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Farm Przegl Nauk, 2009,2, 28-34 Depression of β 1 -integrin signalling and IGF-I receptor expression is responsible for oxidative stress – induced inhibition of collagen biosynthesis and cell growth in cultured fibroblasts. Osłabienie sygnału generowanego przez receptor β1 integrynowy oraz ekspresji receptora IGF-I jest odpowiedzialne za indukowane przez stres oksydacyjny upośledzenie biosyntezy kolagenu i wzrostu fibroblastów. paweł sienkiewicz, wojciech Miltyk, jerzy pałka* department of Medicinal chemistry Medical university in bialystok head of the department: prof. jerzy pałka ABSTRACT The molecular mechanism of oxidative stress dependent inhibition of collagen biosynthesis and cell growth was studied in cultured human skin fibroblasts. Insulin-like growth factor-I (IGF-I) and prolidase play an important role in collagen biosynthesis and cell growth. IGF-I stimulates collagen and prolidase gene expression. Prolidase [EC 3.4.13.9] is involved in the recycling of proline for the protein biosynthesis and cell growth. Prolidase expression is under control of signaling by β 1 - integrin receptor. The endpoint of signal transduced by β 1 -integrin receptor, as well as IGF-IR, is activation of MAP-kinases (ERK 1 , ERK 2 ). The effects of oxidative stress on collagen and DNA biosynthesis, expression of prolidase, β 1 -integrin receptor, focal adhesion kinase pp125 FAK (FAK), IGF-IR and MAP- kinases (ERK 1 , ERK 2 ) were evaluated. Subconfluent cells were submitted to oxidative stresses with 30 µM t-butylhydroperoxide (t-BHP) for 1h per day for 3 days. It was found, that oxidative stres induced inhibition of collagen biosynthesis, prolidase expression and cell growth in human dermal fibroblasts. The mechanism of this phenomenon was found at the level of IGFR and β 1 -integrin signaling. It explains the inhibition of collagen and DNA biosynthesis in fibroblasts submitted to oxidative stress. Key words: collagen, cell growth, IGF-I receptor, β 1 - integrins, intracellular signaling, oxidative stress, prolidase 28 STRESZCZENIE Przedmiotem badań jest ocena molekularnego mechanizmu upośledzenia biosyntezy kolagenu i podziałów komórkowych w przebiegu doświadczalnego stresu oksydacyjnego w hodowli fibroblastów. Insulino-podobny czynnik wzrostowy I (IGF-I) i prolidaza pełnią ważną rolę w biosyntezie kolagenu i wzroście komórek. IGF-I pobudza ekspresję genów kolagenu i prolidazy. Prolidaza [EC 3.4.13.9] uczestniczy w recyklingu proliny do biosyntezy białek i wzrostu komórek. Ekspresja prolidazy jest pod kontrolą szlaku sygnałowego indukowanego przez receptor β1 integrynowy. Końcowym etapem szlaku sygnałowego indukowanego przez receptor β1 integrynowy i receptor IGF-I jest aktywacja MAP kinaz, ERK1 i ERK2. Zbadano wpływ doświadczalnego stresu oksydacyjnego na biosyntezę kolagenu i DNA, ekspresję prolidazy, receptorów β1 integrynowego i IG- F-I, białek FAK, ERK1 i ERK2. Fibroblasty przed osiągnięciem stanu inhibicji kontaktowej poddano stresowi oksydacyjnemu przez dodatek do podłoża hodowlanego 30 μM t-butylo-nadtlenku wodoru (tBHP) na okres 1 godziny/dzień powtarzając tą procedurę przez 3 dni. Wykazano, że stres oksydacyjny indukował obniżenie biosyntezy kolagenu, ekspresji prolidazy i wzrostu komórek w hodowli fibroblastów. Wykazano, że mechanizm tego zjawiska obejmuje upośledzenie aktywacji szlaków sygnałowych generowanych przez receptor β1 integrynowy i receptor IGF-I. Wyjaśnia to mechanizm upośledzenia biosyntezy kolagenu i DNA w fibroblastach poddanych stresowi oksydacyjnemu. Słowa kluczowe: fibroblasty, kolagen, prolidaza, receptor IGF-I, receptor β1 integrynowy, szlaki sygnałowe, stres oksydacyjny
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 <strong>numer</strong>ous 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
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