Enhancement of ATRA-induced cell differentiation by inhibition of calcium accumulation into the endoplasmic reticulum: cross-talk between RAR alpha and calcium-dependent signaling.

Sarco-endoplasmic reticulum calcium ATPase (SERCA) enzymes control calcium-induced cellular activation by accumulating calcium from the cytosol into the endoplasmic reticulum (ER). To better understand the role of SERCA proteins and cellular calcium homeostasis in all-trans retinoic acid (ATRA)-induced differentiation, we investigated the effect of pharmacologic inhibition of SERCA-dependent calcium uptake into the ER on ATRA-induced differentiation of the HL-60 myelogenous and the NB4 promyelocytic cell lines. SERCA inhibitors di-tert-butyl-benzohydroquinone (tBHQ), thapsigargin, and cyclopiazonic acid significantly enhanced the induction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and CD11b marker expression induced by suboptimal concentrations of ATRA (50 nM) in both cell lines. Analysis of cellular calcium homeostasis revealed that a 60% mobilization of the total SERCA-dependent intracellular calcium pool was necessary to obtain enhancement of ATRA-dependent differentiation by tBHQ. Moreover, after 3 days of ATRA treatment in combination with tBHQ, NB4 cells showed a significantly decreased calcium mobilization compared with treatments with tBHQ or ATRA alone, suggesting that enhanced differentiation and calcium mobilization are causally related. Interestingly, several ATRA-resistant NB4-derived cell lines were partially responsive to the differentiation-inducing effect of the combination of the 2 drugs. In addition, we found that retinoic acid receptor alpha (RAR alpha) and PML-RAR alpha proteins are protected from ATRA-induced proteolytic degradation by SERCA inhibition, indicating that cellular calcium homeostasis may interact with signaling systems involved in the control of ATRA-dependent transcriptional activity. By linking calcium to ATRA-dependent signaling, our data open new avenues in the understanding of the mechanisms of differentiation-induction therapy of leukemia.