Implication of extracellular zinc exclusion by recombinant human calprotectin (MRP8 and MRP14) from target cells in its apoptosis‐inducing activity

Abstract

BACKGROUND: Calprotectin is a calcium-binding and zinc-binding protein complex that is abundant in the cytosol of neutrophils. This factor is composed of 8 and 14 kDa subunits, which have also been termed migration inhibitory factor-related proteins MRP8 and MRP14. We previously reported that rat calprotectin purified from inflammatory neutrophils induces apoptosis of various tumor cells or normal fibroblasts in a zinc-reversible manner. AIM: The present study was undertaken to elucidate which subunit is responsible for the apoptosis-inducing activity, and to explore the mechanism of zinc-reversible apoptosis induction. METHODS: The apoptosis-inducing activity of recombinant human MRP8 (rhMRP8) and recombinant human MRP14 (rhMRP14) was examined against EL-4 lymphoma cells in vitro. To determine whether zinc deprivation by calprotectin was essential for the cytotoxicity, the activity of calprotectin was tested under conditions where physical contact between the factor and the cells was precluded by a low molecular weight cut-off dialysis membrane. RESULTS: The cytotoxicity of rhMRP14 against EL-4 cells was first detected at 10 microM in a standard medium, whereas rhMRP8 caused only marginal cytotoxicity at 40 microM. A mixture of both proteins showed higher specific activity (onset of cytotoxicity at 5 microM). When the cells were cultured in divalent cation-depleted medium, each dose-response curve was shifted to about a four-fold lower concentration range. Calprotectin was found to induce cell death even when the complex and the target cells were separated by dialysis membrane. A membrane-impermeable zinc chelator, diethylenetriamine pentaacetic acid (DTPA), also induced target cell apoptosis in a similar time-course as calprotectin. Moreover, the activities of calprotectin and DTPA were completely inhibited by the presence of zinc ions. CONCLUSION: These data indicate that calprotectin has higher specific activity to induce apoptosis than the Individual subunits, and that the mechanism is exclusion of zinc from target cells.

Calprotectin (S100A8/S100A9), an Inflammatory Protein Complex from Neutrophils with a Broad Apoptosis-Inducing Activity

“…60) As a result, the cytotoxicity of the 14 kD subunit was detected from 10 mM, whereas that of the 8 kD subunit was marginal at the same concentration. On the other hand, the mixture of both induced almost complete cell death at 5 mM: calprotectin has higher specific activity than either subunit individually.…”

“…[66][67][68] To examine this possibility, the cytotoxic activity of calprotectin against EL-4 cells was tested under conditions where physical contact between the factor and the cells was precluded by a dialysis membrane. 60) As the result, the factor induced cell death even when contact was hindered. The membrane-impermeable zinc cheletor, Diethylenetriaminepentaacetic acid (DTPA), also induced cell death in a similar way to calprotectin.…”

Mechanism of apoptosis induced by S100A8/A9 in colon cancer cell lines: the role of ROS and the effect of metal ions

“…DTPA, the membrane-impermeable metal ion chelator, induces apoptosis through the depletion of extracellular zinc ion [24]. S100A8/A9 also binds zinc with high affinity, and it has been reported that the antimicrobial activity and the cell death (apoptosis)-inducing activity of S100A8/A9 are inhibited by the presence of zinc [10,24,38].…”

“…DTPA, the membrane-impermeable metal ion chelator, induces apoptosis through the depletion of extracellular zinc ion [24]. S100A8/A9 also binds zinc with high affinity, and it has been reported that the antimicrobial activity and the cell death (apoptosis)-inducing activity of S100A8/A9 are inhibited by the presence of zinc [10,24,38]. However, based on the observations that Zn 2ϩ and Cu 2ϩ did not completely reverse the apoptotic effect of S100A8/A9; the changes in the morphology of both cell lines treated by S100A8/A9 were different from those observed with DTPA; and the time-course of S100A8/A9-induced apoptosis differed from that induced by DTPA, we conclude that S100A8/A9 induces apoptosis by a mechanism that is not simple as a result of zinc sequestration.…”

“…It is interesting that one putative zincbinding site has been associated with the specific binding of polyunsaturated fatty acids, another feature of S100A8/A9 [21][22][23]. Moreover, it has been reported that antimicrobial activity and cell death (apoptosis)-inducing activity of S100A8/A9 are inhibited in the presence of zinc [10,24].…”