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INTRODUCTION
Oxidative stress plays important role in pathogenesis of
many diseases. Oxygen radicals cause oxidation of nucleic
acids [1], lipids [2] and proteins [3] contributing to disturbances
in cellular metabolism. One of the proteins affected
by oxidative stress is collagen [4-7].
Collagen is the most abundant extracellular protein in
mammals, responsible for maintenance of architecture and
integrity of connective tissue. It also plays an important role
in interaction with integrin receptors, through which it participates
in regulation of numerous physiological and pathological
processes [8, 9].
Collagen biosynthesis in human dermal fibroblasts may
depend on the activity of prolidase [10-12]. Prolidase [EC
3.4.13.9] is a cytosolic enzyme which catalyses hydrolysis
of imidodipeptides (mainly derived from collagen degradation)
[13], releasing proline, which is used for collagen resynthesis
[14] and cell growth [15].
Prolidase activity is known to be induced by β 1 -integrin
receptor activation [16]. Stimulated β 1 -integrin receptor induces
phosphorylation of non-receptor focal adhesion kinase
pp125 FAK (FAK) [17], which is then capable of interacting
with several proteins [18] and subsequently, two mitogen
activated protein kinases (MAPK), extracellular-signal-regulated
kinase 1 (ERK 1 ) and kinase 2 (ERK 2 ) [19]. The end
point of the signaling is activation of transcription factors
and gene expression of many proteins involved in regulation
of cell growth and differentiation [20].
Another factor that stimulates collagen biosynthesis and
cell growth is the insulin-like growth factor- I (IGF-I) [21],
that exerts its effects through interaction with IGF-I receptor
(IGF-IR) [22]. Stimulation of this receptor results in the
activation of the Ras-Raf-mitogen activated protein kinase
(MAPK) pathway, which involves the same signaling proteins
and kinases as β 1 -integrin transduced pathway, except
FAK [23]. The effects of IGF-I action is induction of collagen
gene expression [24], up-regulation of prolidase activity
[25], as well as stimulation of mitotic division and apoptosis
prevention [26].
The current study was undertaken to investigate the effects
of oxidative stress on collagen and DNA biosynthesis,
expression of prolidase, β 1 -integrin receptor, FAK, MAP-
kinases (ERK 1 , ERK 2 ) and IGF-I receptor (IGF-IR) in cultured
fibroblasts.
MATERIALS AND METHODS
Materials. Anti–Goat IgG antibody, Anti–Mouse IgG
antibody, bacterial collagenase, Dulbecco’s phosphate buffered
saline (DPBS), 5-bromo-4-chloro-3-indolyl phosphate/
nitro blue tetrazolium liquid substrate reagent (BCIP/NBT),
L-glycyl-proline, L-proline, monoclonal (rabbit) anti-focal
adhesion kinase pp125 FAK antibody, monoclonal (goat) anti-
IGF-IR antibody, monoclonal (mouse) anti-MAPK antibody,
p-nitrophenol and p-nitrophenyl-β-D-galactopyranoside
were provided by Sigma Corp., USA., as were most other
chemicals and buffers used. t-Butylhydroperoxide (t-
BHP) was the product of Fluka Chemie AG (Germany).
Dulbecco’s minimal essential medium (DMEM) and fetal
copyright © 2009 Grupa dr. A. R. Kwiecińskiego ISSN 1425-5073
Fig. 1. β-galactosidase activity in control human dermal fibroblasts
and the cells submitted to oxidative stress by treatment with 30 µM
t-butylhydroperoxide (t-BHP) as described in the methods section.
Mean values from three independent experiments done in duplicates
are presented. P