FoxA1 regulates sweat secretion through Best2 and Nkcc1 ion transporters

Body temperature is maintained in a narrow range in mammals, primarily controlled by sweating. In humans, the dynamic thermoregulatory organ, comprised of 2-4 million sweat glands distributed over the body, can secrete up to 4 liters of sweat per day1, thereby making it possible to withstand high temperatures and run long distances. The genetic basis for sweat gland function, however, is largely unknown. We find that a forkhead transcription factor, FoxA1, is required to generate mouse sweating capacity. When FoxA1 is ablated, mice are otherwise healthy and sweat gland morphogenesis occurs, but no sweating ensues, with the Nkcc1 sodium/potassium/chloride co-transporter and a specialized Ca2+-activated bicarbonate channel protein, Best2, both sharply down-regulated, and glycoprotein accumulating in gland lumens and ducts. Furthermore, Best2 knockout mice display comparable anhidrosis and glycoprotein accumulation. These findings link earlier observations that both sodium/potassium/chloride exchange and Ca2+ are required for sweat production. FoxA1 is inferred to regulate two corresponding features of sweat secretion. One, via Best2, catalyzes a bicarbonate gradient that could help to drive calcium-associated ionic transport; the other, requiring Nkcc1, facilitates monovalent ion exchange into sweat. These mechanistic components can be pharmaceutical targets to defend against hyperthermia and alleviate defective thermoregulation in the elderly2, and may provide a model relevant to more complex secretory processes. For expression profiling of FoxA1, hairless fore footpad skin (6) was collected from FoxA1 knockouts and wild-type littermates at P10, P14 and P31. Three skin samples from 3 embryos for each genotype at each time point were used for biological replicates. Total RNAs were isolated with Trizol (Invitrogen), precipitated by 7.5M LiCl (Ambion), and cyanine-3-labeled cRNAs were hybridized to the NIA Mouse 44K Microarray v3.0 (Agilent Technologies). Triplicate data were analyzed by ANOVA (6). Genes with FDR<0.05, fold difference>1.5 and mean log intensity>2.0 were considered to be significant.哺乳动物的体温始终维持在狭窄的生理范围内，该过程主要通过出汗实现调控。在人体中，作为动态体温调节核心器官的皮肤，全身分布着200万至400万个汗腺，每日可分泌多达4升汗液¹，这使得人类能够耐受高温环境并完成长距离奔跑。然而，目前关于汗腺功能的遗传基础仍知之甚少。 本研究发现，叉头框转录因子FoxA1（forkhead transcription factor FoxA1）是小鼠汗腺分泌功能得以建立的必需因子。当FoxA1被敲除后，小鼠整体健康状态未受明显影响，汗腺形态发生过程亦可正常完成，但小鼠无法产生汗液；此时钠钾氯协同转运蛋白Nkcc1（sodium/potassium/chloride co-transporter Nkcc1）以及特异性钙激活碳酸氢盐通道蛋白Best2（specialized Ca²⁺-activated bicarbonate channel protein Best2）的表达均显著下调，且糖蛋白在腺泡腔与导管中出现异常蓄积。 进一步实验显示，Best2敲除小鼠同样表现出相似的无汗症表型及糖蛋白异常蓄积现象。上述发现印证了此前的研究结论——钠钾氯交换与钙离子信号均为汗液生成所必需的核心环节。我们推断FoxA1通过两类对应机制调控汗液分泌：其一，通过Best2催化形成碳酸氢盐梯度，以助力钙依赖的离子转运过程；其二，依赖Nkcc1促进单价离子向汗液中的转运。这些机制相关的分子组分可作为防治体温过高并改善老年人体温调节障碍的药物靶点，同时也可为更复杂的分泌过程研究提供极具参考价值的模型。 为开展FoxA1的表达谱分析，我们分别在出生后第10天（P10）、第14天（P14）及第31天（P31）三个时间点，从FoxA1敲除小鼠及其野生型同窝仔鼠的无毛前足垫皮肤中采集样本⁶。每个基因型在每个时间点均使用3个胚胎来源的皮肤样本作为生物学重复。总RNA通过Trizol（Invitrogen）试剂提取，经7.5M氯化锂（LiCl，Ambion）沉淀后，将花青-3（cyanine-3）标记的互补RNA（cRNAs）与NIA小鼠44K微阵列v3.0（安捷伦科技（Agilent Technologies））进行杂交。采用方差分析（ANOVA）对三次重复数据进行分析⁶，筛选标准为错误发现率（FDR）<0.05、倍数变化>1.5且平均对数强度>2.0的基因。