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Abstract

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  2. Abstract
  3. REFERENCES

Transforming growth factor β (TGF-β) and secreted protein acidic rich cysteine (SPARC) have been associated with the rapid remodeling of connective tissues that occurs in wound healing and developmental processes. To study the temporal and mechanistic aspects of TGF-β-regulated extracellular-protein gene expression in human fibroblasts, confluent cells were pulse labeled for 30 min with [35S]methionine at various times following the single addition of 1.0 ng/ml TGF-β. After a 4-h chase period, specific radiolabeled media proteins were isolated by either immunoprecipitation or affinity chromatography and quantitated. Stimulation of SPARC synthesis was first apparent 5 h after addition of TGF-β, reached a maximum (3.5-fold increase) at 24 h and persisted for at least 96 h. A similar temporal response to TGF-β was observed for the extracellular matrix proteins collagen and fibronectin. In contrast, TGF-β induced a strong (> sixfold increase at 9 h after addition of TGF-β), but transient stimulation of the synthesis of endothelial-type plasminogen activator inhibitor. Northern blot analysis showed that SPARC mRNA levels were increased by TGF-β in parallel with increase in SPARC synthesis; a maximum 3.9-fold increase in SPARC mRNA being reached at 24 h. Similarly, the levels of both collagen and fibronectin mRNA were increased by TGF-β treatment. In each case the stimulation of mRNA was blocked by the presence of the translation inhibitor, cycloheximide. Stability of SPARC mRNA (half-life of approximately 50 h) was not significantly altered by TGF-β. In contrast, the stability of collagen and fibronectin mRNA were both increased in the presence of TGF-β; the increased stability being pronounced in less dense cells. In addition to effects on stability, transcription of the collagen and fibronectin genes was increased 7 h after TGF-β addition, but returned to control levels by 24 h. However, transcription of the SPARC gene was unaffected by TGF-β at both time points and, together with the stability data, indicates that TGF-β regulates SPARC expression via a nuclear post-transcriptional mechanism. Differential regulation of gene expression by TGF-β in a precise temporal pattern via transcriptional and post-transcriptional pathways may be an important aspect of the response of fibroblast cells in a wound environment.

Abbreviations
α-MEM

α minimal essential medium

BSA

bovine serum albumin

DRB

5,6-dichloro-1-β-d-ribofuranosyl benzimidazole

FBS

fetal bovine serum

IL-1

interleukin-1

PAI

endothelialtype plasminogen activator inhibitor

SPARC

secreted protein acidic rich in cysteine

TGF-β

transforming growth factor β

TPA

12-O-tetradecanoylphorbol 13-acetate

Enzymes
 

Bacterial collagenase (EC 3.4.24.3)

 

pepsin A (EC 3.4.23.13)

REFERENCES

  1. Top of page
  2. Abstract
  3. REFERENCES