Regulation of serine protease inhibitor Kazal type-5 (SPINK5) gene expression in the keratinocytes.
Regulation of serine protease inhibitor Kazal type-5 (SPINK5) gene expression in the keratinocytes.
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DOI：
10.1007/s12199-014-0393-7
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发表时间：
2014-07
影响因子：
4.7
通讯作者：
Saijoh, Kiyofumi
中科院分区：
文献类型：
--
作者：
Ngoc Anh Le;Katsuyama, Midori;Demura, Masashi;Tanii, Hideji;Katsuyama, Hironobu;Saijoh, Kiyofumi
丝氨酸蛋白酶抑制剂Kazal 5型（SPINK 5）在决定皮肤脱屑的时间方面起着至关重要的作用。其基因表达仅限于颗粒层（SG）的最表面，而编码蛋白酶的激肽释放酶（KLK）的表达通常在棘层和SG中发现。为了探索这些蛋白酶/抑制剂的表达调节的差异，使用荧光素酶测定来检查SPINK 5启动子的功能。靶向SPINK 5启动子（主要转录起始位点的核苷酸-676/-532和-318/-146）的荧光素酶测定在NHEK人角质形成细胞中显示出高强度。这两个位点既没有共同的顺式元件，也没有GATA 3元件，但电泳迁移率变动分析显示了相似的阻滞带。此外，DNA足迹没有显示特定的保护带。因此，我们无法鉴定控制这些元件的顺式元件。高Ca ~（2+）诱导分化不能改变其荧光素酶活性。GATA 3表达载体的转染显著但略有增加，而表达其显性阴性形式的载体的转染降低。尽管据报道GATA 3对于抑制角质形成细胞的增殖和诱导分化是重要的，但其对SPINK 5表达的作用是间接的，并且单独的GATA 3不足以使角质形成细胞最终分化，其中观察到SPINK 5的完全表达。
Serine protease inhibitor Kazal type-5 (SPINK5) plays a crucial role in deciding the timing of desquamation of the skin. Its gene expression is limited at the very surface of the stratum granulosum (SG), whereas expression of kallikreins (KLKs) encoding proteases is usually found throughout the stratum spinosum and SG. To explore the difference in expression regulation of these proteases/inhibitors, the function of SPINK5 promoter was examined using luciferase assay. Luciferase assay targeting the SPINK5 promoters (nucleotide −676/−532 and −318/−146 from the major transcription start site) showed high intensity in NHEK human keratinocyte. These two sites had neither common cis-elements nor GATA3 element but electrophoretic mobility shift assay showed similar retardation bands. Moreover, DNA footprinting did not display specific protected bands. Thus, we could not identify cis-element(s) that controlled these elements. Differentiation induced by high Ca2+ medium failed to alter their luciferase activities. Transfection of GATA3 expressing vector significantly but slightly increased them and that of vector expressing its dominant negative form decreased. Although GATA3 is reportedly important for inhibition of proliferation and induction of differentiation of keratinocytes, its effect on SPINK5 expression was indirect and GATA3 alone was insufficient for final differentiation of keratinocytes where full SPINK5 expression was observed.
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