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Farm Przegl Nauk, 2009,8, 32-38 Oxygen regulated protein 150 prevents collagen degradation in glucose-deprived fibroblasts Białko ORP150 zapobiega degradacji kolagenu w hodowlach fibroblastów skóry ludzkiej w przebiegu głodzenia Marzanna Cechowska-Pasko Department of Pharmaceutical Biochemistry Medical University of Białystok Abstract Glucose is one of the most common therapeutic agents used in treatment of patients with various diseases. Recent studies demonstrated the effect of glucose on protein synthesis /degradation balance. We decided to study the effect of glucose deprivation on collagen synthesis and degradation in fibroblast cultures and a correlation of these processes with the expression of oxygen/glucose regulated proteins (ORP150/GRP170) and gelatinolytic activity. The incorporation of radiolabeled proline into collagenase-sensitive and hydroxyproline-containing proteins was used as an index of collagen synthesis, whereas pulse-chase technique was employed to evaluate the degradation of newly synthesised proteins. It was demonstrated that fibroblasts incubated in high glucose medium synthesised detectable amounts of collagenous proteins. The shortage of glucose resulted in about 30% reduction in synthesis of collagenous proteins. These phenomena were accompanied by an increase in the expression of chaperon - ORP150 in cultures growing in low glucose medium. Furthermore, the appearance of ORP150 in glucose deprived cultures coexisted with an increase of gelatinolytic activity. The pulse-chase experiments demonstrated that the reduced amount of newly synthesised collagen was protected against intracellular degradation. Proportionally less collagen was degraded in cultures incubated in low glucose than in high glucose media. It may be suggested that the increased expression of ORP150 is a factor which protects collagen against intracellular degradation induced by glucose deprivation. Keywords: glucose deprivation; collagen synthesis; collagen degradation; oxygen-regulated protein 150; fibroblasts Introduction Glucose is one of the most common therapeutic agents used in treatment of patients with various diseases. In most cases it is administered intravenously to supply energetic substrate. It was reported recently by several authors that glucose regulates protein synthesis/degradation balance, stimulating the biosynthesis and protecting proteins against Streszczenie Glukoza jest jednym z najbardziej popularnych składników wielu preparatów o działaniu farmakologicznym stosowanych w leczeniu różnych schorzeń. Ostatnie badania wykazały wpływ glukozy na syntezę/degradację białek. Zdecydowano ocenić wpływ niedoboru glukozy na syntezę i degradację kolagenu w fibroblastach skóry ludzkiej oraz korelację tych procesów z ekspresją białka regulowanego przez tlen/glukozę (ORP150/GRP170) i aktywnością żelatynolityczną. Syntezę kolagenu zbadano na podstawie oceny wbudowywania radioaktywnej proliny do białek wrażliwych na kolagenazę oraz białek zawierających hydroksyprolinę, podczas gdy degradację nowo powstałego białka oceniono przy pomocy techniki „pulse-chase”. Wykazano, że fibroblasty inkubowane w medium o wysokim stężeniu glukozy syntetyzują kolagen. Niedobór glukozy spowodował obniżenie syntezy białek kolagenowych o około 30%, zwiększenie syntezy białka opiekuńczego – ORP150 oraz nasilenie aktywności żelatynolitycznej. Badania „pulse-chase” wykazały, że obniżona ilość nowo zsyntetyzowanego kolagenu była chroniona przed wewnątrzkomórkową degradacją. Proporcjonalnie mniej kolagenu zostało zdegradowane w hodowlach komórek pozbawionych glukozy w porównaniu do inkubowanych w medium o wysokim stężeniu glukozy. Sugeruje się, że ORP150 jest czynnikiem, który chroni kolagen przed wewnątrzkomórkową degradacją, indukowaną przez niedobór glukozy. Słowa kluczowe: niedobór glukozy; synteza kolagenu; degradacja kolagenu; białko ORP 150; fibroblasty intracellular degradation [1-3]. For this reason it was decided to study the effect of glucose on collagen synthesis/ degradation in fibroblast cultures. Collagen is the most abundant extracellular protein in mammals, responsible for maintenance of architecture and integrity of connective tissue. It was described in our previous paper that glucose deprivation resulted in a marked decrease of collagen content in fibroblast cultures [4]. This 32

copyright © 2009 Grupa dr. A. R. Kwiecińskiego ISSN 1425-5073 phenomenon may be evoked by a decrease of collagen biosynthesis, increased degradation of this protein or by coexistence of both phenomena. Obviously collagen biosynthesis requires energy supply. Glucose is the main energetic substrate and glycolysis is the main process which supplies energy for fibroblasts grown in vitro [5]. For these reasons the glucose shortage may reduce intracellular pool of ATP required for the functions of these cells, including collagen biosynthesis. Furthermore, the shortage of glucose in culture medium enhances proteolytic (and gelatinolytic) activity in these cells [1,6] and may promote collagen degradation. On the other hand glucose deprivation appeared to be a factor which induces the expression of oxygen-regulated protein (ORP) 150 in fibroblast cultures [4]. Oxygen-regulated protein of molecular weight 150 kDa (ORP150) and glucose - regulated protein of molecular weight 170 (GRP170) are endoplasmic reticulum (ER) chaperones, which facilitate protein folding [2]. The GRP170 is a glycosylated form of ORP150. The ORP150/GRP170 system is a part of the ER machinery that assists in the folding and assembly of secretory and membrane proteins within the ER [3]. The expression of ORP150 increases in a range of pathologic situations such as brain ischaemia [7], atherosclerotic plaques [8] and malignant tumours [9-11] suggesting that ORP150 is a contributory factor for the cellular response to environmental stress. The role of ORP150 in cellular physiology remains unclear. Some observations indicate that ORP150, like GRP78 and GRP94, is involved in the processing of proteins in the secretory pathway [12]. This allows suggesting that ORP150 is a chaperon, which protects intracellular collagen against proteolytic effects exerted by glucose shortage. In order to test such a hypothesis we decided to study the effect of glucose deprivation in culture medium on proline incorporation into total proteins and specifically into collagenase-sensitive and hydroxyproline-containing proteins. Furthermore, the degradation of newly synthesised collagen and correlation of this process with the expression of glucose-regulated proteins (ORP150/GRP170) was studied. Materials and methods Reagents The DMEM was provided by Invitrogen (San Diego, USA). Passive lysis buffer (Promega, Madison, USA), monoclonal anti-human ORP 150 (IBL, Gunma, Japan), highly purified bacterial collagenase (type VII), Sigma-Fast BCIP/ NBT reagent, alkaline phosphatase-labelled anti-mouse immunoglobulin G were purchased from Sigma (St Louis, USA), as were most other chemicals and buffers used. [2,3,4,5 – 3 H] L-proline was obtained from Hartmann Analytic (Braunschweig, Germany). Nitrocellulose membranes (0.2 μm), Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) molecular weight standards and Coomassie Brilliant Blue R-250 were purchased from Bio-Rad Laboratories (Hercules, CA). Cell cultures The experiments were performed on the human skin fibroblast cell line (CRL-1474) purchased from American Type Culture Collection (ATCC). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), containing glucose at 4.5 mg/ml (high glucose DMEM) supplemented with 10% heat-inactivated foetal calf serum (FCS), 2 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml). The cells were seeded at a density of 5 x 10 5 cells in 2 ml medium and grown on six-well plates, in a 5 % CO 2 incubator, at 37 O C. Experimental procedure 5 x 10 5 cells in 2 ml medium were seeded in six-well plates and incubated for seven days in the high glucose DMEM. In these conditions the cultured cells reached 70-80% confluency. After this time the medium was removed and replaced with 2 ml of the fresh DMEM (without calf serum): 1. the high glucose DMEM contained 4.5 mg of glucose per ml (25mM), 2. the low glucose DMEM contained 0.5 mg of glucose per ml (2.8 mM). Each was supplemented with [2,3,4,5– 3 H] L-proline, ascorbate (50 µg/ml), 2 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml). The incubation was continued for 12, 24, 48 hours. The fibroblast did not divide during the incubation in serum-free medium. No increase in cell density was observed in this period. After incubation the culture media were removed, the cell layers were washed with PBS and submitted to the action of lysis buffer. It allowed separating the cells and extracellular matrix from the bottom of culture vessels and suspending them in the buffer. Filtration Assay Each hydrophilic Durapore membrane (0.65-µm pore size) was soaked with 250µl of 25% TCA. The cell lysates and culture media were treated with 50% TCA (final TCA concentration 25%) and next incubated at 4 o C for 1h. The formed precipitate was collected on the filter membranes and separated from the supernatant using a vacuum source attached to the 1225 Sampling Manifold (Millipore). To remove all unincorporated [ 3 H]-proline, the filters were washed 3 times with 5 ml of 10% TCA by vacuum-filtration through the membrane. After removing the underdrain and drying, the membranes were then punched out into scintillation vials and the filter-bound radioactivity was quantified by liquid scintillation counting [13,14]. The assay of collagenase-sensitive protein The collagenase-sensitive protein was measured using the method described by Petrkofsky and Diegelman [15]. The cell lysates and culture media were treated with high purity bacterial collagenases (type VII-Sigma). N-ethylmaleimide was applied as an inhibitor of unspecific proteolytic activity associated with collagenase. The amount of newly synthesised collagen was expressed in dpm of [ 3 H]-proline, incorporated into protein susceptible to the action of bacterial collagenases [15]. Determination of radioactive hydroxyproline Cell lysate or incubation medium was submitted to hydrolysis in 6 M HCl, at 100 O C, for 16 h. The hydrolysates were evaporated to dryness in a rotary glass evaporator. The 33

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