Analysis of K-Ras Nuclear Expression in Fibroblasts and Mesangial Cells

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al[Background]: Ras GTPases are considered cytoplasmic proteins that must be localized to cell membranes for activation, and there are few evidences of the presence of any Ras isoform in nuclei of eukaryotic cells. [Methodology/Principal Findings]: Using conventional antibodies and inmunocytochemistry, differential centrifugation and western blot, we have observed the putative presence of K-Ras isoform in the nuclei of fibroblasts and mesangial cells. In order to avoid cross-reactions with other Ras isoforms, and using antibodies against K-Ras (R-3400, H3845-M01, sc-30) or pan-Ras (05-516, OP40) in cells that only expressed the K-Ras isoform (fibroblasts obtained from H-ras−/−,N-ras−/− mice) we also detected some nuclear positive expression. To further probe the identity of nuclear K-Ras, we have generated K-Ras knockout (K-ras−/−) embrionary fibroblasts by mating of K-ras+/− heterozygote mice. Using specific antibodies, only H- and N-Ras isoforms were observed in the cytoplasm of K-ras−/− fibroblasts. However, both K-Ras4A and K-Ras4B positive signals were detected by immunocytochemistry and Western blot with two commercial antibodies (sc-522 and sc-521 against each isoforms, respectively) in both cytoplasm and nuclei from K-ras−/− fibroblasts. [Conclusions/Significance]: We show that the presence of K-Ras4B in fibroblast nuclei, already described by other authors, is probably due to a cross-reaction of the antibody with an undetermined nucleolar protein. Although this study also shows the possible nuclear expression of K-Ras isoform in fibroblasts or in mesangial cells, it also reveals the importance of being cautious in these studies about distribution of protein isoforms due to some important limitations imposed by the unspecificity of the antibodies or contaminations in cellular preparations.This work was supported by grants from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, CP03/00094, PI041817, PI021570 and C03/10), Comisión Interministerial de Ciencia y Tecnologí­a (BFU-2004-00285/BFI; SAF2003-04177), and Junta de Castilla y León (SA001/C05; SA029A05; GRS 167/A/07 and excellence group GR100).Peer reviewe

Increased plasma soluble endoglin levels as an indicator of cardiovascular alterations in hypertensive and diabetic patients

<p>Abstract</p> <p>Background</p> <p>Endoglin is involved in the regulation of endothelial function, but there are no studies concerning its relation with hypertension- and diabetes-associated pathologies. Thus, we studied the relationship between plasma levels of soluble endoglin and cardiovascular alterations associated with hypertension and diabetes.</p> <p>Methods</p> <p>We analyzed 288 patients: 64 with type 2 diabetes, 159 with hypertension and 65 healthy patients. We assessed the relationship of soluble endoglin plasma levels measured by enzyme-linked immunosorbent assay with basal glycemia, glycosylated hemoglobin, blood pressure, endothelial dysfunction (assessed by pressure wave velocity), hypertensive retinopathy (by Keith-Wagener classification), left ventricular hypertrophy (by Cornell and Sokolow indexes), cardiovascular risk and target organ (heart, vascular, kidney) damage.</p> <p>Results</p> <p>There are significant correlations between endoglin and glycemia, systolic blood pressure, pulse pressure, pressure wave velocity and electrocardiographically assessed left ventricular hypertrophy. Endoglin levels were significantly higher in patients with diabetes who had nondipper and extreme dipper circadian blood pressure patterns than in dipper circadian patterns, in patients with hypertension and diabetes who had riser pattern than in the other patients, and in patients with diabetes but not hypertension who had extreme dipper pattern than in dipper, nondipper and riser groups. There was also a significant correlation between plasma-soluble endoglin and lower levels of systolic night-day ratio. Higher endoglin levels were found in patients with diabetes who had retinopathy, in patients with diabetes who had a high probability of 10-year cardiovascular risk, and in patients with diabetes and hypertension who had three or more damaged target organs (heart, vessels, kidney) than in those with no organs affected.</p> <p>Conclusions</p> <p>This study shows that endoglin is an indicator of hypertension- and diabetes-associated vascular pathologies as endothelial dysfunction and cardiovascular damage.</p

Noggin depletion in adipocytes promotes obesity in mice.

ObjectiveObesity has increased to pandemic levels and enhanced understanding of adipose regulation is required for new treatment strategies. Although bone morphogenetic proteins (BMPs) influence adipogenesis, the effect of BMP antagonists such as Noggin is largely unknown. The aim of the study was to define the role of Noggin, an extracellular BMP inhibitor, in adipogenesis.MethodsWe generated adipose-derived progenitor cells and a mouse model with adipocyte-specific Noggin deletion using the AdiponectinCre transgenic mouse, and determined the adipose phenotype of Noggin-deficiency.ResultsOur studies showed that Noggin is expressed in progenitor cells but declines in adipocytes, possibly allowing for lipid accumulation. Correspondingly, adipocyte-specific Noggin deletion in&nbsp;vivo promoted age-related obesity in both genders with no change in food intake. Although the loss of Noggin caused white adipose tissue hypertrophy, and whitening and impaired function in brown adipose tissue in both genders, there were clear gender differences with the females being most affected. The females had suppressed expression of brown adipose markers and thermogenic genes including peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1alpha) and uncoupling protein 1 (UCP1) as well as genes associated with adipogenesis and lipid metabolism. The males, on the other hand, had early changes in a few BAT markers and thermogenic genes, but the main changes were in the genes associated with adipogenesis and lipid metabolism. Further characterization revealed that both genders had reductions in VO2, VCO2, and RER, whereas females also had reduced heat production. Noggin was also reduced in diet-induced obesity in inbred mice consistent with the obesity phenotype of the Noggin-deficient mice.ConclusionsBMP signaling regulates female and male adipogenesis through different metabolic pathways. Modulation of adipose tissue metabolism by select BMP antagonists may be a strategy for long-term regulation of age-related weight gain and obesity

H-Ras isoform modulates extracellular matrix synthesis, proliferation, and migration in fibroblasts

Ras GTPases are ubiquitous plasma membrane transducers of extracellular stimuli. In addition to their role as oncogenes, Ras GTPases are key regulators of cell function. Each of the Ras isoforms exhibits specific modulatory activity on different cellular pathways. This has prompted researchers to determine the pathophysiological roles of each isoform. There is a proven relationship between the signaling pathways of transforming growth factor-β1 (TGF-β1) and Ras GTPases. To assess the individual role of H-Ras oncogene in basal and TGF-β1-mediated extracellular matrix (ECM) synthesis, proliferation, and migration in fibroblasts, we analyzed these processes in embryonic fibroblasts obtained from H-Ras knockout mice (H-ras -/-). We found that H-ras -/-fibroblasts exhibited a higher basal phosphatidylinositol-3-kinase (PI3K)/Akt activation than wild-type (WT) fibroblasts, whereas MEK/ERK 1/2 activation was similar in both types of cells. Fibronectin and collagen synthesis were higher in H-ras -/-fibroblasts and proliferation was lower in Hras -/-than in WT fibroblasts. Moreover, H-Ras appeared indispensable to maintain normal fibroblast motility, which was highly restricted in H-ras -/-cells. These results suggest that H-Ras (through downregulation of PI3K/Akt activation) could modulate fibroblast activity by reducing ECM synthesis and upregulating both proliferation and migration. TGF-β1 strongly increased ERK and Akt activation in WT but not in H-ras -/-fibroblasts, suggesting that H-Ras is necessary to increase ERK 1/2 activation and to maintain PI3K downregulation in TGF-β1-stimulated fibroblasts. TGF-β1 stimulated ECM synthesis and proliferation, although ECM synthesis was higher and proliferation lower in H-ras -/-than in WT fibroblasts. Hence, H-Ras activation seems to play a key role in the regulation of these effects. © 2012 the American Physiological Society.This work was supported by grants from European Commission Research Directorates General (ERG-Mobility-11, no. 508782), Instituto de Salud Carlos III (PS09/01067, PS09/01979 and Retics RD06/0016 and RD06/0020), Ministerio de Ciencia e Innovación (BFU-2004-00285/BFI and SAF2010-15881), and Junta de Castilla y León (SA001/C05; SA029A05; GRS167/A/07 and Excellence Group GR100). Ana M. Blázquez-Medela is supported by Fondo de Investigaciones Sanitarias (FIS) from the Institute Carlos III, Ministerio de Ciencia e Innovación.Peer Reviewe

Immunofluorescence analysis of cellular distribution of Ras isoforms in H-<i>ras</i>^−/−/N-<i>ras</i>^−/− fibroblasts.

<p>Pictures show H-Ras, N-Ras, K-Ras4A and K-Ras4B expression in fibroblasts. The rhodamine red-labeled proteins are Ras isoforms, while the blue-labeled staining is Hoechst-stained DNA. Magnification: 630×. Scale bar: 30 µm. Antibodies: H-Ras, sc-520; K-Ras4A, sc-522; K-Ras4B, sc-521; N-Ras, sc-519.</p

Immunofluorescence analysis of cellular distribution of Ras isoforms in K-<i>ras</i>^+/+ fibroblasts.

<p>Pictures show H-Ras, N-Ras, K-Ras4A and K-Ras4B expression) in K-<i>ras</i>^+/+ fibroblasts. The rhodamine red-labeled proteins are Ras isoforms, while the blue-labeled staining is Hoechst-stained DNA. Magnification: 630×. Scale bar: 30 µm. Antibodies: H-Ras, sc-520; K-Ras4A, sc-522; K-Ras4B, sc-521; N-Ras, sc-519.</p

Immunofluorescence analysis of cellular distribution of Ras isoforms in human mesangial cells.

<p>Pictures show H-Ras, N-Ras, K-Ras4A and K-Ras4B expression in human mesangial cells. The rhodamine red-labeled proteins are Ras isoforms, while the blue-labeled staining is Hoechst-stained DNA. Magnification: 630×. Scale bar: 30 µm. Antibodies: H-Ras, sc-520; K-Ras4A, sc-522; K-Ras4B, sc-521; N-Ras, sc-519.</p