Scara5 is a ferritin receptor mediating non-transferrin iron delivery

Abstract

Developing organs require iron for a myriad of functions, but embryos deleted of the major adult transport proteins, transferrin or its receptor transferrin receptor1 (TfR1(-/-)), still initiate organogenesis, suggesting that non-transferrin pathways are important. To examine these pathways, we developed chimeras composed of fluorescence-tagged TfR1(-/-) cells and untagged wild-type cells. In the kidney, TfR1(-/-) cells populated capsule and stroma, mesenchyme and nephron, but were underrepresented in ureteric bud tips. Consistently, TfR1 provided transferrin to the ureteric bud, but not to the capsule or the stroma. Instead of transferrin, we found that the capsule internalized ferritin. Since the capsule expressed a novel receptor called Scara5, we tested its role in ferritin uptake and found that Scara5 bound serum ferritin and then stimulated its endocytosis from the cell surface with consequent iron delivery. These data implicate cell type-specific mechanisms of iron traffic in organogenesis, which alternatively utilize transferrin or non-transferrin iron delivery pathways.

Figure 1

Initial stages of renal development were preserved in TfR1^−/− mice. (A) Pax2⁺ Wolffian ducts and mesonephros and (B) Pax2⁺ ureteric buds surrounded by mesenchymal cells were detected in TfR1^−/− E11 embryos. Crosses with Hoxβ7-GFP mice distinguished the ureteric bud (GFP⁺Pax-2⁺) from mesenchyme (GFP⁻Pax-2⁺). Bar=10 μm.

Figure 2

Development of GFP-TfR1^−/− cells in E15 kidney. (A) GFP-TfR1^−/− cells were readily detected in capsule and stroma, mesenchyme, renal vesicles, and S-shaped bodies, but detected with limited frequency in UB tips (bar=10 μm). (B) GFP-TfR1^−/− cells (42%) reside in the capsule, interstitium, and podocalyxin⁺ vascular progenitors (confocal section; bar=10 μm). (C, D) GFP-TfR1^−/− cells (58%) also resided in proximal and distal nephrons (Ecadherin⁺; dark blue), in podocytes (podocalyxin⁺; red, Glm=glomerulus) and in the UB stalk (Ecadherin⁺, dark blue; Troma⁺, pink). In some cases, entire segments of the S-shaped body were composed of GFP-TfR1^−/− cells (confocal section; bar=10 μm). (E, F) Genes specific for UB tip or mesenchymal-stromal compartments were assayed in GFP-TfR1^−/− and wild type cells and presented as the log₂ ratio of microarray values. UB tip specific genes were under represented in GFP-TfR1^−/− cells, whereas mesenchymal-stromal genes were expressed. (G) Both GFP-TfR1^−/− and wild type cells (two sections are shown) contained cytoplasmic ferritin. (H) Wild type cells captured Alexa568-transferrin, but neighboring GFP-TfR1^−/− were unlabeled. Bar=10 μm.

Figure 3

Ferritin capture, cell proliferation and Scara5 expression in the capsule of the E15 kidney. (A, B) Both wild type and GFP-TfR1^−/− capsular cells captured rhodamine coupled “Exogenous L-Ferritin”. Bars=20 μm. (C) Holoferritin (+HF; 1.25 μg/ml) induced phosphohistone⁺ nuclei and expanded layers of capsular cells, (D) whereas culture with apoferritin (−HF; 1.25 μg/ml) failed to preserve the capsule. (E) “Endogenous Ferritin” was present throughout the kidney, particularly in the capsule. E-cadherin marked the UB. (F) Scara5 RNA was expressed by spindle-shaped cells encapsulating the kidney (arrowheads) and more faintly by cortical stroma.

Figure 4

Localization of the transferrin pathway. (A, B, C) TfR1 was initially localized to UB tips and cap mesenchyme. By E13, renal vesicles also expressed TfR1 (*B, C). TfR1 expression was reduced or absent in glomeruli (“G”), stroma and capsule. (D) Rhodamine- and (E) fluorescein-transferrin endocytosis paralleled TfR1 expression. Stroma and capsule were weakly labeled despite active fluid-phase endocytosis (rhodamine-dextran; E). (*Presumptive distal nephron; Bar= 10 μm).

Figure 5

Interaction between Scara5 and L-ferritin. (A) Transient transfection of Scara5-V5 (anti-V5, green) and (B) stable expression of Scara5-V5 resulted in the capture of Alexa568-ferritin (A, B right panel) whereas parental Trvb, which are Scara5⁻TfR1⁻ cells, transfected with an empty vector were negative (B left panel). Tris-quenched Alexa568 dye (no ferritin) was an additional control (middle panel). (C) Expression of Scara5-V5 (59KDa, 70KDa) was detected by V5 monoclonal antibody. (D) Endocytosis of Alexa568-ferritin was analyzed in Scara5⁻ (Trvb, B2 clones) and Scara5⁺ (D2 and H2) stable clones. Nearly all D2 and H2 cells captured apo- and L-ferritins, but not H-ferritin. Bar=10 μm.

Figure 6

Interaction between Scara5 and ferritin at the cell surface. (A) Scara5-V5 (anti-V5; Cy2; green) and ferritin (anti-ferritin; Cy3; red) co-localized at the surface (Z-stack; inset: single confocal section) of Scara5-V5 transfected MSC-1 cells incubated with untagged ferritin (2hrs; 4°C). Toto3 marked nuclei. Bar=10 μm. (B) Endocytosis of rhodamine-ferritin (30hrs; 37°C; top panel) was blocked by a 100 fold excess untagged ferritin (bottom panel), Bar=20 μm. (C) V5 antibodies precipitated Scara5-V5 with avidin tetramer-ferritin monomer (85KDa) or multimers (200KDa, >250KDa), but not with unconjugated avidin. Irrelevant isotype matched antibodies did not immunoprecipitate ferritin-avidin nor were ferritin-avidin complexes recovered without cell extracts. (D) Scara5-V5 was detected in cell extracts (clone D2) and in pull-downs with ferritin-avidin-biotin-agarose beads but not with avidin-biotin-agarose-beads.

Figure 7

Scara5 mediates cellular uptake of ferritin-bound iron and represents an endogenous pathway in MSC-1 cells. (A) Capture of ⁵⁹Fe-ferritin by Scara5⁺ at 37°C (red line) but not at 4°C (black line) nor by parental Trvb cells (blue line). (B) Lysosomes of Scara5⁺ Trvb cells were marked with fluorescein-dextran (green). They contained endocytosed Alexa568-ferritin (red). Original magnification 40X. (C) Transfer of iron to the cytoplasm was detected using two IRE-based iron reporters and FACS analysis in MSC-1 cells. The maximal 5′ IRE-CFP signal (designated 100%-black bars) was generated by iron loading with holotransferrin (20 μg/ml) and ferric ammonium citrate (20 μM) (HT+Fe) and the maximal 3′ IRE-YFP signal (designated 100%-stippled bars) was generated by DFO (20 μM). These treatments produced a ~7-fold range in the ratio of 5′/3′ IRE signals. Treatment with holoferritin (HF) increased 5′ IRE-CFP but decreased the 3′ IRE-YFP signal. (D) Holoferritin induces cell growth. Scara5⁺ clone H responded to holo- but not to apo-ferritin, whereas Trvb cells failed to respond. Cell protein was detected by GAPDH immunoblots. (E) Alexa488-ferritin endocytosis was blocked by Poly I >Poly G >Poly C (50 μM). Similar data were obtained in Scara5⁺ Trvb and MSC1 cells. Bar=20 μM.