RESUMEN
Vesicular trafficking of hyaluronan synthases (HAS1-3) from endoplasmic reticulum (ER) through Golgi to plasma membrane (PM), and either back to endosomes and lysosomes, or out into extracellular vesicles, is important for their activities. We studied how post-translational modifications affect the trafficking of HAS2 by mutagenesis of the sites of ubiquitination (K190R), phosphorylation (T110A) and O-GlcNAcylation (S221A), using Dendra2- and EGFP-HAS2 transfected into COS1 cells. Confocal microscopy showed HAS2 wild type (wt) and its K190R and S221A mutants in ER, Golgi and extracellular vesicles, while the T110A mutant remained mostly in the ER. HA synthesis was reduced by S221A, while completely blocked by K190R and T110A. Cell-surface biotinylation indicated that T110A was absent from PM, while S221A was close to the level of wt, and K190R was increased in PM. TIRF microscopy analysis gave similar results. Rab10 silencing increased HA secretion by HAS2, likely by inhibiting endocytosis of the enzyme from PM, as reported before for HAS3. Green-to-red photo-conversion of Dendra2-HAS2 constructs suggested slower decay of K190R and S221A than HAS2 wt, while T110A was barely degraded at all. S221D and S221E, the phosphomimetic mutants of this site, decayed faster and blocked hyaluronan synthesis, suggesting alternative O-GlcNAc/-PO4 substitution to regulate the stability of the enzyme. Probing the role of dynamic O-GlcNAcylation at S221 by adding glucosamine increased the half-life of only HAS2 wt. The Dendra2·HAS2 disappearance from Golgi was slower for K190R. Of the two inactive constructs, K190R co-transfected with HAS2 wt suppressed, whereas T110A had no effect on HA synthesis. Interestingly, the HAS2-stimulated shedding of extracellular vesicles was dependent on HAS residence in PM but independent of HA synthesis. The results indicate that post-translational modifications control the trafficking of HAS2, and that trafficking is an integral part of the post-translational regulation of HAS2 activity.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Hialuronano Sintasas/metabolismo , Mutación , Animales , Células COS , Chlorocebus aethiops , Regulación de la Expresión Génica , Glicosilación , Humanos , Hialuronano Sintasas/genética , Fosforilación , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , UbiquitinaciónRESUMEN
BACKGROUND: Hyaluronan is a large, linear glycosaminoglycan present throughout the narrow extracellular space of the vital epidermis. Increased hyaluronan metabolism takes place in epidermal hypertrophy, wound healing and cancer. Hyaluronan is produced by hyaluronan synthases and catabolized by hyaluronidases, reactive oxygen species and KIAA1199. OBJECTIVES: To investigate the changes in hyaluronan metabolism during epidermal stratification and maturation, and the impact of vitamin C on these events. METHODS: Hyaluronan synthesis and expression of the hyaluronan-related genes were analysed during epidermal maturation from a simple epithelium to a fully differentiated epidermis in organotypic cultures of rat epidermal keratinocytes using quantitative reverse transcriptase polymerase chain reaction, immunostaining and Western blotting, in the presence and absence of vitamin C. RESULTS: With epidermal stratification, both the production and the degradation of hyaluronan were enhanced, resulting in an increase of hyaluronan fragments of various sizes. While the mRNA levels of Has3 and KIAA1199 remained stable during the maturation, Has1, Has2 and Hyal2 showed a transient upregulation during stratification, Hyal1 transcription remained permanently increased and transcription of the hyaluronan receptor, Cd44, decreased. At maturation, vitamin C downregulated Has2, Hyal2 and Cd44, whereas it increased high-molecular-mass hyaluronan in the epidermis, and reduced small fragments in the medium, suggesting stabilization of epidermal hyaluronan. CONCLUSIONS: Epidermal stratification and maturation is associated with enhanced hyaluronan turnover, and release of large amounts of hyaluronan fragments. The high turnover is suppressed by vitamin C, which is suggested to enhance normal epidermal differentiation in part through its effect on hyaluronan.
Asunto(s)
Ácido Ascórbico/farmacología , Epidermis/efectos de los fármacos , Ácido Hialurónico/metabolismo , Queratinocitos/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Epidermis/química , Epidermis/metabolismo , Perfilación de la Expresión Génica , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Hialuronano Sintasas/genética , Hialuronano Sintasas/metabolismo , Ácido Hialurónico/análisis , Hialuronoglucosaminidasa/genética , Hialuronoglucosaminidasa/metabolismo , Queratinocitos/química , Queratinocitos/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
AIM: Acyl-CoAs are important intermediates and regulators of lipid metabolism. Binding proteins like acyl-CoA binding protein (ACBP) can influence their regulatory functions. ACBP has also been shown to exert direct effects on gene regulation in vitro. As the physiological relevance of ACBP in the regulation of lipid metabolism under high fat diets is unclear, we investigated the influence of such diets on the metabolic responses in ACBP-overexpressing rats. METHODS: A transgenic rat line overexpressing the ACBP gene was used to study the effects of 4 weeks of feeding with medium- (MC) or long-chain (LC) fatty acid-containing diets. Glucose tolerance tests were performed. Expression of transcription factors was measured by quantitative RT-PCR and protein levels of AMP-activated protein kinase were determined by western blotting. RESULTS: Transgenic animals fed the MC diet had an improved glucose tolerance and lower serum insulin levels compared with controls. Their liver PPARgamma (by 43%) and SREBP-1 (by 35%) mRNA levels were found to be decreased, while adipose tissue PPARgamma expression was increased by 31%. Tg animals fed the LC diet did not exhibit changes in glucose or insulin levels but exhibited increased mRNA levels of liver PPARs and SREBP-1 (1.5-3.5 times) and decreased protein levels of AMPKalpha (by 48%). CONCLUSIONS: Our results demonstrate that ACBP overexpression affects metabolic responses to diets with distinct difference in their fatty acid chain lengths. The molecular regulatory mechanism behind these effects seems to be an ACBP-induced tissue-specific regulation of expression of PPARs and SREBP.