Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

<div><p>Siderocalin (also lipocalin 2, NGAL or 24p3) binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid) substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.</p> </div

Stably-induced expression of Scn does not drive apoptosis in FL5.12 cells.

<p>(A) FL5.12 cells were transduced with the pCVL-SFFV-muScn-IRES-GFP lentivirus and GFP mean fluorescence intensity was determined one-week post-transduction by cytometry, confirming transgene functionality. (B) A Western blot of supernatants, concentrated from 32 µL, from FL5.12 cells shows that the transduced cells constitutively express Scn, while parental cells in the presence or absence of IL-3 do not secrete detectable amounts of Scn after 72 h in culture. (C) Transduced FL5.12 were incubated with a variety of siderophores in order to assess the role of exogenous siderophores on cell viability (NT: no treatment). The hexadentate chelators DFO and Ent at 100 µM produce robust apoptosis, while the bidentate chelators at 300 µM do not affect viability.</p

Added Scn does not affect the expression of iron responsive genes.

<p>Expression of <i>H-ferritin</i> (<i>FTH-1</i>) and <i>NDRG1</i> in HeLa/X7 and HeLa/24p3R-L cells was assayed by RT-PCR (A) and Western blot (B). Cells were untreated or treated with 2 µM murine Scn or DFO (100 µM or 250 µM) for 24 h. Densitometry results (right) were calculated relative to β-actin; error was calculated from the standard deviation among three experiments; a typical result from three experiments is shown in (A) and (B).</p

Scn has no effect on iron release or iron uptake from HeLa cells.

<p>Control HeLa/X7 (transfected with empty vector) or HeLa/24p3R-L cells were labeled with either (A) 2.5 µM ⁵⁹FeCl₃ or (B) 0.75 µM ⁵⁹FeTf and re-incubated with 2 µM murine Scn or control medium for 5 h (dotted columns) or 24 h (checked columns); 100 µM DFO was used as a positive control. Expression of <i>BOCT</i> in transfected HeLa/24p3R-L cells was confirmed by RT-PCR (C). In (C), a typical result from three experiments is shown. In (D), control HeLa/X7 (white columns) and HeLa/24p3R-L cells (black columns) were incubated for 4 h in the presence of 2 µM ⁵⁹FeCl₃, 2 µM ⁵⁹FeEnt, 2 µM murine Scn with bound ⁵⁹FeEnt (⁵⁹FeEnt+Scn) or in the presence of 2 µM ⁵⁹FeEnt plus 2 µM human albumin (⁵⁹FeEnt+Alb). Internalized ⁵⁹Fe was determined by γ-counting. Albumin was added in (D) as an additional control for non-specific binding. Error was calculated as the standard deviation among three experiments.</p

Steric clashes imposed by the Scn calyx preclude binding of ferric SA and GA complexes.

<p>(A) Hexadentate siderophore structures are shown with iron liganding atoms colored blue. (B) Structures of 2,3-DHBA, GA (2,5-DHBA), 3,4-DHBA and SA (2-hydroxybenzoic acid) are shown in the left column and complexes with iron in the center column (only two of three bidentate groups are shown for clarity). The Scn calyx is represented at top by a gray cylinder and the size constraint imposed by the calyx diameter is represented by dashed lines, schematically showing clashes with all iron complexes except 2,3-DHBA. (C) A section of the Scn/carboxymycobactin complex structure (PDB accession code 1X89) showing a GA moiety superimposed on the phenolate ring of carboxymycobactin. The steric clash of the 5-OH is indicated by penetrating the molecular surface of Scn (dashed red circle) and the short distance to neighboring atoms (green line).</p

BOCT N-and C-terminal domains do not bind Scn.

<p>Predicted BOCT membrane topologies are shown, either as determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043696#pone.0043696-Devireddy2" target="_blank">[15]</a> (A) or calculated here (B), with transmembrane-spanning helices shown as blue cylinders. The sequence lengths of the NTD (green), CTD (red) and connecting loops are indicated; loops synthesized as peptides for binding analyses are indicated with numbered black circles, corresponding to the numbering in the <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043696#s4" target="_blank">Materials & Methods</a></b> section. The amino termini of fragments used to originally identify BOCT as a Scn receptor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043696#pone.0043696-Devireddy2" target="_blank">[15]</a> are indicated with orange arrows in (A). PAGE analyses of bacterially-expressed soluble, purified NTD (C) and CTD (D) are shown. SEC analysis of NTD/Scn is shown in (E). Complex formation would have been indicated by a shift in the Scn+NTD peak to lower elution volumes; in this case, the Scn/NTD mixture runs as the simple summation of the Scn and NTD alone peaks, indicating no binding under these conditions. (F) SPR analysis of Scn/CTD binding, with Scn analyte concentrations indicated. The bar indicates the sample injection period (association phase); gaps in the sensorgrams cover transients associated with injections.</p

Scn does induce apoptosis in murine 32D.3 or FL5.12 cells.

<p>FL5.12 (A) and 32D.3 (C) cells were incubated with 10 µM Scn and DFO for 48 h (NT: no treatment; -IL-3: in the absence of added IL-3). Apoptosis was assayed by annexin V-FITC staining and DAPI was used as a vital stain; percentages of cells positive for annexin staining are indicated. Average annexin V-positivity from three independent experiments are shown for FL5.12 (B) and 32D.3 (D) cells; error was calculated as the standard deviation of three replicates. Statistical significance is indicated as *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. Note that while the effect of adding Scn was significant, the effect was <i>anti</i>-apoptotic.</p

An anti-Scn antibody does not block IL-3 withdrawal-induced apoptosis.

<p>(A) 32D.3 and FL5.12 cells in the presence (NT) or absence (-IL3) of IL-3 were incubated for 48 h with 10 µM of the anti-Scn antibody MAB1857; percent annexin-V positivity is indicated. (B) The structure of Fab MAB1857 with Scn, shown in a ribbon representation (Fab in gray and Scn in orange), reveals the interface that is occluded in the complex. Had Scn had an effect on apoptosis through receptor-mediated uptake, the effect of the antibody on the process would have identified a potential receptor-interacting surface on Scn, the rationale for this approach. However, since Scn does not affect apoptosis, an anti-Scn antibody cannot reveal a receptor-interacting surface by blocking a non-existent effect.</p