RESUMEN
Generation of oxylipins (oxygenated metabolites of fatty acids) by lipoxygenases may be responsible for the beneficial effects of 20- and 22-carbon n-3 fatty acids on adipose tissue dysfunction in obesity, but the potential actions of oxylipins derived from 18-carbon fatty acids, which are generally at higher levels in the diet, are unknown. We therefore compared the effects of select lipoxygenase-derived oxylipins produced from α-linolenic acid (ALA, C18:3 n-3), linoleic acid (LA, C18:2 n-6), and arachidonic acid (AA, C20:4 n-6) on key adipocyte functions that are altered in obesity. Individual oxylipins were added to the culture medium of differentiating 3T3-L1 preadipocytes for 6days. Lipid accumulation was subsequently determined by Oil Red O staining, while Western blotting was used to measure levels of proteins associated with lipid metabolism and characteristics of adipocyte functionality. Addition of all oxylipins at 30nM was sufficient to significantly decrease triglyceride accumulation in lipid droplets, and higher levels completely blocked lipid production. Our results establish that lipoxygenase-derived oxylipins produced from 18-carbon PUFA differentially affect multiple adipocyte processes associated with lipid storage and adipokine production. However, these effects are not due to the oxylipins blocking adipocyte maturation and thus globally suppressing all adipocyte characteristics. Furthermore, these oxylipin species decrease the lipid content of adipocytes regardless from which precursor fatty acid or lipoxygenase they were derived. Consequently, adipocyte characteristics can be altered through the ability of oxylipins to selectively modulate levels of proteins involved in both lipid metabolism and adipokine production.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adipoquinas/biosíntesis , Gotas Lipídicas/efectos de los fármacos , Lipooxigenasa/metabolismo , Oxilipinas/metabolismo , Oxilipinas/farmacología , Células 3T3-L1 , Adipocitos/citología , Animales , Diferenciación Celular/efectos de los fármacos , Gotas Lipídicas/metabolismo , Ratones , Oxilipinas/químicaRESUMEN
Cardiac fibrosis accompanies a variety of myocardial disorders, and is induced by myofibroblasts. These cells may be composed of a heterogeneous population of parent cells, including interstitial fibroblasts and circulating progenitor cells. Direct comparison of human bone marrow-derived mesenchymal stem cells (BM-MSCs) and cardiac myofibroblasts (CMyfbs) has not been previously reported. We hypothesized that BM-MSCs readily adopt a myofibroblastic phenotype in culture. Human primary BM-MSCs and human CMyfbs were isolated from patients undergoing open heart surgery and expanded under standard culture conditions. We assessed and compared their phenotypic and functional characteristics by examining their gene expression profile, their ability to contract collagen gels and synthesize collagen type I. In addition, we examined the role of non-muscle myosin II (NMMII) in modulating MSC myogenic function using NMMII siRNA knockdown and blebbistatin, a specific small molecule inhibitor of NMMII. We report that, while human BM-MSCs retain pluripotency, they adopt a myofibroblastic phenotype in culture and stain positive for the myofibroblast markers α-SMA, vimentin, NMMIIB, ED-A fibronectin, and collagen type 1 at each passage. In addition, they contract collagen gels in response to TGF-ß1 and synthesize collagen similar to human CMyfbs. Moreover, inhibition of NMMII activity with blebbistatin completely attenuates gel contractility without affecting cell viability. Thus, human BM-MSCs share and exhibit similar physiological and functional characteristics as human CMyfbs in vitro, and their propensity to adopt a myofibroblast phenotype in culture may contribute to cardiac fibrosis.
Asunto(s)
Células Madre Mesenquimatosas/metabolismo , Miocardio/citología , Miofibroblastos/metabolismo , Secuencia de Bases , Colágeno Tipo I/biosíntesis , Cartilla de ADN , Humanos , Técnicas In Vitro , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
To identify genes involved in etoposide drug response, we used promoter trap mutagenesis to isolate an etoposide-resistant Chinese hamster ovary (CHO) cell line. This resistant CHO-K1 line, named E91, showed cross-resistance to C(2)-ceramide (N-acetylsphingosine). The promoter trap retrovirus was found integrated into intron 1-2 of the Dlc-2 (Stard13) RhoGap gene. The E91 cells showed elevated guanosine triphosphate (GTP)-bound RhoA levels compared with the parental line, suggesting that retrovirus integration had inactivated one of the Dlc-2 RhoGap alleles. To test whether E91 cells were impaired in an intracellular ceramide-regulated process not directly related to cell killing, we measured mitochondrial phosphatidylglycerolphosphate (PGP) synthase and phospholipase A2 enzyme activities in cells after C(2)-ceramide addition. Parental cells showed elevated enzyme activities after treatment with C(2)-ceramide or tumor necrosis factor alpha, but not the E91 cells. These results suggested that intracellular ceramide signaling was defective in E91 cells due to increased levels of active GTP-bound RhoA. RNA knockdown experiments of the Dlc2 RhoGap resulted in increased GTP-bound RhoA and reduced induction of PGP synthase after C(2)-ceramide addition compared with controls. Expression of a dominant-negative RhoA in the E91 cell line allowed induction of PGP synthase by ceramide. The RNA interference knockdown cell line also showed increased etoposide resistance. This study is the first report for the regulation of a phospholipid biosynthetic enzyme through RhoGap expression.