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The mechanism of oxythiamine-induced collagen biosynthesis in cultured fibroblasts.

Szoka L, Karna E, Palka J - Mol. Cell. Biochem. (2015)

Bottom Line: It was found that OXY-dependent increase in collagen biosynthesis was accompanied by parallel increase in prolidase activity and level, compared to untreated cells.The exposure of the cells to OXY contributed to decrease in IGF-IR, α2β1 integrin receptor, pERK1/2, and NF-κB p65 expressions.The data suggest that OXY-dependent increase of collagen biosynthesis in cultured human skin fibroblasts results from activation of prolidase activity and level, induction in pAkt expression and down-regulation of pERK1/2 and NF-κB p65, the known inhibitor of collagen gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, Medical University of Bialystok, Mickiewicza 2 D, 15-222, Bialystok, Poland.

ABSTRACT
The oxythiamine (OXY) is antivitamin of thiamine. The finding that OXY increases the cytoplasmic concentration of pyruvate, known to enhance collagen biosynthesis, led us to investigate the mechanism of this antivitamin action on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with micromolar concentrations (30-1,000 µM) of OXY for 24 and 48 h. It was found that OXY-dependent increase in collagen biosynthesis was accompanied by parallel increase in prolidase activity and level, compared to untreated cells. Since phosphoenolpyruvate (PEP) is known as an inhibitor of prolidase-the enzyme that plays important role in collagen biosynthesis, the mechanism of pyruvate interconversion was considered as a regulatory switch in collagen biosynthesis. In fact, 3-MPA, specific inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), contributed to up-regulation of prolidase activity, suggesting that down-regulation of PEP formation is an underlying mechanism. Since collagen biosynthesis and prolidase activity are regulated by signal induced by activated α2β1 integrin receptor as well as insulin-like growth factor-I receptor (IGF-IR), the expression of these receptors was measured by Western immunoblot analysis. The exposure of the cells to OXY contributed to decrease in IGF-IR, α2β1 integrin receptor, pERK1/2, and NF-κB p65 expressions. It was accompanied by increase in total ERK1/2 expression and induction of phosphorylation of Akt protein. The data suggest that OXY-dependent increase of collagen biosynthesis in cultured human skin fibroblasts results from activation of prolidase activity and level, induction in pAkt expression and down-regulation of pERK1/2 and NF-κB p65, the known inhibitor of collagen gene expression.

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Viability a of confluent human skin fibroblasts incubated for 24 and 48 h with different concentrations of oxythiamine (OXY). The mean values ± SD from three independent experiments done in duplicates are presented. *P ≤ 0.01. Collagen biosynthesis measured as 5[3H] proline incorporation into proteins susceptible to the action of bacterial collagenase in fibroblasts treated for 24 and 48 h with different concentrations of (OXY) (b) and sodium pyruvate (c). The results present the mean values from 6 assays ± SD *P ≤ 0.01
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Fig1: Viability a of confluent human skin fibroblasts incubated for 24 and 48 h with different concentrations of oxythiamine (OXY). The mean values ± SD from three independent experiments done in duplicates are presented. *P ≤ 0.01. Collagen biosynthesis measured as 5[3H] proline incorporation into proteins susceptible to the action of bacterial collagenase in fibroblasts treated for 24 and 48 h with different concentrations of (OXY) (b) and sodium pyruvate (c). The results present the mean values from 6 assays ± SD *P ≤ 0.01

Mentions: Since oxythiamine (OXY) was found to inhibit proliferation of tumor cells [34–36], the cytotoxicity assay for different doses of OXY in cultured human fibroblasts was performed. Cell viability was measured by the method of Carmichael et al. [26] using tetrazolium salt. The viability of OXY-treated fibroblasts is presented in Fig. 1a. OXY at studied concentrations did not influence the viability of the cells at 24 and 48 h incubation.Fig. 1


The mechanism of oxythiamine-induced collagen biosynthesis in cultured fibroblasts.

Szoka L, Karna E, Palka J - Mol. Cell. Biochem. (2015)

Viability a of confluent human skin fibroblasts incubated for 24 and 48 h with different concentrations of oxythiamine (OXY). The mean values ± SD from three independent experiments done in duplicates are presented. *P ≤ 0.01. Collagen biosynthesis measured as 5[3H] proline incorporation into proteins susceptible to the action of bacterial collagenase in fibroblasts treated for 24 and 48 h with different concentrations of (OXY) (b) and sodium pyruvate (c). The results present the mean values from 6 assays ± SD *P ≤ 0.01
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4383821&req=5

Fig1: Viability a of confluent human skin fibroblasts incubated for 24 and 48 h with different concentrations of oxythiamine (OXY). The mean values ± SD from three independent experiments done in duplicates are presented. *P ≤ 0.01. Collagen biosynthesis measured as 5[3H] proline incorporation into proteins susceptible to the action of bacterial collagenase in fibroblasts treated for 24 and 48 h with different concentrations of (OXY) (b) and sodium pyruvate (c). The results present the mean values from 6 assays ± SD *P ≤ 0.01
Mentions: Since oxythiamine (OXY) was found to inhibit proliferation of tumor cells [34–36], the cytotoxicity assay for different doses of OXY in cultured human fibroblasts was performed. Cell viability was measured by the method of Carmichael et al. [26] using tetrazolium salt. The viability of OXY-treated fibroblasts is presented in Fig. 1a. OXY at studied concentrations did not influence the viability of the cells at 24 and 48 h incubation.Fig. 1

Bottom Line: It was found that OXY-dependent increase in collagen biosynthesis was accompanied by parallel increase in prolidase activity and level, compared to untreated cells.The exposure of the cells to OXY contributed to decrease in IGF-IR, α2β1 integrin receptor, pERK1/2, and NF-κB p65 expressions.The data suggest that OXY-dependent increase of collagen biosynthesis in cultured human skin fibroblasts results from activation of prolidase activity and level, induction in pAkt expression and down-regulation of pERK1/2 and NF-κB p65, the known inhibitor of collagen gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, Medical University of Bialystok, Mickiewicza 2 D, 15-222, Bialystok, Poland.

ABSTRACT
The oxythiamine (OXY) is antivitamin of thiamine. The finding that OXY increases the cytoplasmic concentration of pyruvate, known to enhance collagen biosynthesis, led us to investigate the mechanism of this antivitamin action on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with micromolar concentrations (30-1,000 µM) of OXY for 24 and 48 h. It was found that OXY-dependent increase in collagen biosynthesis was accompanied by parallel increase in prolidase activity and level, compared to untreated cells. Since phosphoenolpyruvate (PEP) is known as an inhibitor of prolidase-the enzyme that plays important role in collagen biosynthesis, the mechanism of pyruvate interconversion was considered as a regulatory switch in collagen biosynthesis. In fact, 3-MPA, specific inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), contributed to up-regulation of prolidase activity, suggesting that down-regulation of PEP formation is an underlying mechanism. Since collagen biosynthesis and prolidase activity are regulated by signal induced by activated α2β1 integrin receptor as well as insulin-like growth factor-I receptor (IGF-IR), the expression of these receptors was measured by Western immunoblot analysis. The exposure of the cells to OXY contributed to decrease in IGF-IR, α2β1 integrin receptor, pERK1/2, and NF-κB p65 expressions. It was accompanied by increase in total ERK1/2 expression and induction of phosphorylation of Akt protein. The data suggest that OXY-dependent increase of collagen biosynthesis in cultured human skin fibroblasts results from activation of prolidase activity and level, induction in pAkt expression and down-regulation of pERK1/2 and NF-κB p65, the known inhibitor of collagen gene expression.

Show MeSH
Related in: MedlinePlus