Dataset Information

MicroRNA-7 inhibition rescues age-associated loss of epidermal growth factor receptor and hyaluronan-dependent differentiation in fibroblasts.

ABSTRACT: Age-related defects in fibroblast differentiation were previously shown to be associated with impaired hyaluronan synthase 2 (HAS2) and epidermal growth factor receptor (EGFR) function, with both required for normal fibroblast functionality. In fibroblasts, transforming growth factor-beta 1 (TGF-?1)-dependent phenotypic activation uses two distinct but co-operating pathways that involve TGF-? receptor (TGF-?R)/Smad2 activation and HA-mediated CD44-EGFR co-localization and signalling through extracellular signal-regulated kinase 1/2 (ERK1/2). The HA-mediated CD44-EGFR pathway was found to be compromised with in vitro aging, through loss of EGFR expression and a reduced movement of CD44 throughout the cellular membrane. Here, we also investigate the involvement of microRNAs (miRNAs) in age-related loss of differentiation, through investigation of miRNA-7 (miR-7) regulation of the HA-mediated EGFR-signalling pathway. The transcription of miR-7 was found to be upregulated in aged cells. In young cells, age-related loss of differentiation could be mimicked through transfection of pre-miR-7, and in aged cells, could be reversed through transfection of locked nucleic acids (LNA) targeting miR-7. Additionally, miR-7 was found to be involved in the regulation of CD44 membrane motility, which was downregulated in instances of miR-7 upregulation, and partially restorable through either miR-7 inhibition or HAS2 overexpression. The altered dynamics of CD44 in the cell membrane demonstrated a further action of miR-7 in regulating the HA-dependent CD44/EGFR pathway. We explain this novel mechanism of age-associated functional consequence due to miR-7 upregulation and demonstrate that it is reversible; highlighting miR-7 as a potential target for restoring the healing capabilities in chronic wounds in the elderly.

SUBMITTER: Midgley AC

PROVIDER: S-EPMC4331777 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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MicroRNA-7 inhibition rescues age-associated loss of epidermal growth factor receptor and hyaluronan-dependent differentiation in fibroblasts.

Midgley Adam C AC Bowen Timothy T Phillips Aled O AO Steadman Robert R

Aging cell 20131112 2

Age-related defects in fibroblast differentiation were previously shown to be associated with impaired hyaluronan synthase 2 (HAS2) and epidermal growth factor receptor (EGFR) function, with both required for normal fibroblast functionality. In fibroblasts, transforming growth factor-beta 1 (TGF-β1)-dependent phenotypic activation uses two distinct but co-operating pathways that involve TGF-β receptor (TGF-βR)/Smad2 activation and HA-mediated CD44-EGFR co-localization and signalling through extr ...[more]

PMID: 24134702

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Project description:While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX) in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month) effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days), and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII) proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days) post-PNX in 3 versus 9 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a) and more alpha smooth muscle actin (αSMA) positive myofibroblasts were present after PNX in 9 month than 3 month mice. Microarray analyses of mRNA expression patterns were performed on lung tissue from 9 month versus 3 month mice, before and after PNX. First, gene expression was compared in whole lungs tissues of young (3 month) vs. middle age (9 month) animals obtained at surgery (left, control lung). Second, the global gene expression responses of 3 vs. 9 mo mice were compared after PNX (1, 3 and 7 days) in the right lung. For each animal in the surgical study, the left (excised) lung lobe was used as a control for the corresponding right lung at the end of the study. This design was intended to increase the statistical power for detecting differences in gene expression by comparing pre- and post-PNX lung tissue from the same biological pools (2 animals/pool, 3 pools per group).

Project description:1 st experiment: 293T and 293T cells expressing a deletion form of FBLN7 lacking the coiled-coil domain, were grown to confluence and the resulting serum-free conditioned medium (CM) was harvested every 2 days. The collected CM was concentrated by protein precipitation with 80%-saturated ammonium sulfate and dissolved in 50 mM Tris-HCl (pH 7.4) containing 150 mm NaCl supplemented with 5 mM CaCl2, 0.1% Triton X-100, and a proteinase inhibitor mixture [0.2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), 0.16 M aprotinin, 0.025 mM bestatin, 7.5 M E-64, 0.01 mM leupeptin, and 5 M pepstatin] . The precipitants were dialyzed against a 400 times volume of diluent buffer. The dialyzed 293T CM was passed through a Cobalt-affinity column once to eliminate non-specific bound proteins, and the flow-through fraction was collected and mixed with deltaCC, and then applied to the column chromatography. deltaCC and its affinity-binding proteins bound to the column, and 1200 mM NaCl was used to elute the proteins that were associated with deltaCC. The eluted samples were subjected to SDS-PAGE and separated proteins were visualized by Coomassie staining and excised. 2nd experiment: CHO cells expressing full-length FBLN7 were grown to confluence and the resulting serum-free conditioned medium (CM) was harvested every 2 days. The collected CM was concentrated by protein precipitation with 80%-saturated ammonium sulfate and dissolved in phosphate-buffered saline without calcium and magnesium [PBS (-)] supplemented with 0.1% Triton X-100 and a proteinase inhibitor mixture [0.2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), 0.16 M aprotinin, 0.025 mM bestatin, 7.5 M E-64, 0.01 mM leupeptin, and 5 M pepstatin]. The precipitants were dialyzed against a 400 times volume of PBS (-) plus Triton X-100 and then applied to a Nickel (Ni)-Sepharose Histidine-affinity column. Bound proteins were eluted with PBS (-) supplemented with 500 mM NaCl, 500 mM imidazole, and 0.01% Triton X-100. To exclude non-specific bound proteins, the fractions containing FBLN7 were applied to a heparin-Sepharose-6B column. FBLN7 bound to the column and mainly eluted at 0.6 M NaCl. This fraction was further purified by Ni-Sepharose column chromatography and the final elute fraction containing FBLN7 and its putative high-affinity binding proteins was subjected to SDS-PAGE. Separated proteins were detected by Coomassie staining and protein bands were excised.

Dataset Information

MicroRNA-7 inhibition rescues age-associated loss of epidermal growth factor receptor and hyaluronan-dependent differentiation in fibroblasts.

Publications

MicroRNA-7 inhibition rescues age-associated loss of epidermal growth factor receptor and hyaluronan-dependent differentiation in fibroblasts.

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