Figure 9.

Holo-Tf blocks MT2 cleavage of Tfr2 (A), but not ALK2 (B), ALK3 (C), ActRIIA (D), Bmpr2 (E), or Hfe (F). pcDNA3-Tfr2, pCMV6-ALK2, ALK3, ActRIIA, Bmpr2, or Hfe/B2M were co-transfected into HEK293 cells with an equal amount of pEGFP-N1 or pCMV6-MT2. At about 24 h post-transfection, medium was changed to Opti-MEM, 1% FCS with or without 30 μm holo-Tf (Tf). After another 24 h of incubation, cell lysate was collected for immunodetection of MT2, ALK2, ALK3, ActRIIA, Bmpr2, and Hfe by using an anti-FLAG antibody, and Tfr2, β-actin, and ferritin by using specific antibodies. All experiments were repeated at least three times (technical replicate = 1; independent biological replicates ≥3) with consistent results. G, a model for MT2 suppression of hepcidin expression. Bmp receptors, Hjv, Hfe, and Tfr2 form a complex with BMP6 at the plasma membrane to induce hepcidin expression through phosphorylation of SMAD1, -5, and -8. MT2 suppresses hepcidin expression by cleaving the extracellular portion of these membrane proteins.