The Putative Role of the Non-Gastric H⁺/K⁺-ATPase ATP12A (ATP1AL1) as Anti-Apoptotic Ion Transporter: Effect of the H⁺/K⁺ATPase Inhibitor SCH28080 on Butyrate-Stimulated Myelomonocytic HL-60 Cells

Abstract

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. Methods: 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 (pH_i) 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). Results: 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 pH_i 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. Conclusion: 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.

The transcriptional profiling of human in vivo-generated plasma cells identifies selective imbalances in monoclonal gammopathies

“…The only gene specifically up-regulated in BMPC is ATP12A . Its product, an H + /K + ATPase with proton pump activity, has been demonstrated recently to act as an anti-apoptotic factor in human myelomonocytic cells [ 68 ]. Whether or not this is the function of ATP12A in human BMPC remains to be proven.…”

The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells

“…We also tested SCH-28080 on RIN-5F rat pancreatic islet cells and NIH-3T3 fibroblasts: in RIN-5F cells, the dose-response curve was shifted towards higher concentrations with significantly reduced viability ≥50 µM, whereas in NIH-3T3 fibroblasts the drug had no effect on cell viability within 24 h (data not shown). The viability of myelomonocytic HL-60 cells was also not affected by the drug up to a concentration of 100 µM [17]. Whether SCH-28080 acts specifically on β-cells, however, needs continuative studies on further insulinoma cell lines and primary β-cells.…”

“…A gravity-driven perfusion system (ALA Scientific Instruments) with a flow-rate of 3–5 ml/min was used for extracellular solution exchange. NH 4 Cl prepulsing experiments were performed to test for the cells’ ability to recover from an acute acid load as described earlier [17]. Briefly, after achieving stable baseline signals under 135 Na + /5.6 K + control conditions, cells were exposed for ∼5 min to a solution containing 20 mM NH 4 Cl.…”

“…Previously, we have identified a role for H + /K + ATPases in cell migration and apoptosis – processes which are also dependent on pH i and cell volume regulation [16, 17]. H + /K + ATPases comprise the gastric and non-gastric isoforms.…”

“…In prostate tissue we demonstrated deranged protein sorting in benign hyperplasia and protein upregulation in prostate cancer [26]. In HL-60 cells ATP12A seems to exert an anti-apoptotic function by counteracting apoptotic cation gradient reversal during apoptotic volume decrease [17]. …”