The influence of cellular differentiation on colony-stimulating factor gene expression was examined in myogenically and adipogenically determined cell lines derived from 5-azacytidine-treated C3H10T1/2 C18 (10T1/2) mouse embryo fibroblasts. These studies demonstrate that colony-stimulating factor gene expression can be modulated by myogenic and adipogenic determination and terminal differentiation.

Colony-stimulating factors (CSF) are important factors in the proliferation and differentiation of hematopoietic progenitor cells (HPC), and in the survival and activation of mature blood cells. Interleukin-1 (IL-1) combined with fetal bovine serum (FBS) strongly induces the expression of macrophage-CSF (M-CSF), granulocyte-CSF (G- CSF), and granulocyte-macrophage-CSF (GM-CSF) in fibroblasts. Here, we report on the regulation of CSF gene expression in murine fibroblasts following IL-1 and FBS stimulation. We demonstrate that 10T1/2 murine fibroblasts induced by FBS or IL-1 accumulate M-CSF messenger RNA (mRNA). G-CSF mRNA expression was induced by IL-1, and not by FBS. For GM-CSF expression, induction with both FBS and IL-1 was required. Blocking studies with actinomycin-D showed that active transcription is essential for accumulation of all three CSF mRNAs. After blocking protein synthesis with cycloheximide, IL-1- or FBS-induced M-CSF expression and IL-1 plus FBS-induced GM-CSF expression still occurred and was increased. IL-1-induced G-CSF expression was completely prevented in these cells by pretreatment with cycloheximide, illustrating that, for this effect, intermediate protein synthesis was required. The half-lives of M-CSF transcripts were not substantially altered by addition of IL-1, FBS, or FBS plus IL-1. Using nuclear run- on assays, we demonstrated that the transcription rate of M-CSF was increased up to 20-fold by the addition of FBS, IL-1, or FBS plus IL-1. After blocking protein synthesis with cycloheximide, IL-1-or FBS- induced increase in M-CSF transcription rate was also observed. GM-CSF transcription increased up to fourfold after induction with FBS or IL- 1. G-CSF transcription rate was not altered by FBS or IL-1. Our results indicate that M-CSF expression induced by FBS or IL-1 in these fibroblasts is primarily regulated at the transcriptional level. GM-CSF expression appears to be regulated both transcriptionally and posttranscriptionally, and G-CSF expression is regulated mainly at the posttranscriptional level.

Colony-stimulating factors (CSF) are important factors in the proliferation and differentiation of hematopoietic progenitor cells (HPC), and in the survival and activation of mature blood cells. Interleukin-1 (IL-1) combined with fetal bovine serum (FBS) strongly induces the expression of macrophage-CSF (M-CSF), granulocyte-CSF (G- CSF), and granulocyte-macrophage-CSF (GM-CSF) in fibroblasts. Here, we report on the regulation of CSF gene expression in murine fibroblasts following IL-1 and FBS stimulation. We demonstrate that 10T1/2 murine fibroblasts induced by FBS or IL-1 accumulate M-CSF messenger RNA (mRNA). G-CSF mRNA expression was induced by IL-1, and not by FBS. For GM-CSF expression, induction with both FBS and IL-1 was required. Blocking studies with actinomycin-D showed that active transcription is essential for accumulation of all three CSF mRNAs. After blocking protein synthesis with cycloheximide, IL-1- or FBS-induced M-CSF expression and IL-1 plus FBS-induced GM-CSF expression still occurred and was increased. IL-1-induced G-CSF expression was completely prevented in these cells by pretreatment with cycloheximide, illustrating that, for this effect, intermediate protein synthesis was required. The half-lives of M-CSF transcripts were not substantially altered by addition of IL-1, FBS, or FBS plus IL-1. Using nuclear run- on assays, we demonstrated that the transcription rate of M-CSF was increased up to 20-fold by the addition of FBS, IL-1, or FBS plus IL-1. After blocking protein synthesis with cycloheximide, IL-1-or FBS- induced increase in M-CSF transcription rate was also observed. GM-CSF transcription increased up to fourfold after induction with FBS or IL- 1. G-CSF transcription rate was not altered by FBS or IL-1. Our results indicate that M-CSF expression induced by FBS or IL-1 in these fibroblasts is primarily regulated at the transcriptional level. GM-CSF expression appears to be regulated both transcriptionally and posttranscriptionally, and G-CSF expression is regulated mainly at the posttranscriptional level.

Myogenic determination factor 1 (MyoD1) expression is modulated by a variety of agents including growth factors and activated cellular proto-oncogenes. However, little is known about the effect of cell-cell contact, which increases as myoblasts terminally differentiate, on the control of MyoD1 expression. Steady-state levels of MyoD1 transcripts decline over a 6-12 hour time period when myoblasts plated at a single cell density are incubated in media supplemented with 0.2% serum; by 48 hours MyoD1 mRNA levels have returned to the initial basal level. The decline in MyoD1 transcripts is diminished, but not prevented in myoblasts which maintain cell-cell contacts (at least 50% of cells with two or more sites of contact). MyoD1 transcript levels do not change if single cell cultures are maintained in 10% serum or are cocultured with fibroblasts. Analysis of conditioned media revealed that myoblasts plated at the single cell density or at a density which allowed multiple sites of cell-cell contact are not producing an activity(s) responsible for modulating MyoD1 mRNA levels. The changes in MyoD1 expression are mediated at the transcriptional level. Thus changes in the degree of cell-cell contact in cultures of myogenically determined cell lines effect changes in MyoD1 gene expression. Consequently when the influence of cytokines or other pharmacological agents on commitment to terminal myogenic differentiation is examined, the degree of cell-cell contact within the culture system may affect the response elicited.

No abstract