Multiple mechanisms for the regulation of haem synthesis during erythroid cell differentiation. Possible role for coproporphyrinogen oxidase

Abstract

Murine erythroleukaemia (MEL) cells are virus-transformed erythroid precursor cells that, when induced to differentiate by dimethyl sulphoxide (DMSO), will initiate haem biosynthesis by the induction and synthesis de novo of all of the enzymes of the haem-biosynthetic pathway. The activities of porphobilinogen (PBG) deaminase (EC 4.3.1.8), coproporphyrinogen oxidase (EC 1.3.3.3), protoporphyrinogen oxidase (EC 1.3.3.4), ferrochelatase (EC 4.99.1.1) and NADH:ferric iron reductase, as well as the synthesis of the enzyme ferrochelatase and the levels of excreted porphyrins, were monitored during DMSO-induced differentiation of MEL cells in culture. The data demonstrate that PBG deaminase and protoporphyrinogen oxidase activities rise rapidly and early, in comparison with ferrochelatase activity, which rises more slowly, and coproporphyrinogen oxidase activity, which decreases by 60% within 24 h of induction before returning to initial levels by 72 h. NADH:ferric iron reductase activity increases slightly, but is always present at levels higher than needed for haem synthesis. Total immunoprecipitable ferrochelatase also rises slowly and parallels the increase in its activity, suggesting that it is not synthesized early in a slowly processed precursor form. Examination of culture media demonstrated that, whereas excretion of protoporphyrin and coproporphyrin occurs within 24 h of induction, coproporphyrin is excreted in amounts 4-15 times greater than protoporphyrin.

Examination of mitochondrial protein targeting of haem synthetic enzymes: in vivo identification of three functional haem-responsive motifs in 5-aminolaevulinate synthase

“…These data indicate that haem-regulated CPO translocation in situ must be of minimal, if any, significance. It should be noted that this does not rule out a role for CPO in overall pathway regulation as has been suggested by other studies [19,20], but only indicates that haem regulation of CPO translocation through interaction with the leader sequence is not of physiological consequence.…”

“…Of these three constructs, only the full-length sequence targeted the GFP reporter into the mitochondrion. Since the leader sequence was quite large and because there have been several reports on the potential regulation of haem biosynthesis at the site of CPO [19,20], we examined the effects of culture haem content on translocation of the 120 residue targeting sequence. The addition of haem or ALA had no effect on the targeting of the CPO-GFP reporter.…”

Methotrexate used in combination with aminolaevulinic acid for photodynamic killing of prostate cancer cells

“…Likewise, human erythroleukaemia K562 cells undergoing differentiation in the presence of TGF-b, also displayed increased CPO expression (Taketani et al, 2001). In erythroid cells, differentiation-inducing agents induced several of the haeme-synthetic enzymes simultaneously (Taketani et al, 1995), but even under such circumstances, CPO may be rate-limiting and therefore important in the regulation of haeme and PpIX production (Conder et al, 1991). Our own demonstrations of CPO upregulation in differentiating keratinocytes (Ortel et al, 1998) and LNCaP cells (Ortel et al, 2002) were among the first to demonstrate such a link in nonhaematopoietic cells.…”

Molecular and Genetic Characterization of Ferrochelatase.

“…Considering that multiple steps, which include iron uptake into cells and mitochondria as well as reduction of ferric ion, are involved in the incorporation of exogenous iron into heme, the iron supply may be a rate-limiting step of heme synthesis during erythroid differentiation. Conder et al (1991) recently reported that coproporphyrinogen oxidase may be rate-limiting on the induction of heme synthesis of MEL cells. If so, supply of the substrate for ferrochelatase would control the production of heme.…”