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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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Western blot analysis for α2-integrin (a), β1-integrin (b) receptor subunits, IGF-I receptor (c), pERK1/2 (d), ERK1/2 (e), pAkt (f), and NF-κB p 65 (g) in control human skin fibroblasts and cultured for 24 and 48 h in the medium containing different concentrations of OXY. The mean values of six pooled cell homogenate extracts from six separate experiments are presented. The intensity of the bands was quantified by densitometric analysis. Densitometry was done with BioSpectrum Imaging System and presented as arbitrary units b. The same amount of supernatant protein (20 μg) was run in each lane. The expression of β-actin served as a control for protein loading (h)
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Fig4: Western blot analysis for α2-integrin (a), β1-integrin (b) receptor subunits, IGF-I receptor (c), pERK1/2 (d), ERK1/2 (e), pAkt (f), and NF-κB p 65 (g) in control human skin fibroblasts and cultured for 24 and 48 h in the medium containing different concentrations of OXY. The mean values of six pooled cell homogenate extracts from six separate experiments are presented. The intensity of the bands was quantified by densitometric analysis. Densitometry was done with BioSpectrum Imaging System and presented as arbitrary units b. The same amount of supernatant protein (20 μg) was run in each lane. The expression of β-actin served as a control for protein loading (h)

Mentions: Collagen biosynthesis and prolidase activity were previously shown to be regulated due to the signal induced by activated α2β1 integrin receptor [17] as well as IGF-IR [12, 24]. Therefore, the expression of α2β1 integrin receptor (receptor for type I collagen) and IGF-IR was measured by Western immunoblot analysis. As can be seen in Fig. 4a, 24 and 48 h treatment of fibroblasts with 30, 100, 300 and 1,000 µM of OXY contributed to a distinct decrease in the expression of α2 integrin subunit to about 94, 81, 79, and 46 % respectively, at 24 h, and to about 84, 75, 72, and 67 %, respectively, at 48 h, compared to the control cells. 24 and 48 h treatment of fibroblasts with 1,000 µM of OXY reduced expression of β1 integrin subunit to about 79 and 85 %, respectively, compared to the control (Fig. 4b). As shown in Fig. 4c, IGF-I receptor expression was decreased for 1,000 µM OXY-treated cells to about 76 and 82 % at 24 and 48 h incubation, respectively, compared to the control cells. We found that the highest concentration of OXY decreases expression of phosphorylated ERK1/2 to about 64 and 81 % at 24 and 48 h incubation, respectively (Fig. 4d) and stimulates expression of total ERK1/2 by about 14 % at 24 h and by about 36 % at 48 h, compared with control (Fig. 4e). Simultaneously, 1,000 µM of OXY-dependent stimulation of total ERK1/2 expressions (Fig. 4e) was accompanied by similar induction of phosphorylation of Akt by about 19 and 20 % at 24 and 48 h (Fig. 4f), suggesting that in the experimental conditions, ERK1/2 and Akt proteins represent signaling molecules that respond to OXY action.Fig. 4


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

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

Western blot analysis for α2-integrin (a), β1-integrin (b) receptor subunits, IGF-I receptor (c), pERK1/2 (d), ERK1/2 (e), pAkt (f), and NF-κB p 65 (g) in control human skin fibroblasts and cultured for 24 and 48 h in the medium containing different concentrations of OXY. The mean values of six pooled cell homogenate extracts from six separate experiments are presented. The intensity of the bands was quantified by densitometric analysis. Densitometry was done with BioSpectrum Imaging System and presented as arbitrary units b. The same amount of supernatant protein (20 μg) was run in each lane. The expression of β-actin served as a control for protein loading (h)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Western blot analysis for α2-integrin (a), β1-integrin (b) receptor subunits, IGF-I receptor (c), pERK1/2 (d), ERK1/2 (e), pAkt (f), and NF-κB p 65 (g) in control human skin fibroblasts and cultured for 24 and 48 h in the medium containing different concentrations of OXY. The mean values of six pooled cell homogenate extracts from six separate experiments are presented. The intensity of the bands was quantified by densitometric analysis. Densitometry was done with BioSpectrum Imaging System and presented as arbitrary units b. The same amount of supernatant protein (20 μg) was run in each lane. The expression of β-actin served as a control for protein loading (h)
Mentions: Collagen biosynthesis and prolidase activity were previously shown to be regulated due to the signal induced by activated α2β1 integrin receptor [17] as well as IGF-IR [12, 24]. Therefore, the expression of α2β1 integrin receptor (receptor for type I collagen) and IGF-IR was measured by Western immunoblot analysis. As can be seen in Fig. 4a, 24 and 48 h treatment of fibroblasts with 30, 100, 300 and 1,000 µM of OXY contributed to a distinct decrease in the expression of α2 integrin subunit to about 94, 81, 79, and 46 % respectively, at 24 h, and to about 84, 75, 72, and 67 %, respectively, at 48 h, compared to the control cells. 24 and 48 h treatment of fibroblasts with 1,000 µM of OXY reduced expression of β1 integrin subunit to about 79 and 85 %, respectively, compared to the control (Fig. 4b). As shown in Fig. 4c, IGF-I receptor expression was decreased for 1,000 µM OXY-treated cells to about 76 and 82 % at 24 and 48 h incubation, respectively, compared to the control cells. We found that the highest concentration of OXY decreases expression of phosphorylated ERK1/2 to about 64 and 81 % at 24 and 48 h incubation, respectively (Fig. 4d) and stimulates expression of total ERK1/2 by about 14 % at 24 h and by about 36 % at 48 h, compared with control (Fig. 4e). Simultaneously, 1,000 µM of OXY-dependent stimulation of total ERK1/2 expressions (Fig. 4e) was accompanied by similar induction of phosphorylation of Akt by about 19 and 20 % at 24 and 48 h (Fig. 4f), suggesting that in the experimental conditions, ERK1/2 and Akt proteins represent signaling molecules that respond to OXY action.Fig. 4

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