scholarly journals Altered beta-actin gene expression in phorbol myristate acetate-treated chondrocytes and fibroblasts.

1985 ◽  
Vol 5 (6) ◽  
pp. 1425-1433 ◽  
Author(s):  
L C Gerstenfeld ◽  
M H Finer ◽  
H Boedtker
Keyword(s):  
Steady State ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Tumor Promoter ◽  
Type I ◽  
Mrna Levels ◽  
Collagen Mrna ◽  
Beta Actin ◽  
Steady State Levels ◽  
Actin Mrna

Phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, was shown to have opposite effects on the cellular morphology and steady-state levels of beta-actin mRNA in embryonic chicken muscle fibroblasts and sternal chondrocytes. When fibroblasts were treated with PMA, they formed foci of densely packed cells, ceased to adhere to culture plates, and had significantly reduced levels of beta-actin mRNA and protein. Conversely, when treated with PMA, floating chondrocytes attached to culture dishes, spread out, and began to accumulate high levels of beta-actin mRNA and proteins. In the sternal chondrocytes the stimulation of the beta-actin mRNA production was accompanied by increased steady-state levels of fibronectin mRNAs and protein. These alterations were concomitant with a fivefold reduction in type II collagen mRNA and a cessation in its protein production. After fibronectin and actin mRNAs and proteins reached their maximal levels, type I collagen mRNA and protein synthesis were turned on. Removal of PMA resulted in reduced beta-actin mRNA levels in chondrocytes and in a further alteration in the cell morphology. These observed correlations between changes in cell adhesion and morphology and beta-actin expression suggest that the effect of PMA on cell shape and adhesion may result in changes in the microfilament organization of the cytoskeleton which ultimately lead to changes in the extracellular matrix produced by the cells.

Altered beta-actin gene expression in phorbol myristate acetate-treated chondrocytes and fibroblasts

1985 ◽  
Vol 5 (6) ◽  
pp. 1425-1433
Author(s):  
L C Gerstenfeld ◽  
M H Finer ◽  
H Boedtker
Keyword(s):  
Steady State ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Tumor Promoter ◽  
Type I ◽  
Mrna Levels ◽  
Collagen Mrna ◽  
Beta Actin ◽  
Steady State Levels ◽  
Actin Mrna

Phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, was shown to have opposite effects on the cellular morphology and steady-state levels of beta-actin mRNA in embryonic chicken muscle fibroblasts and sternal chondrocytes. When fibroblasts were treated with PMA, they formed foci of densely packed cells, ceased to adhere to culture plates, and had significantly reduced levels of beta-actin mRNA and protein. Conversely, when treated with PMA, floating chondrocytes attached to culture dishes, spread out, and began to accumulate high levels of beta-actin mRNA and proteins. In the sternal chondrocytes the stimulation of the beta-actin mRNA production was accompanied by increased steady-state levels of fibronectin mRNAs and protein. These alterations were concomitant with a fivefold reduction in type II collagen mRNA and a cessation in its protein production. After fibronectin and actin mRNAs and proteins reached their maximal levels, type I collagen mRNA and protein synthesis were turned on. Removal of PMA resulted in reduced beta-actin mRNA levels in chondrocytes and in a further alteration in the cell morphology. These observed correlations between changes in cell adhesion and morphology and beta-actin expression suggest that the effect of PMA on cell shape and adhesion may result in changes in the microfilament organization of the cytoskeleton which ultimately lead to changes in the extracellular matrix produced by the cells.

scholarly journals Regulation of type I collagen mRNA in lung fibroblasts by cystine availability

1998 ◽  
Vol 331 (2) ◽  
pp. 417-422 ◽  
Author(s):  
David C. RISHIKOF ◽  
Ping-Ping KUANG ◽  
Christine POLIKS ◽  
Ronald H. GOLDSTEIN
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Amino Acid ◽  
Type I Collagen ◽  
State Level ◽  
Lung Fibroblasts ◽  
Type I ◽  
Mrna Levels ◽  
Collagen Mrna ◽  
Amino Acid Availability

The steady-state level of α1(I) collagen mRNA is regulated by amino acid availability in human lung fibroblasts. Depletion of amino acids decreases α1(I) collagen mRNA levels and repletion of amino acids induces rapid re-expression of α1(I) mRNA. In these studies, we examined the requirements for individual amino acids on the regulation of α1(I) collagen mRNA. We found that re-expression of α1(I) collagen mRNA was critically dependent on cystine but not on other amino acids. However, the addition of cystine alone did not result in re-expression of α1(I) collagen mRNA. Following amino acid depletion, the addition of cystine with selective amino acids increased α1(I) collagen mRNA levels. The combination of glutamine and cystine increased α1(I) collagen mRNA levels 6.3-fold. Methionine or a branch-chain amino acid (leucine, isoleucine or valine) also acted in combination with cystine to increase α1(I) collagen mRNA expression, whereas other amino acids were not effective. The prolonged absence of cystine lowered steady-state levels of α1(I) collagen mRNA through a mechanism involving decreases in both the rate of gene transcription as assessed by nuclear run-on experiments and mRNA stability as assessed by half-life determination in the presence of actinomycin D. The effect of cystine was not mediated via alterations in the level of glutathione, the major redox buffer in cells, as determined by the addition of buthionine sulphoximine, an inhibitor of γ-glutamylcysteine synthetase. These data suggest that cystine directly affects the regulation of α1(I) collagen mRNA.

Collagen expression in embryonic chicken chondrocytes treated with phorbol myristate acetate

1985 ◽  
Vol 5 (6) ◽  
pp. 1415-1424
Author(s):  
M H Finer ◽  
L C Gerstenfeld ◽  
D Young ◽  
P Doty ◽  
H Boedtker
Keyword(s):  
Morphological Change ◽  
Type I Collagen ◽  
Late Phase ◽  
Tumor Promoter ◽  
Type I ◽  
Mrna Levels ◽  
Type Ii ◽  
Collagen Mrna ◽  
Type I Procollagen ◽  
Embryonic Chicken

Growth of embryonic chicken sternal chondrocytes in the presence of phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, resulted in a dramatic morphological change from spherical floating cells to adherent fibroblastic cells. This morphological change was accompanied by a quantitative switch from synthesis of cartilage-specific type II procollagen to type I procollagen. Type II procollagen mRNA levels decreased 10-fold in PMA-treated cells. Activation of type I collagen genes led to the accumulation of type I procollagen mRNA levels comparable to those of type II mRNA in these cells. However, only type I procollagen mRNA was translated. In addition to gene activation, unprocessed pro alpha 1(I) transcripts present at low levels in control chondrocytes were processed to mature mRNA species. Redifferentiation of PMA-treated chondrocytes was possible if cells were removed from PMA after the morphological change and cessation of type II procollagen synthesis but before detectable amounts of type I procollagen were synthesized. Production of type I collagen thus marks a late phase of chondrocyte "dedifferentiation" from which reversion is no longer possible. Redifferentiated cell populations contained 24-fold more pro alpha 1(II) collagen mRNA than pro alpha 1(I) collagen mRNA, but the rates of procollagen synthesis were comparable. This suggests that the PMA-mediated dedifferentiation of chondrocytes as well as their redifferentiation is under both transcriptional and posttranscriptional regulation.

scholarly journals Lack of a phenotype in transgenic mice aberrantly expressing COL2A1 mRNA because of highly selective post-transcriptional down-regulation

2000 ◽  
Vol 345 (2) ◽  
pp. 377-384 ◽  
Author(s):  
Constance M. YUAN ◽  
Leena ALA-KOKKO ◽  
Dominique LE GUELLEC ◽  
Suzanne FRANC ◽  
Andrzej FERTALA ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Type I ◽  
Mrna Levels ◽  
Type Ii ◽  
Col2a1 Gene ◽  
Type I Procollagen ◽  
Steady State Levels ◽  
Hybridization In Situ

We reported previously that a 1.9-kb 5ʹ-fragment from the human COL1A1 gene drove transcription of a promoterless human COL2A1 gene in tissues of transgenic mice that normally express the COL1A1 but not the COL2A1 gene. In the present study, we have established that the aberrant transcription of the COL2A1 gene did not produce any gross or microscopic phenotype, because the transcripts were not efficiently translated in cells that do not normally express the COL2A1 gene. In two lines of transgenic mice, the mRNA levels from the transgene were 30% to 45% of the mRNA for the proα1(I) chain of type I procollagen, the most abundant mRNA in the same tissues. Analysis of collagens extracted from skin of the transgenic mice indicated that triple-helical type II collagen, with the normal pattern of cyanogen bromide peptides, was synthesized from the transgene. However, the level of type II collagen in skin was less than 2% of the level of type I collagen. Hybridization in situ indicated the presence of mRNA for both COL2A1 and COL1A1 in the same cells. Immunofluorescence staining for type II collagen, however, was negative in the same tissues. The results, therefore, indicated that many mesenchymal cells in the transgenic mice had high steady-state levels of the homologous mRNAs for type I and type II procollagen, but only the mRNAs for type I procollagen were efficiently translated.

scholarly journals Collagen expression in embryonic chicken chondrocytes treated with phorbol myristate acetate.

1985 ◽  
Vol 5 (6) ◽  
pp. 1415-1424 ◽  
Author(s):  
M H Finer ◽  
L C Gerstenfeld ◽  
D Young ◽  
P Doty ◽  
H Boedtker
Keyword(s):  
Morphological Change ◽  
Type I Collagen ◽  
Late Phase ◽  
Tumor Promoter ◽  
Type I ◽  
Mrna Levels ◽  
Type Ii ◽  
Collagen Mrna ◽  
Type I Procollagen ◽  
Embryonic Chicken

Growth of embryonic chicken sternal chondrocytes in the presence of phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, resulted in a dramatic morphological change from spherical floating cells to adherent fibroblastic cells. This morphological change was accompanied by a quantitative switch from synthesis of cartilage-specific type II procollagen to type I procollagen. Type II procollagen mRNA levels decreased 10-fold in PMA-treated cells. Activation of type I collagen genes led to the accumulation of type I procollagen mRNA levels comparable to those of type II mRNA in these cells. However, only type I procollagen mRNA was translated. In addition to gene activation, unprocessed pro alpha 1(I) transcripts present at low levels in control chondrocytes were processed to mature mRNA species. Redifferentiation of PMA-treated chondrocytes was possible if cells were removed from PMA after the morphological change and cessation of type II procollagen synthesis but before detectable amounts of type I procollagen were synthesized. Production of type I collagen thus marks a late phase of chondrocyte "dedifferentiation" from which reversion is no longer possible. Redifferentiated cell populations contained 24-fold more pro alpha 1(II) collagen mRNA than pro alpha 1(I) collagen mRNA, but the rates of procollagen synthesis were comparable. This suggests that the PMA-mediated dedifferentiation of chondrocytes as well as their redifferentiation is under both transcriptional and posttranscriptional regulation.

scholarly journals Differential regulation of transcription and transcript stability of pro-α 1(I) collagen and fibronectin in activated fibroblasts derived from patients with systemic scleroderma

1996 ◽  
Vol 315 (2) ◽  
pp. 549-554 ◽  
Author(s):  
Beate ECKES ◽  
Cornelia MAUCH ◽  
Gabriele HÜPPE ◽  
Thomas KRIEG
Keyword(s):  
Steady State ◽  
Three Dimensional ◽  
Systemic Scleroderma ◽  
Regulation Of Transcription ◽  
Mrna Levels ◽  
Transcript Stability ◽  
Collagen Mrna ◽  
Steady State Levels ◽  
Activated Fibroblasts ◽  
Fibronectin Mrna

Activated fibroblasts were derived from the skin of patients with systemic scleroderma (SSc), used as a model for fibrosis. Such cells are characterized by increased production of collagens and other matrix constituents. Increased collagen and fibronectin production has been correlated with similarly elevated mRNA steady-state levels. In the present study we analysed the contribution of transcriptional activity and post-transcriptional transcript stability to the increases in pro-α1(I) collagen and fibronectin mRNA steady-state levels in activated (scleroderma) fibroblasts. Fibroblasts, when cultured in close contact with a three-dimensional collagenous matrix, down-regulate collagen synthesis. Culture of skin fibroblasts from two patients with SSc in three-dimensional collagen lattices, however, showed 4-fold elevated pro-α1(I) collagen mRNA levels over fibroblasts from healthy donors. Transcription of the COL1A1 gene in SSc fibroblasts was induced 2–3-fold over that in controls in both monolayer and lattice cultures, accounting in part for the elevated steady-state level. A 50% decrease in transcription rate in lattice compared with monolayer culture occurred, as in control cells. In contrast, whereas control cells in lattices responded with decreased (50%) pro-α1(I) collagen mRNA stability, in SSc cells these transcripts were found to be more stable (half-life of 5 h compared with 2 h in control cells). Fibronectin steady-state mRNA levels, in contrast, were not significantly regulated by the three-dimensional environment. In SSc fibroblasts, fibronectin mRNA levels were induced 1.5–4.9-fold over controls. In part, this increase appears to be due to elevated transcription, and an increase in fibronectin transcript stability was also detected. We therefore conclude that activated fibroblasts such as those derived from scleroderma patients utilize transcriptional and post-transcriptional mechanisms to maintain increased collagen and fibronectin production, which contribute to the pathogenesis of the disease.

scholarly journals Regulation of type I collagen mRNA levels in fibroblasts

1986 ◽  
Vol 157 (2) ◽  
pp. 433-439 ◽  
Author(s):  
Tilman VOSS ◽  
Paul BORNSTEIN
Keyword(s):  
Type I Collagen ◽  
Type I ◽  
Mrna Levels ◽  
Collagen Mrna

Expression of type I and III collagen genes during differentiation of embryonic chicken myoblasts in culture

1984 ◽  
Vol 4 (8) ◽  
pp. 1483-1492
Author(s):  
L C Gerstenfeld ◽  
D R Crawford ◽  
H Boedtker ◽  
P Doty
Keyword(s):  
Steady State ◽  
Cell Cultures ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Myoblast Differentiation ◽  
Type I ◽  
Mrna Levels ◽  
Embryonic Chicken ◽  
Steady State Levels ◽  
Alpha Actin

Expression of type I and III procollagen genes was studied in embryonic chicken myoblast cell cultures, obtained from thigh muscles of 11-day-old embryos. Differentiation initiated by the addition of ovotransferrin (30 micrograms/ml) was followed visually by phase-contrast microscopy. Myoblast fusion and myotube formation were detected by day 3 and appeared to be complete by day 7. The synthesis of procollagens was monitored by labeling cell cultures for 1 h with [3H]proline and determining the radioactivity in procollagen chains by scanning densitometry of the fluorograms of the sodium dodecyl sulfate-polyacrylamide gels. A 10- to 20-fold increase in the rate of pro alpha-1(I), pro alpha-2(I), and pro alpha-1(III) collagen synthesis was observed, with the greatest increase occurring between days 3 and 9. Collagen mRNA levels in the myoblast cultures were examined by Northern blot and dot blot hybridization assays. The 10- to 20-fold increased rate of protein synthesis was accompanied by a 15-fold increase in the steady-state levels of pro alpha-1(I) and pro alpha-2(I) mRNAs and a 10-fold increase in the steady-state levels of pro alpha-1(III). As a correlate to the studies of collagen expression during myoblast differentiation, the expression of actin mRNAs was examined. Although alpha actin could be detected by day 4, a complete switch from lambda and beta to alpha actin was not observed in the time periods examined. Similar results were obtained in the analysis of RNA extracted from embryonic legs at days 12 and 17 of gestation. Myoblast differentiation is manifested by the accumulation of both muscle-specific mRNAs, such as actin, and type I and III procollagen mRNAs.

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