Differentiated keratinocyte-releasable stratifin (14-3-3 sigma) stimulates MMP-1 expression in dermal fibroblasts
Differentiated keratinocyte-releasable stratifin (14-3-3 sigma) stimulates MMP-1 expression in dermal fibroblasts
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DOI：
10.1111/j.0022-202x.2004.23521.x
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发表时间：
2005-01-01
影响因子：
6.5
通讯作者：
Weinfeld, M
中科院分区：
文献类型：
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作者：
Ghahary, A;Marcoux, Y;Weinfeld, M
通过使用角质形成细胞/成纤维细胞共培养系统，我们最近发现了一种有效的角质形成细胞来源的抗成纤维因子（KDAF），用于真皮成纤维细胞。对角质形成细胞条件培养基（KCM）的活性组分进行了连续色谱分析，并对候选蛋白进行了肽图绘制，确定了KDAF是角质形成细胞可释放的14-3-3sigma （14-3-3sigma）蛋白，也被称为stratifin。在这项研究中，我们假设分化但不增殖的角质形成细胞是条件培养基中可释放14-3-3sigma的主要来源。为了验证这一假设，在一项纵向研究中，通过在含50%无血清角质细胞培养基（KSFM）和50%不含任何添加剂的Dulbecco改良eagle培养基中培养这些细胞，诱导角质细胞分化长达20天。当每隔一天收集KCM并将其添加到成纤维细胞中时，基质金属蛋白酶(MMP)-1 mRNA的表达水平在接受KCM的成纤维细胞中显著升高，而在较晚时间点接受条件培养基的细胞中，这种升高幅度更大。western blot分析结果进一步显示，从第4天到第10天，在试验培养基中生长的角质形成细胞中14-3-3sigma蛋白的表达明显增加。这一发现与14-3-3sigma mRNA在分化角质形成细胞中的表达水平一致。与角化细胞中14-3-3sigma mRNA的高水平表达相反，对14-3-3sigma高度敏感的成纤维细胞无法表达该因子。有趣的是，与成纤维细胞共培养的角质形成细胞中，14-3-3sigma mRNA的表达水平明显高于单独培养的角质形成细胞。综上所述，本研究证明角质形成细胞在mRNA和蛋白水平上高水平表达14-3-3 - sigma。但14-3-3 -3sigma蛋白的释放形式仅存在于分化角质形成细胞的条件培养基中。此外，我们最近纯化的重组14-3-3sigma蛋白模拟了KCM在真皮成纤维细胞中的胶原酶刺激作用。
Through the use of a keratinocyte/fibroblast co-culture system, we have recently identified a potent keratinocyte-derived anti-fibrogenic factor (KDAF) for dermal fibroblasts. A sequential chromatography of the active fractions of keratinocyte-conditioned medium (KCM) and peptide mapping of the candidate proteins identified KDAF as being the keratinocyte-releasable 14-3-3 sigma (14-3-3sigma) protein, which is also known as stratifin. In this study, we hypothesize that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3sigma in conditioned medium. To address this hypothesis, in a longitudinal study, keratinocyte differentiation was induced by growing these cells in a medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% Dulbecco's modified eagle's medium without any additives for up to 20 d. When KCM was collected every other day and added to fibroblasts, the level of matrix metalloproteinase (MMP)-1 mRNA expression was markedly increased in fibroblasts receiving KCM and this increase was even greater in cells receiving conditioned media collected at later time points relative to that of controls. The results of a western blot analysis further showed a marked increase in the expression of 14-3-3sigma protein in keratinocytes grown in test medium from day 4 to day 10. This finding was consistent with the levels of 14-3-3sigma mRNA expression in differentiated keratinocytes. In contrast to a very high level of 14-3-3sigma mRNA expression seen in keratinocytes, fibroblasts that are highly responsive to14-3-3sigma were unable to express this factor. Interestingly, the level of 14-3-3sigma mRNA expression was markedly higher in keratinocytes co-cultured with fibroblasts relative to that of mono-cultured keratinocytes. In conclusion, this study provides evidence that keratinocytes express a high level of 14-3-3sigma at the levels of mRNA and protein. But the releasable form of 14-3-3sigma protein was only found in conditioned medium derived from differentiated keratinocytes. Further, our recently purified recombinant 14-3-3sigma protein mimics the collagenase stimulatory effect of KCM in dermal fibroblasts.