Abstract
We have compared regulation of the serglycin gene in human erythroleukemia (HEL) and CHRF 288-11 cells, which have megakaryocytic characteristics, with promyelocytic HL-60 cells. Deletion constructs were prepared from the region -1123/+42 to -20/+42, and putative regulatory sites were mutated. In all three cell lines, the two major regulatory elements for constitutive expression were the (-80)ets site and the cyclic AMP response element (CRE) half-site at -70. A protein from HEL and CHRF, but not HL60, nuclear extracts bound to the (-80)ets site. Another protein from all three cell lines bound to the (-70)CRE. Phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP (dbcAMP) increased expression of the reporter in HEL cells 2.5-3- and 4.5-fold, respectively, from all constructs except those with (-70)CRE mutations. PMA virtually eliminated expression of serglycin mRNA and promoter constructs, but dbcAMP increased expression in HL-60 cells. The effects of PMA and dbcAMP on promoter expression correlated with mRNA expression. The strengths of two DNase I-hypersensitive sites in the 5'-flanking region and the first intron in all three cells correlated with relative endogenous serglycin mRNA expression. An additional DNase I-hypersensitive site in HL60 DNA in the first intron may be related to the high serglycin expression in HL60 relative to HEL or CHRF cells.
Highlights
The serglycin proteoglycan is synthesized by a number of hematopoietic cells, including platelets [1] and their parent megakaryocytes [2], granulocytes, macrophages, and lymphocytes [3,4,5,6], mast cells [4], and a large number of hematopoietic tumor cell lines [3, 7,8,9,10]
We have reported that the serglycin proteoglycan of platelets of Wistar-Furth rats, which have a severe defect in ␣ granule structure and protein content, has abnormally shortened glycyosaminoglycan chains [15]
Treatment with dibutyryl cyclic AMP (dbcAMP) resulted in increased serglycin mRNA expression in human erythroleukemia (HEL) and HL60 cells but not in CHRF cells (Fig. 5b), and the same effect was seen with 50 M forskolin
Summary
The human cell lines were human erythroleukemia (HEL) cells (American Type Culture Collection, Manassas, VA), which have erythroid and megakaryocytic potential; CHRF-288-11 megakaryoblastic cells (donated by Dr Michael Lieberman, University of Cincinnati) [40]; and HL-60 promyelocytic leukemia cells (American Type Culture Collection). The murine mastocytoma cells were donated by Dr Jeffrey Esko [41]. HEL and HL-60 cells were cultured in RPMI 1640 with 10% fetal bovine serum, 2 mM glutamine. CHRF cells were cultured in Fischer’s medium with 20% horse serum. The murine mastocytoma cells were cultured in 45% Ham’s F-12 medium, 45% Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum. Fusenig (German Cancer Research Center, Heidelberg, Germany), were grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Fetal bovine serum was from Hyclone (Logan, UT). The cells were maintained in the presence of 5% CO2 and 95% humidity
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