Constitutive expression of steel factor gene by human stromal cells

Michael Heinrich, Douglas C. Dooley, Alison C. Freed, Louise Band, Maureen Hoatlin, Winifred W. Keeble, Sandra T. Peters, Kirsten V. Silvey, Frederick (Fred) Ey, David Kabat, Richard Maziarz, Grover C. Bagby

Research output: Contribution to journalArticle

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Abstract

Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1α, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in long-term bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1α neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1α. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.

Original languageEnglish (US)
Pages (from-to)771-783
Number of pages13
JournalBlood
Volume82
Issue number3
StatePublished - Aug 1 1993

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Stem Cell Factor
Stromal Cells
Genes
Endothelial cells
Fibroblasts
Diploidy
Hematopoiesis
Bone
Endothelial Cells
Bone Marrow
Interleukin-1
Cell culture
Gene expression
Northern Blotting
Messenger RNA
Exons
Gametogenesis
Membranes
Gene Expression
Umbilical Veins

ASJC Scopus subject areas

  • Hematology

Cite this

Heinrich, M., Dooley, D. C., Freed, A. C., Band, L., Hoatlin, M., Keeble, W. W., ... Bagby, G. C. (1993). Constitutive expression of steel factor gene by human stromal cells. Blood, 82(3), 771-783.

Constitutive expression of steel factor gene by human stromal cells. / Heinrich, Michael; Dooley, Douglas C.; Freed, Alison C.; Band, Louise; Hoatlin, Maureen; Keeble, Winifred W.; Peters, Sandra T.; Silvey, Kirsten V.; Ey, Frederick (Fred); Kabat, David; Maziarz, Richard; Bagby, Grover C.

In: Blood, Vol. 82, No. 3, 01.08.1993, p. 771-783.

Research output: Contribution to journalArticle

Heinrich, M, Dooley, DC, Freed, AC, Band, L, Hoatlin, M, Keeble, WW, Peters, ST, Silvey, KV, Ey, FF, Kabat, D, Maziarz, R & Bagby, GC 1993, 'Constitutive expression of steel factor gene by human stromal cells', Blood, vol. 82, no. 3, pp. 771-783.
Heinrich M, Dooley DC, Freed AC, Band L, Hoatlin M, Keeble WW et al. Constitutive expression of steel factor gene by human stromal cells. Blood. 1993 Aug 1;82(3):771-783.
Heinrich, Michael ; Dooley, Douglas C. ; Freed, Alison C. ; Band, Louise ; Hoatlin, Maureen ; Keeble, Winifred W. ; Peters, Sandra T. ; Silvey, Kirsten V. ; Ey, Frederick (Fred) ; Kabat, David ; Maziarz, Richard ; Bagby, Grover C. / Constitutive expression of steel factor gene by human stromal cells. In: Blood. 1993 ; Vol. 82, No. 3. pp. 771-783.
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abstract = "Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1α, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in long-term bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1α neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1α. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.",
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AU - Keeble, Winifred W.

AU - Peters, Sandra T.

AU - Silvey, Kirsten V.

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AU - Maziarz, Richard

AU - Bagby, Grover C.

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N2 - Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1α, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in long-term bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1α neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1α. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.

AB - Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1α, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in long-term bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1α neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1α. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.

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