Elevated IGFBP3 levels in diabetic tears: a negative regulator of IGF-1 signaling in the corneal epithelium.
Elevated IGFBP3 levels in diabetic tears: a negative regulator of IGF-1 signaling in the corneal epithelium.
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DOI：
10.1016/j.jtos.2012.01.004
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发表时间：
2012-04
期刊：
影响因子：
--
通讯作者：
Robertson DM
中科院分区：
文献类型：
--
作者：
Wu YC;Buckner BR;Zhu M;Cavanagh HD;Robertson DM
测定正常和糖尿病人泪液中IGFBP3:IGF-1的比例，以及在高葡萄糖条件下培养的端粒酶永生化人角膜上皮细胞（hTCEpi）中IGFBP3:IGF-1的比例，并将这些变化与IGF-1R总水平和磷酸化水平相关联。无创采集糖尿病受试者和非糖尿病对照组的泪液样本；使用Cochet-Bonnet美感计评估角膜敏感度。hTCEpi细胞在正常（5 mM）和升高（25 mM）葡萄糖条件下培养后收集条件培养基；甘露醇作为渗透控制剂。ELISA检测IGFBP3、IGF-1和磷酸化IGF-1R水平。采用实时聚合酶链反应（real - time polymerase chain reaction， PCR）检测IGFBP3和IGF-1R mRNA水平。western blot检测全细胞裂解物中IGF-1R的总表达和磷酸化表达。与非糖尿病对照组相比，糖尿病患者泪液中IGFBP3增加2.8倍（P=0.006）；IGF-1水平没有明显改变。两组间角膜敏感性无差异。泪液中IGFBP3的浓度与IGF-1无关。与人体内的泪液测量结果一致，体外培养hTCEpi细胞后，IGFBP3的分泌增加了2.2倍（P<0.001）。葡萄糖治疗和甘露醇对照降低了IGFBP3 mRNA （P<0.001）。总的IGF-1R水平没有变化。在体外测试中，与正常对照相比，糖尿病患者泪液中IGFBP3:IGF-1比值的增加阻断了IGF-1对IGF-1R的磷酸化（P<0.001）。综上所述，这些体内和体外的验证性研究结果表明，在人泪液中发现的IGFBP3的增加可能会减弱糖尿病角膜中的IGF-1R信号。据报道，IGFBP3的长期升高可能导致糖尿病眼表并发症的发病机制和上皮损害。
To determine the ratio of IGFBP3:IGF-1 in normal and diabetic human tears, and in telomerase-immortalized human corneal epithelial cells (hTCEpi) cultured under elevated glucose conditions and to correlate these changes with total and phosphorylated levels of IGF-1R. Tear samples were collected noninvasively from diabetic subjects and non-diabetic controls; corneal sensitivity was assessed using a Cochet-Bonnet Aesthesiometer. Conditioned media were collected following culture of hTCEpi cells in normal (5 mM) and elevated (25 mM) glucose conditions; mannitol was used as an osmotic control. IGFBP3, IGF-1, and phosphorylated IGF-1R levels were assessed by ELISA. IGFBP3 and IGF-1R mRNA were assessed by real time polymerase chain reaction (PCR). Total and phosphorylated IGF-1R expression in whole cell lysates was assessed by western blot. There was a 2.8-fold increase in IGFBP3 in diabetic tears compared to non-diabetic controls (P=0.006); IGF-1 levels were not significantly altered. No difference in corneal sensitivity was detected between groups. The concentration of IGFBP3 in tears was independent of IGF-1. Consistent with human tear measurements in vivo, IGFBP3 secretion was increased 2.2 fold (P<0.001) following culture of hTCEpi cells under elevated glucose conditions in vitro. Treatment with glucose and the mannitol control reduced IGFBP3 mRNA (P<0.001). Total IGF-1R levels were unchanged. The increase in the IGFBP3:IGF-1 ratio detected in diabetic tears compared to normal controls blocked phosphorylation of the IGF-1R by IGF-1 (P<0.001) when tested in vitro. Taken together, these in vivo and confirmatory in vitro findings suggest that the observed increase in IGFBP3 found in human tears may attenuate IGF-1R signaling in the diabetic cornea. A long-term increase in IGFBP3 may contribute to epithelial compromise and the pathogenesis of ocular surface complications reported in diabetes.
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作者：
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