During erythroid differentiation and maturation, the formation of hemoglobin requires coordinated synthesis of globin chains and of heme. the iron containing protoporphyrin IX that serves as a prostetic group. Up-regulation of heme synthesis per se might increase hemoglobin synthesis. The regulatory mechanisms of heme pathway in erythroid cells, which are distinct from those of other tissues are not yet fully understood. In this study we investigated the expression of the mRNA of 0-aminolevulinate .synthase (ALAS-E) and of heme oxygenase-1 (HO-1 ), the inducible form of HO (EC 1.14.993) the rate-limiting enzyme for heme degradation, during normal erythroid differentiation and maturation. A liquid culture derived from normal human peripheral blood stem cells was set up in a two phases system according to Rbach. Total cellular RNA was extracted from harvested cells (10-20xl06 cells) on different days of culture by the acid guanidium thiocyanate-phenolchloroform method and it was subjected to Northern blot analysis. The hybridization probe for erythroid-ALAS-E mRNA was the 869 bp cDNA fragment that we prepared by RT-PCR. The hybridization probe for heme oxygenase-1 (HO-1) mRNA was the Xho I/Xba I fragment (64/693) derived from the human heme oxygenase-1 cDNA, pHHOl, kindly provided by Dr. S. Shibahara. ALAS-E mRNA was detectable from day 10, peaked at day 16 and declined at day 19 of culture, correlating with the extent of basophilic and polychromatophilic erythroblasts. ALAS-E mRNA maximum expression was seen after that of erythroid-specific transcription factor GATA-1 and before βglobin. HO-1 mRNA was very low until day 16 and reached its maximum at day 19 when orthocromatic erythroblasts were the most abundant erythroid cells. Thus ALAS-E and HO-1 have a developmentally specific mRNA expression, suggesting different gene regulation.
|Number of pages||1|
|Publication status||Published - 1998|
ASJC Scopus subject areas
- Cancer Research
- Cell Biology