Briata P, D'Anna F, Franzi A T, Gherzi R
Laboratorio di Immunobiologia, Istituto Nazionale per la Ricerca sul Cancro (I.S.T.), Genoa, Italy.
Exp Cell Res. 1993 Jan;204(1):136-46. doi: 10.1006/excr.1993.1018.
Increased levels of c-fos and c-jun expression have been observed in differentiating epithelial cells. However, no data are available on activator protein 1 (AP-1) activity during keratinocyte differentiation. In this work we investigated c-fos and c-jun gene expression and AP-1-(12-O-tetradecanoylphorbol-13-acetate)-responsive enhancer element (TRE) binding activity during keratinocyte differentiation utilizing both authentic and in culture-reconstituted human epidermis. We demonstrate that: (i) in reconstituted epidermis, non-differentiated and differentiated keratinocytes express equivalent levels of c-Jun, while in reconstituted epidermis permanently grafted onto athymic mice, as well as in authentic epidermis, c-Jun is predominantly expressed in the granular layer of the tissue. Equivalent levels of c-fos expression have been found in all the layers of both reconstituted and authentic epidermis. (ii) Nuclear extracts from cultures enriched in differentiated keratinocytes display an 80-90% reduction of AP-1 activity when compared to extracts from cultures enriched in nondifferentiated cells. (iii) Cytosolic extracts obtained from cultures enriched in differentiated cells reduce, in a concentration-dependent manner, the AP-1 activity present in nuclear extracts of both mammalian and Drosophila cells. (iv) The specific TRE binding activity of a recombinant c-Jun protein is significantly reduced by cytosolic extracts of differentiated keratinocytes, while the specific DNA binding of the purified recombinant human homeoprotein HOX4B is not. (v) The dephosphorylation, by alkaline phosphatase, of cytosolic extracts increases the inhibitory activity already present or makes evident a latent activity.
在分化的上皮细胞中已观察到c-fos和c-jun表达水平升高。然而，关于角质形成细胞分化过程中激活蛋白1（AP-1）的活性尚无相关数据。在这项研究中，我们利用真实的和培养重建的人表皮，研究了角质形成细胞分化过程中c-fos和c-jun基因表达以及AP-1-（12-O-十四烷酰佛波醇-13-乙酸酯）反应增强子元件（TRE）结合活性。我们证明：（i）在重建表皮中，未分化和分化的角质形成细胞表达等量的c-Jun，而在永久移植到无胸腺小鼠的重建表皮以及真实表皮中，c-Jun主要在组织的颗粒层中表达。在重建表皮和真实表皮的所有层中均发现c-fos表达水平相当。（ii）与富含未分化细胞的培养物提取物相比，富含分化角质形成细胞的培养物的核提取物显示AP-1活性降低了80-90%。（iii）从富含分化细胞的培养物中获得的胞质提取物以浓度依赖的方式降低了哺乳动物和果蝇细胞核提取物中的AP-1活性。（iv）分化角质形成细胞的胞质提取物显著降低了重组c-Jun蛋白的特异性TRE结合活性，而纯化的重组人同源蛋白HOX4B的特异性DNA结合活性则未受影响。（v）胞质提取物经碱性磷酸酶去磷酸化后，增强了已有的抑制活性或使潜在活性显现出来。
