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Hyaluronic acid abrogates ethanol-dependent inhibition of collagen biosynthesis in cultured human fibroblasts.

Donejko M, Przylipiak A, Rysiak E, Miltyk W, Galicka E, Przylipiak J, Zaręba I, Surazynski A - Drug Des Devel Ther (2015)

Bottom Line: The application of HA has a protective effect on disturbances caused by the examined substances.It seems that regulatory mechanism of ethanol-induced collagen aberration take place at the level of collagen biosynthesis, since no effect of ethanol and HA was found on process of collagen degradation by MMP-2 and MMP-9.This study provides evidence that ethanol impairs collagen metabolism in human skin fibroblasts, leading to a significant decrease in the amount of produced protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Esthetic Medicine, Medical University of Białystok, Białystok, Poland.

ABSTRACT

Introduction: The aim of the study was to evaluate the effect of ethanol on collagen biosynthesis in cultured human skin fibroblasts, and the role of hyaluronic acid (HA) in this process. Regarding the mechanism of ethanol action on human skin fibroblasts we investigated: expression of β1 integrin and insulin-like growth factor 1 receptor (IGF-IR), signaling pathway protein expression: mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), nuclear factor kappa B (NF-κB) transcription factor, cytotoxicity assay and apoptosis, metalloproteinase activity, as well as the influence of HA on these processes.

Materials and methods: Collagen biosynthesis, activity of prolidase, DNA biosynthesis, and cytotoxicity were measured in confluent human skin fibroblast cultures that have been treated with 25, 50, and 100 mM ethanol and with ethanol and 500 µg/mL HA. Western blot analysis and zymography were performed to evaluate expression of collagen type I, β1 integrin receptor, IGF-IR, NF-κB protein, phospho-Akt protein, kinase MAPK, caspase 9 activity, and matrix metalloproteinases (MMP-9 and MMP-2).

Results: Ethanol in a dose-dependent manner lead to the impairment of collagen biosynthesis in fibroblast cultures through decreasing prolidase activity and expression of β1 integrin and IGF-IR. This was accompanied by an increased cytotoxicity, apoptosis and lowered expression of the signaling pathway proteins induced by β1 integrin and IGF-IR, that is, MAPK (ERK1/2) kinases. The lowered amount of synthesized collagen and prolidase activity disturbance may also be due to the activation of NF-κB transcription factor, which inhibits collagen gene expression. It suggests that the decrease in fibroblast collagen production may be caused by the disturbance in its biosynthesis but not degradation. The application of HA has a protective effect on disturbances caused by the examined substances. It seems that regulatory mechanism of ethanol-induced collagen aberration take place at the level of collagen biosynthesis, since no effect of ethanol and HA was found on process of collagen degradation by MMP-2 and MMP-9.

Conclusion: This study provides evidence that ethanol impairs collagen metabolism in human skin fibroblasts, leading to a significant decrease in the amount of produced protein. This mechanism probably is due to downregulation of prolidase activity, expression of β1 integrin and IGF-IR receptors, and the signaling pathway proteins induced by these receptors.

No MeSH data available.


Related in: MedlinePlus

Western immunoblot analysis for p65 NF-κB (A) protein and β-actin (B) in subconfluent human skin fibroblasts (control), cells treated with or without HA for 24 hours. The intensity of the bands staining was quantified by densitometry analysis.Notes: Student’s t-test: **P<0.01. 1, control; 2, ethanol 25 mM; 3, ethanol 50 mM; 4, ethanol 100 mM; 5, HA; 6, ethanol 25 mM and HA; 7, ethanol 50 mM and HA; 8, ethanol 100 mM and HA. Samples used for electrophoresis consisted of 20 µg protein of pooled cell extracts.Abbreviations: AU, arbitrary units; HA, hyaluronic acid.
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f7-dddt-9-6225: Western immunoblot analysis for p65 NF-κB (A) protein and β-actin (B) in subconfluent human skin fibroblasts (control), cells treated with or without HA for 24 hours. The intensity of the bands staining was quantified by densitometry analysis.Notes: Student’s t-test: **P<0.01. 1, control; 2, ethanol 25 mM; 3, ethanol 50 mM; 4, ethanol 100 mM; 5, HA; 6, ethanol 25 mM and HA; 7, ethanol 50 mM and HA; 8, ethanol 100 mM and HA. Samples used for electrophoresis consisted of 20 µg protein of pooled cell extracts.Abbreviations: AU, arbitrary units; HA, hyaluronic acid.

Mentions: Several data suggested that prolidase-dependent regulation of collagen biosynthesis may take place at the transcriptional level, depending on NF-κB expressions. NF-κB is the transcription factor that acts as inhibitor of expression of alpha 1 and alpha 2 subunits of type I collagen.19,20 We found increased NF-κB expression in fibroblast treated with ethanol (Figure 7A). An addition of HA to cells treated with ethanol contributed to decrease in the expression of this transcriptional factor to the control level. Moreover, expression of β-actin was not affected in the cells when treated with ethanol in the presence or in absence of HA (Figure 7B). It seems that regulatory mechanism of ethanol-induced collagen aberration take place at the point of collagen biosynthesis, since no effect of ethanol and HA was found on process of collagen degradation by MMP-2 and MMP-9 (Figure 8).


Hyaluronic acid abrogates ethanol-dependent inhibition of collagen biosynthesis in cultured human fibroblasts.

Donejko M, Przylipiak A, Rysiak E, Miltyk W, Galicka E, Przylipiak J, Zaręba I, Surazynski A - Drug Des Devel Ther (2015)

Western immunoblot analysis for p65 NF-κB (A) protein and β-actin (B) in subconfluent human skin fibroblasts (control), cells treated with or without HA for 24 hours. The intensity of the bands staining was quantified by densitometry analysis.Notes: Student’s t-test: **P<0.01. 1, control; 2, ethanol 25 mM; 3, ethanol 50 mM; 4, ethanol 100 mM; 5, HA; 6, ethanol 25 mM and HA; 7, ethanol 50 mM and HA; 8, ethanol 100 mM and HA. Samples used for electrophoresis consisted of 20 µg protein of pooled cell extracts.Abbreviations: AU, arbitrary units; HA, hyaluronic acid.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664499&req=5

f7-dddt-9-6225: Western immunoblot analysis for p65 NF-κB (A) protein and β-actin (B) in subconfluent human skin fibroblasts (control), cells treated with or without HA for 24 hours. The intensity of the bands staining was quantified by densitometry analysis.Notes: Student’s t-test: **P<0.01. 1, control; 2, ethanol 25 mM; 3, ethanol 50 mM; 4, ethanol 100 mM; 5, HA; 6, ethanol 25 mM and HA; 7, ethanol 50 mM and HA; 8, ethanol 100 mM and HA. Samples used for electrophoresis consisted of 20 µg protein of pooled cell extracts.Abbreviations: AU, arbitrary units; HA, hyaluronic acid.
Mentions: Several data suggested that prolidase-dependent regulation of collagen biosynthesis may take place at the transcriptional level, depending on NF-κB expressions. NF-κB is the transcription factor that acts as inhibitor of expression of alpha 1 and alpha 2 subunits of type I collagen.19,20 We found increased NF-κB expression in fibroblast treated with ethanol (Figure 7A). An addition of HA to cells treated with ethanol contributed to decrease in the expression of this transcriptional factor to the control level. Moreover, expression of β-actin was not affected in the cells when treated with ethanol in the presence or in absence of HA (Figure 7B). It seems that regulatory mechanism of ethanol-induced collagen aberration take place at the point of collagen biosynthesis, since no effect of ethanol and HA was found on process of collagen degradation by MMP-2 and MMP-9 (Figure 8).

Bottom Line: The application of HA has a protective effect on disturbances caused by the examined substances.It seems that regulatory mechanism of ethanol-induced collagen aberration take place at the level of collagen biosynthesis, since no effect of ethanol and HA was found on process of collagen degradation by MMP-2 and MMP-9.This study provides evidence that ethanol impairs collagen metabolism in human skin fibroblasts, leading to a significant decrease in the amount of produced protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Esthetic Medicine, Medical University of Białystok, Białystok, Poland.

ABSTRACT

Introduction: The aim of the study was to evaluate the effect of ethanol on collagen biosynthesis in cultured human skin fibroblasts, and the role of hyaluronic acid (HA) in this process. Regarding the mechanism of ethanol action on human skin fibroblasts we investigated: expression of β1 integrin and insulin-like growth factor 1 receptor (IGF-IR), signaling pathway protein expression: mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), nuclear factor kappa B (NF-κB) transcription factor, cytotoxicity assay and apoptosis, metalloproteinase activity, as well as the influence of HA on these processes.

Materials and methods: Collagen biosynthesis, activity of prolidase, DNA biosynthesis, and cytotoxicity were measured in confluent human skin fibroblast cultures that have been treated with 25, 50, and 100 mM ethanol and with ethanol and 500 µg/mL HA. Western blot analysis and zymography were performed to evaluate expression of collagen type I, β1 integrin receptor, IGF-IR, NF-κB protein, phospho-Akt protein, kinase MAPK, caspase 9 activity, and matrix metalloproteinases (MMP-9 and MMP-2).

Results: Ethanol in a dose-dependent manner lead to the impairment of collagen biosynthesis in fibroblast cultures through decreasing prolidase activity and expression of β1 integrin and IGF-IR. This was accompanied by an increased cytotoxicity, apoptosis and lowered expression of the signaling pathway proteins induced by β1 integrin and IGF-IR, that is, MAPK (ERK1/2) kinases. The lowered amount of synthesized collagen and prolidase activity disturbance may also be due to the activation of NF-κB transcription factor, which inhibits collagen gene expression. It suggests that the decrease in fibroblast collagen production may be caused by the disturbance in its biosynthesis but not degradation. The application of HA has a protective effect on disturbances caused by the examined substances. It seems that regulatory mechanism of ethanol-induced collagen aberration take place at the level of collagen biosynthesis, since no effect of ethanol and HA was found on process of collagen degradation by MMP-2 and MMP-9.

Conclusion: This study provides evidence that ethanol impairs collagen metabolism in human skin fibroblasts, leading to a significant decrease in the amount of produced protein. This mechanism probably is due to downregulation of prolidase activity, expression of β1 integrin and IGF-IR receptors, and the signaling pathway proteins induced by these receptors.

No MeSH data available.


Related in: MedlinePlus