Takao J, Yudate T, Das A, Shikano S, Bonkobara M, Ariizumi K, Cruz P D
Department of Dermatology, The University of Texas Southwestern Medical Center, and the Dallas Veterans Affairs Medical Center, Dallas, TX 75390, USA.
Br J Dermatol. 2003 Apr;148(4):680-8. doi: 10.1046/j.1365-2133.2003.05285.x.
Nuclear factor-kappaB (NF-kappaB) is a transcription factor involved in a number of signalling pathways in many cell types. NF-kappaB in mice has been implicated as an important regulator of keratinocyte proliferation and differentiation.
To evaluate the role of NF-kappaB in keratinocyte growth in human beings, we examined its expression in keratinocytes both in culture and in situ, and studied the relationship between NF-kappaB activation and the inhibition of keratinocyte proliferation induced by known modulators of keratinocyte growth.
The expression of subunits of the NF-kappaB family was examined in human skin, primary cultured keratinocytes and an immortalized keratinocyte line by immunohistochemistry and reverse transcriptase-polymerase chain reaction analysis. NF-kB activation was examined in keratinocytes treated with various modulating agents by electrophoretic mobility shift assay (for DNA-binding activity) and by immunocytochemistry (nuclear translocation). The proliferative capacity of treated keratinocytes was also examined by 3H-thymidine incorporation, cell cycle analysis, and expression of Ki-67, a nuclear marker for cell proliferation. The involvement of NF-kappaB was assessed using sodium salicylate, which inhibits NF-kappaB activation.
The NF-kappaB subunits, p50, p65, RelB, and c-Rel (but not p52), were detected in keratinocytes and in normal epidermis at mRNA and protein levels. The four subunits were expressed in a cytoplasmic (rather than a nuclear) pattern in both basal and suprabasal keratinocytes. Phorbol myristate acetate (PMA), tumour necrosis factor alpha, and interferon gamma each activated NF-kappaB and inhibited keratinocyte proliferation. Lipopolysaccharide and dexamethasone did not activate NF-kappaB and had the least effect on proliferation. Finally, a high concentration of calcium (Ca2+) and retinoic acid each failed to activate NF-kappaB, but were potent inhibitors of keratinocyte proliferation, respectively. PMA-induced cell cycle arrest of keratinocytes was blocked by pretreatment with sodium salicylate.
NF-kappaB is constitutively expressed in a resting state in both human cultured keratinocytes and the epidermis. Activation of NF-kappaB is required for PMA-induced keratinocyte growth arrest.
核因子-κB（NF-κB）是一种转录因子，参与多种细胞类型中的多条信号通路。小鼠中的NF-κB已被认为是角质形成细胞增殖和分化的重要调节因子。
为了评估NF-κB在人类角质形成细胞生长中的作用，我们检测了其在培养的角质形成细胞和原位角质形成细胞中的表达，并研究了NF-κB激活与已知角质形成细胞生长调节剂诱导的角质形成细胞增殖抑制之间的关系。
通过免疫组织化学和逆转录-聚合酶链反应分析，检测NF-κB家族亚基在人皮肤、原代培养的角质形成细胞和永生化角质形成细胞系中的表达。通过电泳迁移率变动分析（用于DNA结合活性）和免疫细胞化学（核转位）检测用各种调节剂处理的角质形成细胞中NF-κB的激活情况。还用3H-胸腺嘧啶掺入、细胞周期分析以及细胞增殖的核标志物Ki-67的表达检测处理后的角质形成细胞的增殖能力。使用抑制NF-κB激活的水杨酸钠评估NF-κB的参与情况。
在角质形成细胞和正常表皮中，在mRNA和蛋白质水平检测到NF-κB亚基p50、p65、RelB和c-Rel（但不是p52）。这四个亚基在基底和基底上层角质形成细胞中均以细胞质（而非细胞核）模式表达。佛波醇肉豆蔻酸酯乙酸盐（PMA）、肿瘤坏死因子α和干扰素γ各自激活NF-κB并抑制角质形成细胞增殖。脂多糖和地塞米松未激活NF-κB，对增殖的影响最小。最后，高浓度的钙（Ca2+）和视黄酸各自未能激活NF-κB，但分别是角质形成细胞增殖的有效抑制剂。PMA诱导的角质形成细胞细胞周期停滞被水杨酸钠预处理阻断。
NF-κB在人培养的角质形成细胞和表皮中均以静息状态组成性表达。PMA诱导的角质形成细胞生长停滞需要NF-κB的激活。
