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Transcriptional repression of fibronectin gene expression in v-src transformation
1Department of Anatomy and Cell Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Abstract:
V-src-dependent and -independent alterations in the steady-state content, rates of synthesis, and turnover of fibronectin protein and mRNA were identified using rat fibroblasts which are temperature sensitive for p60v-src activity. Activation of p60v-src caused a fivefold reduction in the rate of fibronectin biosynthesis. The v-src-dependent decrease in fibronectin biosynthesis resulted from a similar reduction in the steady-state content of fibronectin mRNA. This change was reversible and required more than 24 h, implying an indirect effect of p60v-src on fibronectin gene expression. The rate of fibronectin mRNA turnover and pattern of alternative splicing were unchanged following p60v-src activation, indicating that these regulatory steps are insensitive to v-src transformation. A v-src-specific reduction of at least threefold was measured for the rate of fibronectin gene transcription, and gene transfer studies using fibronectin promoter-CAT reporter genes indicated that transcriptional repression occurs at the level of initiation. When p60v-src was inactive, CAT reporter genes controlled by 4.9 or 3.2 kb of the rat fibronectin promoter exhibited relatively increased CAT activity (approximately twofold) compared to another CAT reporter construction controlled by only 880 bp of the fibronectin promoter. In contrast, CAT activity was relatively reduced (approximately twofold) for the reporter constructions containing 4.9 or 3.2 kb of the promoter when p60v-src was active. These findings indicate that the distal portion of the fibronectin promoter contains a v-src-sensitive element(s) which mediates a decrease in the rate of fibronectin transcription initiation by negative control.
Insights
Activation of p60v-src significantly reduces fibronectin biosynthesis and mRNA levels in rat fibroblasts. This regulation occurs indirectly, impacting fibronectin gene transcription initiation via a v-src-sensitive promoter element.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Fibronectin is a crucial extracellular matrix protein involved in cell adhesion, migration, and differentiation.
- The viral src (v-src) oncogene, encoded by the Rous sarcoma virus, is a potent activator of cellular signaling pathways.
- Understanding how v-src influences gene expression is vital for cancer research.
Purpose of the Study:
- To investigate the effects of p60v-src activity on fibronectin gene expression in temperature-sensitive rat fibroblasts.
- To elucidate the regulatory mechanisms by which v-src impacts fibronectin biosynthesis and mRNA levels.
- To identify the specific elements within the fibronectin promoter responsive to v-src transformation.
Main Methods:
- Utilized temperature-sensitive rat fibroblasts to control p60v-src activity.
- Quantified fibronectin protein and mRNA levels, synthesis rates, and turnover.
- Employed gene transfer studies with fibronectin promoter-CAT reporter constructs to assess transcriptional regulation.
Main Results:
- Activation of p60v-src led to a fivefold reduction in fibronectin biosynthesis and a similar decrease in fibronectin mRNA steady-state levels.
- Fibronectin mRNA turnover and alternative splicing patterns remained unaffected by v-src activation.
- A v-src-specific reduction in fibronectin gene transcription initiation was observed, mediated by a distal promoter element.
Conclusions:
- p60v-src negatively regulates fibronectin gene expression primarily at the level of transcription initiation.
- A v-src-sensitive element in the distal fibronectin promoter controls transcriptional repression.
- These findings reveal an indirect mechanism of v-src-mediated gene regulation impacting extracellular matrix production.