Jakab Martin, Hofer Sabine, Ravasio Andrea, Huber Felix, Schmidt Sabine, Hitzl Wolfgang, Geibel John P, Fürst Johannes, Ritter Markus
Institute of Physiology and Pathophysiology, Laboratory of Functional and Molecular Membrane Physiology, Paracelsus Medical University Salzburg, Salzburg, Austria.
Cell Physiol Biochem. 2014;34(5):1507-26. doi: 10.1159/000366355. Epub 2014 Oct 9.
BACKGROUND/AIMS: The ATP12A gene codes for a non-gastric H(+)/K(+) ATPase, which is expressed in a wide variety of tissues. The aim of this study was to test for the molecular and functional expression of the non-gastric H(+)/K(+) ATPase ATP12A/ATP1AL1 in unstimulated and butyrate-stimulated (1 and 10 mM) human myelomonocytic HL-60 cells, to unravel its potential role as putative apoptosis-counteracting ion transporter as well as to test for the effect of the H(+)/K(+) ATPase inhibitor SCH28080 in apoptosis.
Real-time reverse-transcription PCR (qRT-PCR) was used for amplification and cloning of ATP12A transcripts and to assess transcriptional regulation. BCECF microfluorimetry was used to assess changes of intracellular pH (pHi) after acute intracellular acid load (NH4Cl prepulsing). Mean cell volumes (MCV) and MCV-recovery after osmotic cell shrinkage (Regulatory Volume Increase, RVI) were assessed by Coulter counting. Flow-cytometry was used to measure MCV (Coulter principle), to assess apoptosis (phosphatidylserine exposure to the outer leaflet of the cell membrane, caspase activity, 7AAD staining) and differentiation (CD86 expression).
We found by RT-PCR, intracellular pH measurements, MCV measurements and flow cytometry that ATP12A is expressed in human myelomonocytic HL-60 cells. Treatment of HL-60 cells with 1 mM butyrate leads to monocyte-directed differentiation whereas higher concentrations (10 mM) induce apoptosis as assessed by flow-cytometric determination of CD86 expression, caspase activity, phosphatidylserine exposure on the outer leaflet of the cell membrane and MCV measurements. Transcriptional up-regulation of ATP12A and CD86 is evident in 1 mM butyrate-treated HL-60 cells. The H(+)/K(+) ATPase inhibitor SCH28080 (100 µM) diminishes K(+)-dependent pHi recovery after intracellular acid load and blocks RVI after osmotic cell shrinkage. After seeding, HL-60 cells increase their MCV within the first 24 h in culture, and subsequently decrease it over the course of the next 48 h. This effect can be observed in the overall- and non-apoptotic fraction of both untreated and 1 mM butyrate-treated HL-60 cells, but not in 1 mM butyrate-stimulated phosphatidylserine-positive cells. These cells do not shrink from 24 h to 72 h and have finally a higher MCV than untreated cells unless they are exposed to SCH28080. 10 mM butyrate induces apoptosis within 24 h.
In summary we show that in HL-60 cells ATP12A is a functionally active H(+)/K(+) ATPase that may counteract events during early apoptosis like intracellular acidosis, loss of intracellular K(+) ions and apoptotic volume decrease. Its expression and/or susceptibility to the H(+)/K(+) ATPase inhibitor SCH28080 becomes most evident in cells exposing phosphatidylserine on the outer leaflet of the cell membrane and therefore during early apoptosis.
背景/目的：ATP12A基因编码一种非胃H(+)/K(+)ATP酶，其在多种组织中表达。本研究的目的是检测非胃H(+)/K(+)ATP酶ATP12A/ATP1AL1在未刺激的和丁酸盐刺激（1和10 mM）的人骨髓单核细胞HL-60细胞中的分子和功能表达，以阐明其作为假定的抗凋亡离子转运体的潜在作用，并检测H(+)/K(+)ATP酶抑制剂SCH28080对细胞凋亡的影响。
实时逆转录PCR（qRT-PCR）用于ATP12A转录本的扩增和克隆，并评估转录调控。BCECF微量荧光测定法用于评估急性细胞内酸负荷（氯化铵预脉冲）后细胞内pH（pHi）的变化。通过库尔特计数评估平均细胞体积（MCV）和渗透细胞收缩后的MCV恢复（调节性容积增加，RVI）。流式细胞术用于测量MCV（库尔特原理），评估细胞凋亡（磷脂酰丝氨酸暴露于细胞膜外小叶、半胱天冬酶活性、7-AAD染色）和分化（CD86表达）。
通过RT-PCR、细胞内pH测量、MCV测量和流式细胞术，我们发现ATP12A在人骨髓单核细胞HL-60细胞中表达。用1 mM丁酸盐处理HL-60细胞导致单核细胞定向分化，而更高浓度（10 mM）诱导细胞凋亡，这通过流式细胞术测定CD86表达、半胱天冬酶活性、磷脂酰丝氨酸在细胞膜外小叶的暴露和MCV测量来评估。在1 mM丁酸盐处理的HL-60细胞中，ATP12A和CD86的转录上调明显。H(+)/K(+)ATP酶抑制剂SCH28080（100 μM）减少细胞内酸负荷后K(+)依赖性pHi恢复，并在渗透细胞收缩后阻断RVI。接种后，HL-60细胞在培养的最初24小时内增加其MCV，随后在接下来的48小时内降低。这种效应在未处理的和1 mM丁酸盐处理的HL-60细胞的总体和非凋亡部分中都可以观察到，但在1 mM丁酸盐刺激的磷脂酰丝氨酸阳性细胞中没有观察到。这些细胞从24小时到72小时不收缩，最终具有比未处理细胞更高的MCV，除非它们暴露于SCH28080。10 mM丁酸盐在24小时内诱导细胞凋亡。
总之，我们表明在HL-
