM PI4P levels and inhibited Ca2+ entry via the store-operated Ca2+ entry pathway. The C-terminal region of ORP3 that follows the strictly defined lipid transfer domain was found to be critical for the proper localization and function of the protein.
Asunto(s)
Retículo Endoplásmico , Oxidorreductasas , Fosfatos de Fosfatidilinositol , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , FosforilaciónRESUMEN
The endocytic pathway plays an instrumental role in recycling internalized molecules back to the plasma membrane or in directing them to lysosomes for degradation. We recently reported a new role of endosomes-the delivery of components from extracellular vesicles (EVs) to the nucleoplasm of recipient cells. Using indirect immunofluorescence, FRET, immunoisolation techniques, and RNAi, we report here a tripartite protein complex (referred to as the VOR complex) that is essential for the nuclear transfer of EV-derived components by orchestrating the specific localization of late endosomes into nucleoplasmic reticulum. We found that the VOR complex contains the endoplasmic reticulum-localized vesicle-associated membrane protein (VAMP)-associated protein A (VAP-A), the cytoplasmic oxysterol-binding protein-related protein 3 (ORP3), and late endosome-associated small GTPase Rab7. The silencing of VAP-A or ORP3 abrogated the association of Rab7-positive late endosomes with nuclear envelope invaginations and, hence, the transport of endocytosed EV-derived components to the nucleoplasm of recipient cells. We conclude that the VOR complex can be targeted to inhibit EV-mediated intercellular communication, which can have therapeutic potential for managing cancer in which the release of EVs is dysregulated.
Asunto(s)
Proteínas Portadoras/fisiología , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Complejos Multiproteicos/química , Membrana Nuclear/metabolismo , Proteínas de Transporte Vesicular/fisiología , Comunicación Celular , Células Cultivadas , Endocitosis , Proteínas de Unión a Ácidos Grasos , Humanos , Complejos Multiproteicos/fisiología , Proteínas R-SNARE , Receptores de Esteroides , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
ORP3 is an R-Ras interacting oxysterol-binding protein homolog that regulates cell adhesion and is overexpressed in several cancers. We investigated here a novel function of ORP3 dependent on its targeting to both the endoplasmic reticulum (ER) and the plasma membrane (PM). Using biochemical and cell imaging techniques we demonstrate the mechanistic requirements for the subcellular targeting and function of ORP3 in control of R-Ras activity. We show that hyperphosphorylated ORP3 (ORP3-P) selectively interacts with the ER membrane protein VAPA, and ORP3-VAPA complexes are targeted to PM sites via the ORP3 pleckstrin homology (PH) domain. A novel FFAT (two phenylalanines in an acidic tract)-like motif was identified in ORP3; only disruption of both the FFAT-like and canonical FFAT motif abolished the phorbol-12-myristate-13-acetate (PMA) stimulated interaction of ORP3-P with VAPA. Co-expression of ORP3 and VAPA induced R-Ras activation, dependent on the interactions of ORP3 with VAPA and the PM. Consistently, downstream AktS473 phosphorylation and ß1-integrin activity were enhanced by ORP3-VAPA. To conclude, phosphorylation of ORP3 controls its association with VAPA. Furthermore, we present evidence that ORP3-VAPA complexes stimulate R-Ras signaling.