Abstract
Native human HL-60 cells do not express CD38, a multifunctional ectoenzyme, which generates cyclic ADP-ribose (cADPR), a potent calcium mobilizer. However, when HL-60 cells are induced to differentiate to granulocytes by treatment with retinoic acid (RA), they express CD38 and accumulate cADPR. Both processes play a causal role in RA-induced differentiation. Other granulocyte differentiation-inducers, including dimethyl sulfoxide (Me(2)SO), fail to induce CD38 expression. We investigated whether treatment of HL-60 cells with Me(2)SO involves any changes in the cADPR/intracellular calcium ([Ca(2+)](i)) signaling system and, specifically, whether Me(2)SO affects those nucleoside transporters (NT) (both equilibrative (ENT) and concentrative (CNT)) that mediate influx of extracellular cADPR. Semiquantitative polymerase chain reaction analysis of transcripts, binding of [(3)H]nitrobenzylthioinosine (NBMPR) to intact cells, and influx experiments of extracellular cADPR (with selective inhibitors of NT as NBMPR or in specific conditions) were performed in native and Me(2)SO-differentiated HL-60 cells. The native cells showed uptake of cADPR across ENT2, whereas influx of cADPR into the Me(2)SO-differentiated cells occurred mostly by concentrative processes mediated by CNT3 and by an NBMPR-inhibitable concentrative NT designated cs-csg. Me(2)SO-differentiated, but not native HL-60 cells, accumulated cADPR and showed increased [Ca(2+)](i) levels when grown in a transwell co-culture setting over CD38-transfected 3T3 fibroblasts where nanomolar cADPR concentrations are present in the medium. NBMPR inhibited both responses of Me(2)SO-induced cells. Thus, concentrative influx of extracellular cADPR across CNT3 and cs-csg NT could substitute in the absence of CD38 in eliciting cADPR-dependent [Ca(2+)](i) increases in granulocyte-differentiated HL-60 cells, as well as in other CD38(-) cells.
Highlights
We investigated whether treatment of HL-60 cells with Me2SO involves any changes in the cyclic ADP-ribose (cADPR)/intracellular calcium ([Ca2؉]i) signaling system and, whether Me2SO affects those nucleoside transporters (NT) (both equilibrative (ENT) and concentrative (CNT)) that mediate influx of extracellular cADPR
Both CD38 and BST-1, sharing significant sequence identity, feature two important properties [9]. (i) They are multifunctional enzymes able to generate cADPR and to hydrolyze cADPR to ADP-ribose. (ii) Both are ectoenzymes. The latter property has prompted studies aimed at defining two major topological inconsistencies of the CD38/cADPR system. (i) The apparent unavailability of the substrate NADϩ to the ectocellular active site of CD38 and (ii) generation of cADPR at sites opposite to cADPR-responsive Ca2ϩ stores and gated by the ryanodine receptors (RyR). These investigations led to the discovery that across ENT2, whereas influx of cADPR into the Me2SO- the CD38/cADPR system is a remarkably compartmentalized differentiated cells occurred mostly by concentrative pro- one and that a number of previously unrecognized transporters cesses mediated by CNT3 and by an NBMPR-inhibitable can translocate NADϩ and cADPR, thereby circumventing both concentrative NT designated cs-csg
HL-60 cells induced to differentiate by treatment with both Me2SO and phorbol 12-myristate 13-acetate (PMA) exhibit a decrease of equilibrative nucleoside transport [30, 38], accompanied by an increase in the rate of concentrative Naϩ-dependent transport of nucleosides, the extent of which correlates with enhanced CNT3 expression in PMA-differentiated HL-60 cells [30]
Summary
(i) The apparent unavailability of the substrate NADϩ to the ectocellular active site of CD38 and (ii) generation of cADPR at sites opposite to cADPR-responsive Ca2ϩ stores and gated by the ryanodine receptors (RyR) (either the extracellular environment or the intravesicular space of intracellular membrane vesicles, Ref. 14) These investigations led to the discovery that across ENT2, whereas influx of cADPR into the Me2SO- the CD38/cADPR system is a remarkably compartmentalized differentiated cells occurred mostly by concentrative pro- one and that a number of previously unrecognized transporters cesses mediated by CNT3 and by an NBMPR-inhibitable can translocate NADϩ and cADPR, thereby circumventing both concentrative NT designated cs-csg. ADP-ribosyl cyclases play a major role in regulating intracellular calcium levels ([Ca2ϩ]i) by converting NADϩ to nicotinafamily of nucleoside transporters, both equilibrative (ENT) and concentrative (CNT) Among these promiscuous transporters, ENT2, CNT2, and a not yet molecularly characterized nitrobenzylthioinosine (NBMPR)-inhibitable nucleo-. Side transporting system proved to mediate translocation of cADPR in CD38Ϫ cells, independent of the concentrative transport catalyzed by CD38 [19]
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