Abstract
Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5'-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization.
Highlights
32000 JOURNAL OF BIOLOGICAL CHEMISTRY fibroblasts involves a complex machinery that implicates at least three transcription proteins, Human c-Krox (hc-Krox), Sp1, and Sp3, which could act in concert to up-regulate COL1A1 transcriptional activity and provide evidence for a pro-fibrotic role of hc-Krox
We have investigated the role of hc-Krox transcription factor on type I collagen expression by human dermal fibroblasts. hc-Krox exerted a stimulating effect on type I collagen protein synthesis and enhanced the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in foreskin fibroblasts (FF), adult normal fibroblasts (ANF), and scleroderma fibroblasts (SF)
We found that hc-Krox increases type I collagen synthesis in human foreskin fibroblasts (FF) obtained from young children, adult normal fibroblasts (ANF), and scleroderma fibroblasts (SF)
Summary
Cell Cultures—Children foreskin samples were provided by Dr P. Fibroblasts were obtained after explant cultures and seeded at 2.5 ϫ 106 cells/cm in 175-cm flasks, in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, glutamine (2 mM), penicillin (100 IU/ml), streptomycin (100 g/ml), and fungizone (0.25 g/ml) in a 5% CO2 environment. They were passaged with a trypsin (0.05%), EDTA (0.25 mM) solution (Invitrogen) after reaching confluency. Collagen Labeling and Assay—Fibroblasts were seeded at 0.17 ϫ 105 cells/9.6-cm wells, in 10% fetal calf serum-containing Dulbecco’s modified Eagle’s medium, and transiently transfected, or not, by the calcium phosphate precipitation method with the hc-Krox expression vector and/or the corresponding insertless plasmid (pSG5/hc-Krox).
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