Heme-bound iron activates placenta growth factor in erythroid cells via erythroid Krüppel-like factor

Abstract

In adults with sickle cell disease (SCD), markers of iron burden are associated with excessive production of the angiogenic protein placenta growth factor (PlGF) and high estimated pulmonary artery pressure. Enforced PlGF expression in mice stimulates production of the potent vasoconstrictor endothelin-1, producing pulmonary hypertension. We now demonstrate heme-bound iron (hemin) induces PlGF mRNA >200-fold in a dose- and time-dependent fashion. In murine and human erythroid cells, expression of erythroid Krüppel-like factor (EKLF) precedes PlGF, and its enforced expression in human erythroid progenitor cells induces PlGF mRNA. Hemin-induced expression of PlGF is abolished in EKLF-deficient murine erythroid cells but rescued by conditional expression of EKLF. Chromatin immunoprecipitation reveals that EKLF binds to the PlGF promoter region. SCD patients show higher level expression of both EKLF and PlGF mRNA in circulating blood cells, and markers of iron overload are associated with high PlGF and early mortality. Finally, PlGF association with iron burden generalizes to other human diseases of iron overload. Our results demonstrate a specific mechanistic pathway induced by excess iron that is linked in humans with SCD and in mice to markers of vasculopathy and pulmonary hypertension. These trials were registered at www.clinicaltrials.gov as #NCT00007150, #NCT00023296, #NCT00081523, and #NCT00352430.

Figures

Figure 1

Relationship of iron to PlGF expression and death. (A) Elevated serum ferritin level is associated with higher mortality in SCD by ferritin tertile (n = 279; Kaplan-Meier plot, P < .0001; log-rank [Mantel-Cox] test, P < .0001; log-rank test for trend). (B) PIGF plasma concentration varies as a function of ferritin level, a recognized iron overload marker in SCD patients. (C) K562 cells were incubated with 20 µM hemin for 24 hours, and cells were harvested for immunoblot with anti-PlGF. Equivalent amount of protein from control and tested cell lysate were loaded into 4% to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and blotted with antihuman PlGF. PlGF enzyme-linked immunosorbent assay was performed with medium and calculated per million cells. (D) Regular RT-PCR showed that PlGF expression is specifically upregulated by hemin. The oligonucleotides used for PCR span all 7 exons of PlGF mRNA (“Materials and methods”). (E-F) K562 cells were incubated with either indicated concentrations of hemin or same amount of vehicle, and cells were harvested and RNA extracted. cDNA was prepared with use of an Applied Biosciences kit, and quantitative PCR was performed. Time course and concentration dependence of (E,G) heme oxygenase 1 (HO-1) expression as a positive control and (F,H) PlGF expression in response to hemin.

Figure 2

Iron is the key factor in promoting PlGF expression. Iron-containing mesoporphyrin, but not mesoporphyrin alone, stimulates PlGF expression in K562 cells. K562 were grown and treated as in Figure 1. (A-B) HO-1 expression and PlGF expression. (C-D) Quantitative PCR results of HO-1 and PlGF expression from K562 cells treated with 20 µM zinc protoporphyrin (ZnPP) or 20 µM hemin or both. Cells were incubated with ZnPP for 30 minutes before hemin was included. (E) Differentially expressed transcripts in the microarray assay. (F) Quantitative PCR of EKLF from K562 cells treated as indicated.

Figure 3

PlGF and EKLF are expressed simultaneously at high levels in vivo in patients with SCD and expression of PlGF follows EKLF expression in isolated erythroid cells. (A) PlGF and (B) EKLF expression is significantly higher among SCD patients than healthy controls, and (C) expression of PlGF and EKLF is correlated with each other, indicating that PlGF may be the target of EKLF. (D) Erythroid progenitor cells were cultured by a 2-phase approach and treated with EPO, cells were harvested, and quantitative PCR performed to detect the expression of EKLF and PlGF. The results show that EKLF and PlGF are expressed during erythroid development, with EKLF expression preceding that of PlGF. (E-F) EKLF and PlGF expression in isolated human erythroid progenitor cells. Erythroid progenitor cells were cultured, and then EKLF was transfected by electroporation. Cells were harvested on day 5. Overexpression of EKLF leads to increased PlGF expression in erythroid progenitor cells.

Figure 4

EKLF is required for PlGF expression, and iron overload is significantly related to mortality. Enforced EKLF expression in EKLF knockout erythroid cells increases (A) hemoglobin and (B) PlGF expression, whereas EKLF knockout erythroid cells are not able to respond in the same manner, suggesting that EKLF is required for PlGF induction. (C-D) Quantitative PCR results from anti-EKLF ChIP. EK-1 cells were treated as described in “Materials and methods.” Immunoprecipitation was carried out with anti-EKLF. After immunoprecipitation, quantitative PCR was performed to determine the amount of DNA precipitated. (E) HO-1 and (F) PlGF expression in K562 with indicated chemicals. ST638 was incubated with K562 for 30 minutes before hemin was included. (G) Hemin-induced PlGF expression was attenuated by NAC treatment. K562 cells were treated with indicated concentrations of NAC 30 minutes before hemin induction. Cells were harvested after 24 hours, RNA was extracted, and quantitative PCR was performed on cDNA preparations. (H) Patients with HH the most common form of iron overload disease, at the time of initial diagnosis have elevated levels of serum ferritin and PlGF (r = 0.241, P = .06, Spearman correlation). When ferritin levels are lowered by phlebotomy therapy, the PlGF concentration significantly decreases (P = .0013).

Figure 5

Proposed model of the iron overload/EKLF/PlGF/ ET-1 pathway. Erythroblast expression of PlGF is affected by erythroblast hyperplasia induced by erythropoietin as a compensatory response to hemolytic anemia. PlGF expression is augmented in some patients by excess iron via EKLF as a consequence of repeated therapeutic transfusion of red blood cells. PlGF induces expression of endothelin-1, which promotes pulmonary artery vasoconstriction and eventual pulmonary hypertension. Additional factors are also involved in dysregulation of endothelin-1 expression, such as expression of inflammatory cytokines, and several more pathways contribute to the development of pulmonary hypertension.