In vitro characteristics of early epidermal progenitors isolated from keratin 14 (K14)-deficient mice: insights into the role of keratin 17 in mouse keratinocytes.

Troy TC; Turksen K

摘要

Keratin 14 (K14) is believed to play a pivotal role in the maintenance of epidermal cell shape and contributing to their resistance to mechanical trauma, thereby protecting the cells from lysing. Mice harboring a K14 null mutation produce phenotypic characteristics of epidermolysis bullosa simplex, a skin blistering disease (Lloyd et al., 1995, J Cell Biol 129:1329-1344). K14 null animals die several days after birth, making the detailed study of the consequences of K14 deletion in epidermal cell physiology in vivo particularly difficult. To define the consequences of K14 loss more precisely, we used an in vitro approach by isolating K14-/- cell lines and studying epidermal differentiation in the K14 null background. Several keratinocyte cell lines were generated from 6-day-old mice homozygous for a targeted disruption of the K14 gene (lines designated MKC-5, MKC-23, and MKC-33) and from their wild-type littermates (lines designated MKC-1 and MKC-6). Under low Ca2+ (0.066 mM) and low serum (2%) conditions, both wild-type and mutant cells were able to adhere to collagen type I-coated dishes and form epithelial sheets. They maintained basal epidermal cell characteristics and continued to proliferate without obvious signs of terminal differentiation; however, K14-/- cells proliferated two- to threefold slower than did their wild-type counterparts. The distribution of K5, the natural partner of K14, at the immunofluorescence level was also normal looking in the K14-/- MKC-5 cells, but with fewer filaments detectable, consistent with the approximately 20% reduction in K5 detectable on immunoblots. K17 expression was increased approximately 40% in the K14-/- cells. The levels of K15 and K16 were not different in the MKC-5 and MKC-6 cell lines, suggesting that they are not contributing factors to the stabilization of K5 in the mutant cells. K8, K19, and vimentin were undetectable in both lines. Both MKC-5 and MKC-6 cells underwent morphological and biochemical differentiation in response to a switch to high Ca2+ medium. These findings indicate that K14-/- MKC-5 cells preserve the morphological, biochemical, and physiological characteristics of epidermal cells for an extensive period of time in vitro, likely due to the compensatory expression of K17. The culturing capacity of these cells also permits the analysis of keratinocyte growth and differentiation in the absence of K14. In addition, the culturing methods we describe will be useful for the generation of epithelial cell lines from a wealth of increasingly available knockout mouse strains with early lethality.

机译：角蛋白14 (K14)被认为发挥的关键在表皮细胞形状的维护作用和贡献他们的阻力机械创伤,从而保护细胞从溶解。产生的表型特征表皮松解大疱单工,皮肤起泡疾病(Lloyd et al ., 1995, J细胞129:1329 - 1344)。出生后,使的详细研究K14删除在表皮细胞的后果体内生理特别困难。定义的后果K14损失更多准确地说,我们使用的体外方法隔离K14 - / -细胞系和学习表皮分化K14 null背景。来自6-day-old老鼠的纯合子针对K14基因(线的干扰指定MKC-5、MKC-23 MKC-33)和从野生型的同胞(指定行MKC-1和MKC-6)。低血清(2%)条件下,野生型和突变细胞能够坚持胶原蛋白类型I-coated菜肴并形成上皮表。保持基底上皮细胞特征并继续增殖没有明显终端分化的迹象;K14 - / -细胞扩散两到三倍慢的比野生型同行。速率分布的天然伙伴K14、在免疫荧光级别也是正常的在K14 - / - MKC-5细胞,但较少丝检测,符合在K5检测减少大约20%免疫印迹。大约40%在K14 - / -细胞。MKC-5 K15和K16没有不同的和MKC-6细胞株,表明不稳定的因素K5突变细胞。两行是无法觉察的。MKC-6细胞进行了形态学和生化分化在回答切换到高Ca2 +中。表明K14 - / - MKC-5细胞保存形态学、生物化学和生理表皮细胞的特征广泛的时间体外,可能由于补偿K17的表情。这些细胞还允许分析的能力角化细胞生长和分化的缺乏K14。方法:我们描述将是有用的代的上皮细胞系的财富越来越多的基因敲除老鼠菌株与早期的杀伤力。

In vitro characteristics of early epidermal progenitors isolated from keratin 14 (K14)-deficient mice: insights into the role of keratin 17 in mouse keratinocytes.

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