Department of Nephropathy, The Third Hospital of Hebei Medical University, Shijiazhuang 050051, China.
BMC Nephrol. 2013 Feb 11;14:33. doi: 10.1186/1471-2369-14-33.
Gremlin, 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.
To 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.
HG 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.
Culture 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.
Gremlin 是一种骨形态发生蛋白拮抗剂，在糖尿病肾病（DN）发病机制中起重要作用。然而，Gremlin 参与 DN 的具体分子机制尚未完全阐明。本研究探讨了 Gremlin 对高糖（HG）诱导的小鼠系膜细胞（MMC）增殖和细胞外基质（ECM）积聚的作用，并研究了 Gremlin 与 ERK1/2 通路的关系。
通过实时聚合酶链反应和 Western blot 分析分别评估 Gremlin 在 HG 暴露后 MMC 中的表达，以确定 Gremlin 在 MMC 中的表达。通过 Western blot 分析评估 pERK1/2、转化生长因子-β1（TGF-β1）和结缔组织生长因子（CTGF）的表达，以确定 Gremlin 对细胞增殖和 ECM 积聚的作用。通过溴脱氧尿苷（BrdU）ELISA 检测细胞增殖，通过放射免疫测定法测量胶原 IV 的积聚。这使我们能够研究 Gremlin 与 ERK1/2 通路激活之间的关系。
HG 暴露诱导 Gremlin 表达，HG 暴露后 12 小时达到峰值。HG 单独暴露或转染正常葡萄糖（NG）暴露的 MMC 质粒（NG + P）可增加细胞增殖。HG 预处理的 MMC 转染 Gremlin 质粒（HG + P）显著增加了这种 HG 诱导的现象。HG 和 NG + P 条件上调 TGF-β1、CTGF 和胶原 IV 积聚的蛋白水平，而 HG + P 则显著增加了这些水平。用 Gremlin siRNA 质粒抑制 Gremlin 逆转了 HG 诱导的现象。这些数据表明 Gremlin 可诱导 MMC 中细胞增殖和 ECM 积聚。HG 还诱导 ERK1/2 通路的激活，HG 暴露后 24 小时达到峰值。HG 和 NG + P 条件诱导 pERK1/2 过表达，而 HG + P 则显著诱导水平进一步增加。用 Gremlin siRNA 质粒抑制 Gremlin 逆转了 HG 诱导的现象。这表明 Gremlin 可诱导 MMC 中 ERK1/2 通路的激活。
在 HG 存在的情况下培养 MMC 可上调 Gremlin 的表达。Gremlin 诱导 MMC 中细胞增殖和 ECM 积聚，并增强 ERK1/2 通路的激活。
