Abstract
BackgroundGremlin, a bone morphogenetic protein antagonist, plays an important role in the pathogenesis of diabetic nephropathy (DN). However, the specific molecular mechanism underlying Gremlin’s involvement in DN has not been fully elucidated. In the present study, we investigated the role of Gremlin on cell proliferation and accumulation of extracellular matrix (ECM) in mouse mesangial cells (MMCs), and explored the relationship between Gremlin and the ERK1/2 pathway.MethodsTo determine expression of Gremlin in MMCs after high glucose (HG) exposure, Gremlin mRNA and protein expression were evaluated using real-time polymerase chain reaction and western blot analysis, respectively. To determine the role of Gremlin on cell proliferation and accumulation of ECM, western blot analysis was used to assess expression of pERK1/2, transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF). Cell proliferation was examined by bromodeoxyuridine (BrdU) ELISA, and accumulation of collagen IV was measured using a radioimmunoassay. This enabled the relationship between Gremlin and ERK1/2 pathway activation to be investigated.ResultsHG exposure induced expression of Gremlin, which peaked 12 h after HG exposure. HG exposure alone or transfection of normal-glucose (NG) exposed MMCs with Gremlin plasmid (NG + P) increased cell proliferation. Transfection with Gremlin plasmid into MMCs previously exposed to HG (HG + P) significantly increased this HG-induced phenomenon. HG and NG + P conditions up-regulated protein levels of TGF-β1, CTGF and collagen IV accumulation, while HG + P significantly increased levels of these further. Inhibition of Gremlin with Gremlin siRNA plasmid reversed the HG-induced phenomena. These data indicate that Gremlin can induce cell proliferation and accumulation of ECM in MMCs. HG also induced the activation of the ERK1/2 pathway, which peaked 24 h after HG exposure. HG and NG + P conditions induced overexpression of pERK1/2, whilst HG + P significantly induced levels further. Inhibition of Gremlin by Gremlin siRNA plasmid reversed the HG-induced phenomena. This indicates Gremlin can induce activation of the ERK1/2 pathway in MMCs.ConclusionCulture of MMCs in the presence of HG up-regulates expression of Gremlin. Gremlin induces cell proliferation and accumulation of ECM in MMCs. and enhances activation of the ERK1/2 pathway.
Highlights
Gremlin, a bone morphogenetic protein antagonist, plays an important role in the pathogenesis of diabetic nephropathy (DN)
Effects of high glucose on the expression of Gremlin in mouse mesangial cells (MMCs) In order to confirm the effects of glucose on the expression of Gremlin, MMCs were cultured in media containing 5.5 mM glucose (NG), stimulated with NG plus 24.5 mM mannitol (M) or 30 mM glucose (HG) for 6, 12, 24, and 48 h
Consistent with the results observed at the mRNA level, western blot analysis showed a significant increase in Gremlin protein expression under high glucose conditions (30 mM) at all time intervals
Summary
A bone morphogenetic protein antagonist, plays an important role in the pathogenesis of diabetic nephropathy (DN). Gremlin is a 184-amino acid protein, which is a member of the structural cysteine knot superfamily [1,2] This protein is evolutionarily conserved and the human Gremlin gene has been mapped to chromosome 15q13-q15 [3,4]. TGF-β, when added to serum-restricted human mesangial cells, has been found to augment Gremlin expression, but the stimulatory effect of high glucose on Gremlin expression can be attenuated by the addition of anti-TGF-β antibody [7]. This evidence indicates that Gremlin expression is induced by TGF-β under diabetic conditions. These data suggest a pathogenetic role for Gremlin in DN
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