The mechanism of hydralazine-induced collagen biosynthesis in cultured fibroblasts

Abstract

The finding that hydralazine (HYD) affects collagen metabolism led us to investigate the mechanism of its action on collagen biosynthesis, prolidase expression and activity, expression of α2β1 integrin, insulin-like growth factor-I receptor (IGF-IR), focal adhesion kinase (FAK), mitogen-activated protein (MAP) kinases (ERK1, ERK2), and transcription factors hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB p65 (NF-κB p65) in human dermal fibroblasts. Confluent fibroblasts were treated with micromolar concentrations (50-500 μM) of HYD for 24 h. HYD had no influence on cell viability. It was found that HYD-dependent increase in collagen biosynthesis was accompanied by a parallel increase in prolidase activity and expression, HIF-1α expression, and decrease in DNA biosynthesis, compared to untreated cells. Since collagen biosynthesis and prolidase activity are regulated by a signal induced by activated α2β1 integrin receptor as well as IGF-IR, the expression of these receptors was measured by Western immunoblot analysis. The exposure of the cells to HYD contributed to the increase in IGF-IR expression without any effect on α2β1 integrin receptor and FAK expressions. It was accompanied by a decrease in expression of MAP kinases and NF-κB p65, the known inhibitor of collagen gene expression. The data suggest that the HYD-dependent increase of collagen biosynthesis in cultured human skin fibroblasts results from activation of IGF-IR expression and prolidase activity and downregulation of NF-κB p65.

Fig. 1

Collagen biosynthesis (a) measured as 5[³H] proline incorporation into proteins susceptible to the action of bacterial collagenase and prolidase activity (b) in confluent human skin fibroblasts incubated for 24 h in the medium containing 10 % FBS and different concentrations of HYD or CAP. The results present the mean values from six assays ± SD. The asterisk indicates P ≤ 0.05. Western blot analysis for prolidase (c), phosphorylated prolidase (d), hypoxia-inducible factor (HIF-1α) (e), in control human skin fibroblasts (lane 1) and cultured in the medium containing 50 μM of HYD (lane 2), 100 μM of HYD (lane 3), 250 μM of HYD (lane 4), and 500 μM of HYD (lane 5). 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. Twenty micrograms of supernatant protein was run in each lane for prolidase and β-actin Western blot analysis. 60 μg of supernatant protein was run in each lane for HIF-1α Western blot analysis. The expression of β-actin served as a control for protein loading (f)

Fig. 2

DNA synthesis (a) and viability (b) of confluent human skin fibroblasts incubated for 24 h with different concentrations of hydralazine. The mean values ± SD from three independent experiments done in duplicates are presented. The asterisk indicates P ≤ 0.05

Fig. 3

Western blot analysis for α₂-integrin (a), β₁-integrin (b) receptor subunits, FAK (c), IGF-I receptor (d), ERK1/ERK2 (e), and NF-κB p65 (f) in control human skin fibroblasts (lane 1) and cultured in the medium containing 50 μM of HYD (lane 2), 100 μM of HYD (lane 3), 250 μM of HYD (lane 4), and 500 μM of HYD (lane 5). 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. 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 (g)