水产品外文文献.pdf

Casein phosphopeptides promote calcium uptake and modulate the differentiation pathway in human primary osteoblast like cells Bianca Maria Donida a Emanuela Mrakb Claudia Gravaghic Isabella Villab Stefania Cosentino a Elena Zacchib Silvia Peregoa Alessandro Rubinaccib Amelia Fiorilli a Guido Tettamantia d Anita Ferrarettoa aDipartimento di Chimica Biochimica e Biotecnologie per la Medicina Universita degli Studi di Milano LITA Via Fratelli Cervi 93 20090 Segrate Italy b Unita Metabolica dell Osso Istituto Scientifi co San Raffaele 20132 Milano Italy cStrang Cancer Research Laboratory Division of Gastroenterology and Hepatology Department of Medicine Weill Medical College of Cornell University 10065 New York USA dIRCCS Policlinico San Donato via R Morandi 30 20097 San Donato Milanese Milano Italy 1 Introduction The possibility to enhance calcium absorptionat intestinal level and subsequently its availability for mineralized tissues has attracted numerous studies at different levels animal and or human In this context the discovery of peptides originating from casein hydrolysis 27 i e casein phosphopeptides or CPPs characterized by the ability to complex and solubilize calcium ions preventing their precipitation has opened the way to the recognition of functional foods and nutraceuticals acting as mineral carriers 12 All CPPs although displaying different amino acid sequence length possess a cluster of three phosphory lated serines followed by two glutamic acids the acidic motif able to bind mono and bivalent metals as calcium but also iron and zinc 12 In vitro studies performed in the rat ileum sacs or ligated segments as well as studies on whole animals demonstrated a positive role on intestinal calcium absorption and bioavailability exerted by CPPs 4 2 8 9 18 19 22 34 although their mechanism of action remained undefi ned The CPP mediated increase in the bioavailable fraction of the mineral at the intestinal level could implement the availability of calcium for bone potentially resulting in an enhancement of bone calcium content and a modulation of the cellular activity The observations that bone weight and calcium content are increased 26 28 30 that bone loss in aged ovariectomized rats is prevented 38 and that bone loss in aged ovariectomized rats is inhibited under dietary CaCPP supplementation 39 fi ts this view When CPP supplementation to rats of increasing age is combined with genistein a phytoestrogen with demonstrated bone remodelling effect 13 35 synergistic anabolic effect on bone formation was observed 24 thus suggesting a potential role of CPPs as cofactors in osteoporosis prevention Peptides 30 2009 2233 2241 A R T I C L EI N F O Article history Received 3 June 2009 Received in revised form 3 August 2009 Accepted 4 August 2009 Available online 12 August 2009 Keywords Casein phosphopeptides Human primary osteoblast like cells SaOS 2 cells Calcium Bone A B S T R A C T Casein phosphopeptides CPPs originating by in vitro and or in vivo casein digestion are characterized by the ability to complex and solubilize calcium ions preventing their precipitation Previous works demonstrated that CPPs improve calcium uptake by human differentiated intestinal tumor cell lines are able to re mineralize carious lesions in a dental enamel and as components of a diet affect bone weight and calcium content in rats The aim of the present study was to evaluate if CPPs can directly modulate bone cells activity and mineralization Primary human osteoblast like cells were established in culture from trabecular bone samples obtained from waste materials during orthopedic surgery Commercial mixtures of bovine casein phosphopeptides were used The CPP dependent intracellular calcium rises were monitored at the single cell level through fura2 fl uorescence assays Results show that CPPs i stimulate calcium uptake by primary human osteoblast like cells ii increase the expression and activity of alkaline phosphatase a marker of human osteoblast differentiation iii affect the cell proliferation rate and the apoptotic level iv enhance nodule formation by human SaOS 2 Taken together these results confi rm the possibility that CPPs play a role as modulator of bone cell activity probably sustained by their ability as calcium carriers Although the exact mechanism by which CPPs act remains not completely clarifi ed they can be considered as potential anabolic factors for bone tissue engineering 2009 Elsevier Inc All rights reserved Corresponding author Tel 39 0250330341 fax 39 0250230365 E mail address anita ferraretto unimi it A Ferraretto Contents lists available at ScienceDirect Peptides journal homepage 0196 9781 see front matter 2009 Elsevier Inc All rights reserved doi 10 1016 j peptides 2009 08 003 Moreover various other studies demonstrated that CPPs are able to re mineralise carious lesions in dental enamel 7 32 stimulate the formation of mineralized bone nodules in an osteosarcoma cell line Saos 2 31 markedly enhance cartilage hypertrophy and provide suffi cient ionic calcium to cartilage calcifi cation in explanted embryonic rat bone rudiments placed inorganculture 15 Alltheseobservationssustainthe hypothesis that CPPs can act as direct modulator of bone cell activity theoretically linked to their mineral carrier ability in situ It has been indeed shown that cytosolic calcium concentra tion in human intestinal cell lines in culture i e HT 29 and Caco2 able to differentiate in vitro toward an enterocytic phenotype is enhanced by CPP exposure 6 10 11 16 It is therefore conceivable that any potential modulation of cytosolic calcium concentration in osteoblast like cells might activate anabolic cell functions Alongthislineofthought wehavedesignedthefollowingstudy to evaluate whether the exposure of primary human osteoblast like cell cultures to CPPs results in the activation of the calcium messenger system with subsequent modulation of cell prolifera tion differentiation and or activity A mixture of peptides coming from a trypsin hydrolysate of bovine casein known as CPP already characterized and used in studies with intestinal cells 6 10 11 16 was here employed 2 Materials and methods Joklik s modifi ed MEM and all other cell culture reagents unless otherwise specifi ed were from Sigma St Louis MO USA Iscove s modifi edDulbecco smedium IMDM wasfromBiowhittaker Lonza Milanosrl Italy FetalbovineserumwasfromEuroclone CelbioSpA Milan Italy while 1 25 DihydroxyvitaminD3 1 25 OH 2D3 Fura 2 acetoxymethyl ester fura 2 AM and fura 2 pentasodium salt were all obtained from Calbiochem La Jolla CA USA Thapsigargin Bay K 8644 and Nifedipine were all obtained from Sigma St Louis MO USA 2 1 Primary human osteoblast like cell hOBs in vitro culture Human bone cells were established in culture by a modifi cation of the Gehron Robey and Termine procedure 14 from trabecular bone samples obtained from waste materials during orthopedic surgery for degenerative diseases or traumatic fractures of the femoralneckrequiringosteotomyprocedures Noneofthepatients 66 83 years old submitted to surgery had any metabolic or malignant bone disease A written consent for the use of the waste material was obtained from each patient Briefl y the trabecular bone was cut into small pieces 2 mm 2 mm 2 mm and washed thoroughly with commercial standardized Joklik s mod ifi ed MEM serum free medium to remove non adherent marrow cells The bone pieces were incubated with the same medium containing 0 5 mg ml type IV collagenase at 37 8C for 30 min with rotation The collagenase digestion was stopped by adding Iscove s modifi ed Dulbecco s medium IMDM containing 10 fetal bovine serum The bone pieces 8 10 for each patient were then placed in 25 cm2 fl asks and cultured in IMDM medium containing 10 FBS 100 U ml penicillin 100mg ml streptomycin 50 U ml mycostatin and 0 25mg ml amphotericin B Cells began to migrate within 1 2 weeks and reached confl uence after 1 month Culture medium was changed every 2 3 days The cell population was tested for alkalinephosphataseandosteocalcinproductiontoensurethatthe cells were endowed with osteoblast characteristics In some experiments alkaline phosphatase and osteocalcin production were evaluated after cell pre treatment 48 h with 10 8M 1 25 OH 2D3 All cells were used at the fi rst passage to reduce the possibility of phenotype changes 2 2 Human SaOS 2 cells in vitro culture Human SaOS 2 cells SaOS 2 HTB 85 ATCC LGC standards Milan Italy weremaintainedbypassagingweeklyin75 cm2 fl asks containing IMDM medium supplemented with 10 FBS 2 mML glutammine 0 1 mg Lstreptomycin 100 000 U Lpenicillin 0 25 mg L amphotericin B and maintained in a humidifi ed atmo sphere of 5 CO2at 37 8C 2 3 Casein phosphopeptides CPPs The most used CPP preparation CPP is a casein derived hydrolysate CE 90 CPP III DMV International Veghel The Netherlands constituted by several components each containing the characteristic CPP cluster sequence Ser P Ser P Ser P Glu Glu displaying the following composition 93 8 as dry matter 96 purity 10 8 total nitrogen content 3 7 phosphor ous content nitrogen phosphorous ratio 3 1 P Ser molar ratio 0 85 average molecular weight 2500 This CPP mixture was assessed to be calcium free as already reported 10 For the intracellular calcium measurement experiments CPP was dis solved in doubly distilled water in stock solutions 1000 concentrated with respect to the fi nal concentration and stored at 20 8C For in vitro mineralization experiments SaOS 2 cells were treated either with CPP DMV or with a different commercial CPP mixture CPP MD by MD Foods Ingredients Amba Videbaek Denmark enriched in calcium 6 6 w w and comprising almost peptidesfrombovinebcaseinasCPPDMV manuscript submitted To distinguish from CPP DMV devoid of calcium ions CPP MD is here mentioned as Calcium CPP 2 4 Measurement of intracellular calcium concentration Ca2 i at a single cell level hOBs were seeded on a glass coverslips 24 mm diameter thickness0 13 0 17 mm VWRInternational WestChester Pennsylvania USA in Petri dishes 35 mm diameter Costar Concorezzo Italy at 5 10 104cells coverslip All the experi ments were performed after 48 h serum starvation Cytoplasmic calcium was measured according to the procedure described by Tsien and Poenie 36 Briefl y cells on glass coverslips were incubated for 15 min at 37 8C with 5mM Fura 2 AM and 2 5mM PluronicF 127in1 mlKrebs Ringer HEPESsolution KRH containing in mM NaCl 125 0 KCl 5 0 KH2PO41 2 CaCl22 0 MgSO41 2 glucose 6 0 and HEPES 25 0 and adjusted to pH 7 4 After incubation cells were extensively rinsed with KRH and maintainedforadditional10 minatroomtemperaturetoallowde esterifi cation of the fl uorescent probe The glass coverslip was then mounted in a thermostatted TC 202 A perfusion chamber PDMI 2 from Medical System Corporation Harvard Apparatus Hollis ton MA USA and placed on the microscope stage TE 200 Nikon Tokyo Japan where the cells incubated in 2 ml of KRH were alternately excited at 340 380 nm through a 40 oil immersion objective NA 1 3 Nikon Tokyo Japan The emitted fl uorescence at 510 nm was measured at 1 2 s intervals by a CCD intensifi ed camera Extended Isis Photonic Science Millham UK and ratio images of single cells averaged over 8 frames within a chosen window of 50 70 cells were collected and analyzed after background subtraction using a Fluorescence image acquisition and data analysis system which was supplied by Applied Imaging High Speed Dynamic Video Imaging Systems Quanticell 700 Sunderland UK The amount of free calcium within the cells Ca2 i was calculated from the 340 380 nm images by means of a calibration performed with external standards of calcium and Fura 2 according to the equation of Grynkiewicz et al 17 The B M Donida et al Peptides 30 2009 2233 22412234 duration of each experiment never exceeded 2 min a period of time during which the cells appeared to maintain full viability The extracellular calcium concentration Ca2 o was kept at 2 mM and the intracellular calcium concentration was continuously mon itoredinsinglecells Forexperimentsintheabsenceof extracellular calcium CaCl2was omitted from the KRH but it wasnot possibletouseEGTAsincethiscalciumchelatordrastically affects hOB viability Under these experimental conditions usually known as nominally calcium free the calcium concentration is about 0 5 1mM and is due to the presence of calcium ions in the doubly distilled water The single cell analysis provided the following results i percentage of responsive cells i e the percentage of cells which responded to CPP administration with Ca2 iincrements equal or above 20 nM ii the mean single maximum Ca2 irise calculated for each single cell subtracting the baseline to the peak value after CPP administration and averaged for all the analyzed cells In experiments performed to characterize calcium entry release cellsweretreatedwith10mMThapsigargin 10mMBay K8644and 10mM Nifedipine according to the following protocols Cells buffered in 2 mM Ca2 owere exposed to 10mM Thapsigargin a specifi cendoplasmiccalciumpumpinhibitorwhichactsbyinducing animmediatereleaseofstoredintracellularcalcium eitherbeforeor after CPP addition while changes in the Ca2 iwere continuously monitored In the case of nominally calcium free conditions Thapsigargin was added to cells before CPPs CPPs were only added after having monitored the return of Ca2 ito the baseline Bay K 8644 a known L type calcium channels agonist evoking calcium infl ux from the extracellular space by activating the L type calcium channels is added to cells in the presence of 2 mM Ca2 o Nifedipine a selective L type calcium channel blocker requiring 10 min to act was added before CPP exposure 2 5 Cell proliferation assay After 48 h serum starvation hOB cells 5 10 103cells well cultured in 96 well plates were incubated for 24 h with either 1 28 mM CPP or 100 nM 1 25 OH 2D3 At the end of the incubation cells were submitted to a 2 h pulse with bromodeoxyuridine BrdU and BrdU incorporation into DNA quantifi ed by the chemiluminiscent immunoassay Roche Applied Science Milan Italy following the manufacturer s instructions The results are expressed as percentage referred to control cells CRT 2 6 Alkaline phosphatase activity ALP determination After 48 h serum starvation hOBs were treated with either 1 28 mM CPP or 100 nM 1 25 OH 2D3for 48 h and alkaline phosphatase activity was determined in the cell layer solubilized with 0 5 ml 0 1 SDS by measuring the p nitrophenol phosphate reductionasalreadydescribed 16 ResultswereexpressedasmU mg protein 1 Unit being defi ned as the enzyme activity that hydrolyses 1mmol of substrate min The protein content was measured following the Lowry et al method 23 The results are reported as percentage referred to control cells CRT 2 7 Reverse transcriptase and real time PCR The relative expression of specifi c osteoblast differentiation markers such as runx2 alkaline phosphatase ALP and osteo calcin OC was evaluatedby real time PCR in hOBs obtained from four different donors After 48 h serum starving hOBs at confl uence in Petri dishes were treated for 4 and 24 h with CPP 1 28 mM 1 25 OH 2D3treated hOBs were used as positive controls Total RNA was extracted using TRIzol according to the manufacturer s instruction Invitrogen Life Technology Inc Paisley UK RNA pellets were dissolved in sterile distilled water and their concentrations were assessed by spectrophotometric analysis OD260 280 One microgram of total RNA was retro transcribed in a total volume of 25ml using an oligodT primer 0 5mM 200 U of M MLV Reverse Transcriptase deoxynucleo tides 0 5 mM M MLV reaction Buffer 1 and rRNasin Ribonu clease Inhibitor 1 U ml Promega Italia srl Milan Italy Relative quantifi cation of runx2 ALP and OC gene expression was performed on an ABI PRISM 7900 sequence detector Applied Biosystems Foster City CA USA using 10 ng cDNA of the RT PCR solution in a fi nal volume of 25ml The primer probe sets were purchasedasAssay on DemandforgeneexpressionfromApplied Biosystems Foster City CA Real time PCR was performed with FAMlabelledspecifi cprobesforthetargetgenesdetectionandVIC labelled probes for the detection of the house keeping gene glyceraldehyde 3 phosphate dehydrogenase GAPDH used as endogenous control All primers were chosen either to span exon junction or to lie in different exons to prevent amplifi cation of genomic DNA Real time PCR was run according to the following protocol an initial step of 2 min at 50 8C and 10 min at 95 8C followed by 40 cycles of 15 s at 95 8C and 1 min at 60 8C mRNA levels were quantifi ed using the comparative threshold cycle Ct method First the amount of target mRNA in each sample was normalized to the amount of the house keeper mRNA GAPDH designated as a calibrator to giveDCt Cttarget CtGAPDH Second the amounts of target mRNA in the samples were expressed using the formula amount of target mRNA 2 DDCt whereDDCt DCt sample 1 D Ct untreated sample assuming that the effi cien cies of the PCR reaction were close to 1 Three replicates were performed for each experimental point and experiments were repeated three times 2 8 Lactate dehydrogenase LDH release assay CytoTox ONETM assay After 48 h serum starvation hOBs 5 103cells well seeded in 96 black multiwell plate were treated with 2 56 mM CPP for 24 h The assay was performed following the manufacturer s instruc tions CytoTox ONETMassay Promega Milan Italy Results were expressedandherereportedaspercentagewithrespecttothetotal cell LDH activity determined by cellular lyses with 9 w w Triton X 100 100 total LDH activity 2 9 Determination of CPP effect on hOB apoptosis activity 2 9 1 Apo ONE1assay The Apo ONE1assay is a practical tool to measure the activities of caspase 3 and 7 which are members of the cysteine aspartic acid specifi c protease caspase family playing a key effector role in the apoptosis of mammalian cells 41 The assay consists in a cell lysis followed by the cleavage of the non fl uorescent caspase substrate Z