Abstract
We examined the effect of basic fibroblast growth factor (bFGF) on alpha 1(I) procollagen mRNA levels, alpha 1(I) collagen gene transcription, and alpha 1(I) collagen promoter activity in osteoblastic MC3T3-E1 cells. Cells were stably transfected with ColCAT 3.6, containing 3521 base pairs of alpha 1(I) collagen promoter DNA, fused to the CAT reporter gene, or an upstream deletion mutant of ColCAT 3.6 designated ColCAT 2.3. After 48 h, bFGF (0.1-10 nM) inhibited the incorporation of [3H]proline into collagenase-digestible protein (CDP). Indomethacin did not alter the inhibitory effect of bFGF on CDP labeling. Aphidicolin, an inhibitor of DNA synthesis, did not block the inhibitory effect of bFGF on CDP. bFGF (1-10 nM) decreased alpha 1(I) procollagen mRNA levels, with maximal inhibition, nearly 99% of control, caused by 10 nM bFGF. After 48 h, bFGF (1 nM) reduced alpha 1(I) procollagen gene transcription by about 92%. ColCAT 3.6 activity was inhibited with 0.1-10 nM bFGF and was maximally repressed by about 83% with 10 nM bFGF. In contrast, bFGF (1 and 10 nM) caused a stimulation of ColCAT 2.3 activity. These data show that bFGF inhibits collagen synthesis by a transcriptional mechanism and the alpha 1(I) collagen promoter contains DNA sequences which mediate bFGF inhibition of type I collagen gene expression in bone.
Highlights
We examined the effect of basic fibroblast growth ylation of the receptor [9], activationof protein kinase A and factor onal[1] procollagen mRNA levels, al[1] collagen gene transcription, and al[1] collagen promoter activity in osteoblastic MC3T3-El cells
The inhibitory effect of bFGF on collagen synthesis appears tobe in part at a pretranslational level since in cell cultures both stimulatory and inhibitory effects of bFGF on procollagen mRNA levels have been reported[23, 24]
To determine whetheerndogenous factors presentin serum are necessary for mediating the effects of bFGF on collagen gene expression, MC3T3-El cells containing ColCAT 3.6 cells were plated a t high density grown to confluence, serum deprived for 24 h and treated with bFGF at l nM
Summary
BFGF at 1and 10nM markedly inhibited CDP labeling and CAT activity in cells plated a t 5,000 or 45,000 cells/cm (Fig. 6). To determine whetheerndogenous factors presentin serum are necessary for mediating the effects of bFGF on collagen gene expression, MC3T3-El cells containing ColCAT 3.6 cells were plated a t high density grown to confluence, serum deprived for 24 h and treated with bFGF at l nM. Collagen gene transcription, the rate of procollagen mRNA synthesis was measured in isolated nuclei from control and treated MC3T3-El cells. After a 48-h treatment, bFGF decreased CDP labeling and ColCAT 3.6 activity in a dose-dependent manner with bFGF at 10 nM reducing CDP by 97%and CAT activitbyy 83% (TableIIA).
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