Cellular zinc homeostasis is a regulator in monocyte differentiation of HL-60 cells by 1α,25-dihydroxyvitamin D3

Abstract

It was reported previously that zinc-deficient mice show impaired lymphopoiesis. At the same time, monocyte numbers in these animals are increased, indicating a negative impact of zinc on monocyte development. Here, we investigate the role of zinc homeostasis in the differentiation of myeloid precursors into monocytes. Reduced gene expression of several zinc transporters, predominantly from the Zip family, was observed during 1 alpha, 25-dihydroxyvitamin D(3) (1,25D(3))-induced differentiation of HL-60 cells. This was accompanied by a reduction of intracellular-free zinc, measured by FluoZin-3. Amplifying this reduction with the zinc chelator TPEN or zinc-depleted cell-culture medium enhanced 1,25D(3)-induced expression of monocytic surface markers CD11b and CD14 on HL-60, THP-1, and NB4 cells. In contrast, differentiation of NB4 cells to granulocytes was not zinc-sensitive, pointing toward a specific effect of zinc on monocyte differentiation. Further, monocyte functions, such as TNF-alpha secretion, phagocytosis, and oxidative burst, were also augmented by differentiation in the presence of TPEN. The second messenger cAMP promotes monocyte differentiation. We could show that zinc inhibits the cAMP-synthesizing enzyme adenylate cyclase, and chelation of zinc by TPEN increases cAMP generation after stimulation with the adenylate cyclase activator forskolin. Based on our in vitro results and the in vivo observations from the literature, we suggest a model in which the intracellular-free zinc concentration limits AC activity, and the decrease of zinc after 1,25D(3) treatment promotes differentiation by relieving AC inhibition. Thus, cellular zinc homeostasis acts as an endogenous modulator of monocyte differentiation.

Differential impact of zinc deficiency on phagocytosis, oxidative burst, and production of pro-inflammatory cytokines by human monocytes

“…Calcium and magnesium were routinely added back to CHELEX-treated medium. 11 Still, magnesium levels were slightly below those of the control, but this seems to be only a minor difference. Moreover, the findings presented in Table 2 differ from the literature with regard to the removal of several other elements, indicating that different protocols can lead to differential depletion of trace elements from cell culture media.…”

“…16 In contrast, CHELEX removes metal ions from the culture medium and calcium and magnesium have to be reconstituted subsequently. 11 Specificity is achieved by adding zinc back to CHELEX-treated medium, making the zinc concentration the only difference between the two kinds of media. 17,18 In Fig.…”

“…This was followed by addition of 20 ml of PBS containing 2 Â 10 7 bacteria transformed to express fluorescent proteins; either DSred-E. coli (strain BL21), 11 GFP-S. aureus (strain ATCC29213) 14 or GFP-S. pneumoniae (strain R6). 15 Additionally, redox sensitive pro-fluorophores were added, either DHR123 (1 mM) for DsRed-E. coli or DHE (2.5 mM) for bacteria expressing GFP, respectively.…”

Differential Regulation of TLR-Dependent MyD88 and TRIF Signaling Pathways by Free Zinc Ions

“…It is conceivable that these enzymes also have a similar reactivity toward Zn 2+ , making them potential molecular targets for an effect of Zn 2+ in addition to PTPs. Dendritic cell maturation in response to LPS and monocytic differentiation of myeloid precursors stimulated by calcitriol both depend on alterations of cellular zinc homeostasis, ultimately leading to reduced free Zn 2+ (20,47). This shows that Zn 2+ exerts regulatory functions not only through short-term spikes, but also via modulation of basal Zn 2+ levels.…”

Bone marrow increases human islets insulin positive cells in co-culture: quantification with flow cytometry

“…This method uses FluoZin-3 staining to reveal higher amounts of chelatable zinc ions in β-cells [18]. However, we did not successfully detect positive FluoZin-3 in dissociated monolayer islet cells in long term culture in vitro because this method also stained some kind bone marrow cell like monocyte [19] and T cell [20]. So this method will result false positive β-cells in islet and bone marrow co-culture.…”