Blimp-1在角質形成細胞和癌細胞中的調節和細胞功能

Abstract

B淋巴細胞誘導的成熟蛋白-1 (Blimp-1）是一種轉錄抑制因子，在調節各種免疫細胞的發育和功能中起著至關重要的作用。目前，關於角質形成細胞和癌細胞中Blimp-1表達和細胞功能的調節的理解有限。在這項研究中，我們發現EGF、PMA、TGF-β、TNF-、H2O2、UVB和TLR激活劑（LPS、polyIC、CpG）可以上調HaCaT角質形成細胞及鱗狀細胞癌(Cal-27和SAS)中Blimp-1的蛋白質和mRNA水平。儘管這些刺激物可以同時激活細胞中的EGFR，但不是所有刺激物誘導Blimp-1表達的作用與EGFR相關。研究顯示PMA在Cal-27細胞及TGF-β在前列腺癌細胞PC3及 LNCaP的作用與內生性EGFR的活化有關，相反的，EGFR的抑制劑易瑞沙 (Iressa) 並未抑制PMA和TNF-在HaCaT引起的Blimp-1誘導作用，也不抑制TNF-α 和TGF-β分別在Cal-27 及 SAS細胞的作用。另一方面，Syk抑制劑可降低PMA在HaCaT細胞中誘導的Blimp-1基因表達，而不影響TNF-的作用。於Blimp-1報告基因的分析得知，AP-1參與EGF刺激HaCaT細胞的Blimp-1基因表達。共軛顯微鏡之研究結果顯示Blimp-1依不同細胞可以表現在細胞核及細胞質中，而SAS細胞在PMA及TNF-α刺激下，Blimp-1會由細胞質轉移到細胞核中。此外，Blimp-1基因靜默時會增強角質細胞和癌細胞的遷移。所有這些結果顯示Blimp-1基因可以受各種刺激劑而增加基因及蛋白表現，且在細胞遷移中起負面的調節作用。值得注意的是，TGF-β活化EGFR的作用方式與其他的刺激劑不同，TGF-β的作用是以增加EGFR的蛋白量所致，而其他的刺激劑則是直接對EGFR產生transactivation。功能研究表明Blimp-1基因靜默不影響細胞存活，但增加癌細胞的增生，促進TGF-β刺激細胞的遷移、侵入及上皮細胞間質轉化。總之，多種刺激劑可以誘導Blimp-1的基因表現，而在某些細胞種類及刺激條件下需要內生性的EGFR活性，且Blimp-1在角質形成細胞和癌細胞中扮演負向調節細胞遷移的角色。

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor, and plays a crucial role in the regulation of development and functions of various immune cells. Currently, there is limited understanding about the regulation of Blimp-1 expression and cellular functions in keratinocytes and cancer cells. In this study, we found that EGF, PMA, TGF-β, TNF-α, H2O2, UVB, and TLR ligands (LPS, polyIC and CpG) can upregulate the protein and mRNA levels of Blimp-1 in HaCaT keratinocytes and/or Cal-27 and SAS squamous cell carcinoma (SCC). Even though all of these stimuli can transactivate EGFR, not all stimuli-induced Blimp-1 upregulation depend on the constitutive EGFR activity. We found that the Blimp-1 responses in PMA-activated Cal-27 cells as well as in TGF-β-activated prostate cancer PC3 and LNCaP cells were inhibited by iressa. In contrast, iressa did not inhibit the Blimp-1 induction responses of PMA and TNF-α in HaCaT cells nor those of TNF-α and TGF-β in Cal-27 and SAS cells, respectively. On the other hands, Syk inhibitor can reduce PMA-, but not TNF-α-induced Blimp-1 gene expression in HaCaT and Cal-27 cells. Data of reporter assay indicate that AP-1 is involved in Blimp-1 gene expression in EGF-stimulated HaCaT keratinocytes. Confocal microscopic data revealed that Blimp-1 is localized in the nuclei and cytosol depending on cell types, and can be translocated from the cytosol to the nuclei in PMA- and TNF-α-stimulated SAS cells. Furthermore, Blimp-1 silencing enhances keratinocytes and cancer cell migration. All these findings suggest that Blimp-1 gene expression can respond to various stimuli and Blimp-1 plays a negative role in cell migration. Of note, different from other stimuli which directly transactivates EGFR activity, the effects of TGF-β in increasing EGFR activity in SAS, PC3 and LNCaP cells are resulting from the upregulation of EGFR protein. Functional study revealed that silencing Blimp-1 can not only increase cell proliferation, but also accelerate cell migration and TGF-β-induced EMT in prostate cancer cells. In conclusion, Blimp-1 can be upregulated via gene transcription by various stimuli, and in some cases depending on the EGFR activity. Blimp-1 may act as a brake on keratinocyte and cancer cell migration.