Molecular mechanisms of suberoylanilide hydroxamic acid in the inhibition of TGF-β1-mediated canine corneal fibrosis.
Molecular mechanisms of suberoylanilide hydroxamic acid in the inhibition of TGF-β1-mediated canine corneal fibrosis.
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DOI：
10.1111/vop.12331
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发表时间：
2016-11
影响因子：
1.6
通讯作者：
Mohan RR
中科院分区：
文献类型：
--
作者：
Gronkiewicz KM;Giuliano EA;Sharma A;Mohan RR
使用体外模型研究SAHA在犬角膜中介导抗纤维化作用的分子机制。我们假设SAHA通过调节TGF-β1激活的Smad依赖性和较小程度上的Smad非依赖性信号通路以及基质金属蛋白酶（MMP）活性来减轻角膜纤维化。将培养的犬角膜成纤维细胞（CCF）在存在/不存在TGF-β1（5 ng/ml）和SAHA（2.5μM）的情况下孵育24小时。Western印迹分析用于定量Smad 2/3、p38 MAP激酶（MAPK）、ERK 1/2和JNK 1的非磷酸化和磷酸化亚型。采用Real-time PCR和酶谱法分别检测MMP 1、MMP 2、MMP 8和MMP 9 mRNA表达及MMP 2和MMP 9蛋白活性。TGF-β1处理引起磷酸化Smad 2/3和磷酸化p38 MAPK显著增加。SAHA处理降低TGF-β1诱导的Smad 2/3磷酸化，但不降低p38 MAPK磷酸化。TGF-β1不调节ERK 1/2或JNK 1的磷酸化。SAHA引起磷酸化ERK 1/2表达的显著降低，而与同时进行的TGF-β1治疗无关。SAHA单独使用或与TGF-β1联合使用都不会改变磷酸化JNK 1的表达。TGF-β1可显著增加MMP 1和MMP 9 mRNA的表达，但对MMP 2 mRNA的表达无明显影响。SAHA处理可减弱TGF-β1诱导的MMP 9 mRNA表达，而显著增强TGF-β1诱导的MMP 1 mRNA表达。酶谱法检测到MMP 2和MMP 9蛋白在未处理的对照CCF中的表达减少。TGF-β1处理没有改变它们的表达，但SAHA处理+/−TGF-β1显著增加MMP 2和MMP 9蛋白表达。SAHA的角膜抗纤维化作用涉及多种机制，包括调节TGF-β1细胞内信号传导和MMP活性的经典和非经典组分。
To investigate molecular mechanisms mediating anti-fibrotic effect of SAHA in the canine cornea using an in vitro model. We hypothesized that SAHA attenuates corneal fibrosis by modulating Smad-dependent and, to a lesser extent, Smad-independent signaling pathways activated by TGF-β1, as well as matrix metalloproteinase (MMP) activity. Cultured canine corneal fibroblasts (CCF) were incubated in the presence/absence of TGF-β1 (5ng/ml) and SAHA (2.5μM) for 24hrs. Western blot analysis was used to quantify non-phosphorylated and phosphorylated isoforms of Smad2/3, p38 MAP kinase (MAPK), ERK1/2 and JNK1. Real-time PCR and zymography were utilized to quantify MMP1, MMP2, MMP8 and MMP9 mRNA expression and MMP2 and MMP9 protein activity, respectively. TGF-β1 treatment caused a significant increase in phospho-Smad2/3 and phospho-p38 MAPK. SAHA treatment reduced TGF-β1-induced phosphorylation of Smad2/3 but not of p38 MAPK. TGF-β1 did not modulate the phosphorylation of ERK1/2 or JNK1. SAHA caused a significant reduction in phospho-ERK1/2 expression regardless of concurrent TGF-β1 treatment. Neither SAHA alone nor in combination with TGF-β1 altered phospho-JNK1 expression. TGF-β1 significantly increased MMP1 and MMP9 mRNA expression but did not alter MMP2 mRNA. SAHA treatment attenuated TGF-β1-induced MMP9 mRNA expression while significantly enhancing TGF-β1-induced MMP1 mRNA expression. Zymography detected reduced expression of MMP2 and MMP9 proteins in untreated control CCF. TGF-β1 treatment did not alter their expression but SAHA treatment +/−TGF-β1 significantly increased MMP2 and MMP9 protein expression. The corneal anti-fibrotic effects of SAHA involve multiple mechanisms including modulation of canonical and non-canonical components of TGF-β1 intracellular signaling and MMP activity.