Gla-Rich Protein Is a Novel Vitamin K-Dependent Protein Present in Serum That Accumulates at Sites of Pathological Calcifications
2009; Elsevier BV; Volume: 175; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.090474
ISSN1525-2191
AutoresCarla Viegas, Sofia Cavaco, Pedro Leão Neves, Ana Carina Ferreira, Alexandre João, Matthew K. Williamson, Paul A. Price, M. Leonor Cancela, Dina C. Simes,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoMineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization. Mineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization. Extracellular matrix (ECM) calcification can be either a physiological or a pathological process depending on site and time of occurrence. Physiological ECM calcification is restricted to bone and to the hypertrophic zones of growth plate cartilage, whereas pathological or ectopic ECM calcification, defined as inappropriate biomineralization occurring in soft tissues and consisting of calcium phosphate salts that include hydroxyapatite, is an abnormal process that can occur virtually in any tissue of the body.1Schinke T McKee MD Karsenty G Extracellular matrix calcification: where is the action?.Nat Genet. 1999; 21: 225-229Crossref PubMed Scopus (126) Google Scholar However, skin, kidney, tendons, and the cardiovascular system appear particularly prone to develop this pathology.2Giachelli CM Ectopic calcification: gathering hard facts about soft tissue mineralization.Am J Pathol. 1999; 154: 671-675Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar First considered to be a passive process occurring as a nonspecific response to tissue injury or necrosis, recent evidence now indicates that ECM calcification is a naturally occurring process that must be actively inhibited and starts to appear as soon as inhibitors are removed from the matrix.1Schinke T McKee MD Karsenty G Extracellular matrix calcification: where is the action?.Nat Genet. 1999; 21: 225-229Crossref PubMed Scopus (126) Google Scholar, 3Wallin R Wajih N Greenwood GT Sane DC Arterial calcification: a review of mechanisms, animal models, and the prospects for therapy.Med Res Rev. 2001; 21: 274-301Crossref PubMed Scopus (202) Google Scholar, 4Danziger J Vitamin K-dependent proteins, warfarin, and vascular calcification.Clin J Am Soc Nephrol. 2008; 3: 1504-1510Crossref PubMed Scopus (195) Google Scholar In a healthy organism, cells appear to synthesize natural inhibitors of mineralization that prevent ectopic calcification, which initiates when disequilibrium occurs between expression of calcification inhibitors and enhancers, emphasizing the need for a tight regulation to prevent ectopic calcifications. Key genes known to be involved in the regulation of this complex process are those acting as calcification inhibitors such as matrix Gla protein (MGP), osteocalcin (BGP), bone sialoprotein (BSP), osteoprotegerin (Opg), and fetuin.1Schinke T McKee MD Karsenty G Extracellular matrix calcification: where is the action?.Nat Genet. 1999; 21: 225-229Crossref PubMed Scopus (126) Google Scholar, 3Wallin R Wajih N Greenwood GT Sane DC Arterial calcification: a review of mechanisms, animal models, and the prospects for therapy.Med Res Rev. 2001; 21: 274-301Crossref PubMed Scopus (202) Google Scholar Among those, MGP, a vitamin K-dependent protein (VKD), is widely accepted as playing a pivotal role in preventing soft tissue calcification, local mineralization of the vascular wall,5Cranenburg EC Schurgers LJ Vermeer C Vitamin K: the coagulation vitamin that became omnipotent.Thromb Haemost. 2007; 98: 120-125Crossref PubMed Scopus (190) Google Scholar and more recently, skin elastic fiber mineralization in pseudoxanthoma elasticum (PXE)6Gheduzzi D Boraldi F Annovi G DeVincenzi CP Schurgers LJ Vermeer C Quaglino D Ronchetti IP Matrix Gla protein is involved in elastic fiber calcification in the dermis of pseudoxanthoma elasticum patients.Lab Invest. 2007; 87: 998-1008Crossref PubMed Scopus (82) Google Scholar, 7Li Q Jiang Q Schurgers LJ Uitto J Pseudoxanthoma elasticum: reduced gammaglutamyl carboxylation of matrix gla protein in a mouse model (Abcc6-/-).Biochem Biophys Res Commun. 2007; 364: 208-213Crossref PubMed Scopus (48) Google Scholar, 8Hendig D Zarbock R Szliska C Kleesiek K Götting C The local calcification inhibitor matrix Gla protein in pseudoxanthoma elasticum.Clin Biochem. 2008; 41: 407-412Crossref PubMed Scopus (29) Google Scholar and in scleroderma with and without calcinosis.9Davies CA Jeziorska1 M Freemont AJ Herrick AL Expression of osteonectin and matrix Gla protein in scleroderma patients with and without calcinosis.Rheumatology. 2006; 45: 1349-1355Crossref PubMed Scopus (42) Google Scholar It is also known that several factors, such as insufficient intake of vitamin K, mutations in the γ-carboxylase enzyme, and warfarin treatment, which can all induce arterial10Price PA Faus SA Williamson MK Warfarin causes rapid calcification of the elastic lamellae in rat arteries and heart valves.Arterioscler Thromb Vasc Biol. 1998; 18: 1400-1407Crossref PubMed Scopus (470) Google Scholar, 11Schurgers LJ Spronk HM Soute BA Schiffers PM DeMey JG Vermeer C Regression of warfarin-induced medial elastocalcinosis by high intake of vitamin K in rats.Blood. 2007; 109: 2823-2831Crossref PubMed Scopus (249) Google Scholar, 12Schurgers LJ Spronk HM Skepper JN Hackeng TM Shanahan CM Vermeer C Weissberg PL Proudfoot D Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification.J Thromb Haemost. 2007; 5: 2503-2511Crossref PubMed Scopus (204) Google Scholar and skin calcifications,7Li Q Jiang Q Schurgers LJ Uitto J Pseudoxanthoma elasticum: reduced gammaglutamyl carboxylation of matrix gla protein in a mouse model (Abcc6-/-).Biochem Biophys Res Commun. 2007; 364: 208-213Crossref PubMed Scopus (48) Google Scholar, 13Li Q Jiang Q Pfendner E Váradi A Uitto J Pseudoxanthoma elasticum: clinical phenotypes, molecular genetics and putative pathomechanisms.Exp Dermatol. 2009; 18: 1-11Crossref PubMed Scopus (125) Google Scholar, 14Li Q Schurgers LJ Smith AC Tsokos M Uitto J Cowen EW Co-existent pseudoxanthoma elasticum and vitamin K-dependent coagulation factor deficiency: compound heterozygosity for mutations in the GGCX gene.Am J Pathol. 2009; 174: 534-540Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 15Vanakker OM Martin L Gheduzzi D Leroy BP Loeys BL Guerci VI Matthys D Terry SF Coucke PJ Pasquali-Ronchetti I De Paepe A Pseudoxanthoma elasticum-like phenotype with cutis laxa and multiple coagulation factor deficiency represents a separate genetic entity.J Invest Dermatol. 2007; 127: 581-587Crossref PubMed Scopus (155) Google Scholar may act by reducing or abolishing γ-carboxylation of VKD proteins. Those pathologies have also been associated with a loss of MGP function, until now considered to be the central Gla protein for prevention of connective tissue mineralization, both in the vascular system and skin. Although many efforts have been made to understand the mechanisms controlling these abnormal calcifications, the existence of other potential, still unknown, calcification inhibitors has been suggested to explain some reported phenotypes and occurrences that are not completely justified by the presence or absence of MGP.1Schinke T McKee MD Karsenty G Extracellular matrix calcification: where is the action?.Nat Genet. 1999; 21: 225-229Crossref PubMed Scopus (126) Google Scholar, 16Nanda A Anim JT Al-Gareeb M Alsaleh QA Keutel syndrome with overlapping features of cutis laxa: a new variant.Am J Med Genet A. 2006; 140: 1487-1489Crossref PubMed Scopus (14) Google Scholar, 17Boer-van den Berg MA Verstijnen CP Vermeer C Vitamin K-dependent carboxylase in skin.J Invest Dermatol. 1986; 87: 377-380Crossref PubMed Scopus (13) Google Scholar We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), with an unprecedented high content of Gla residues and uncommonly high capacity to bind calcium, with orthologs in all taxonomic groups of vertebrates and highly conserved throughout evolution (78% identity between sturgeon and human GRP).18Viegas CS Simes DC Laizé V Williamson MK Price PA Cancela ML Gla-rich Protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates.J Biol Chem. 2008; 283: 36655-36664Crossref PubMed Scopus (93) Google Scholar GRP mRNA was found to be highly expressed in sturgeon cartilaginous tissues, and in rat skeletal tissues, both cartilage and bone, which invalidated the concept that this protein could be solely a specific marker for distal chondrocytes, as previously proposed by others.18Viegas CS Simes DC Laizé V Williamson MK Price PA Cancela ML Gla-rich Protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates.J Biol Chem. 2008; 283: 36655-36664Crossref PubMed Scopus (93) Google Scholar In this study we show, for the first time, that GRP is a circulating protein also expressed and accumulated in soft tissues like skin and vascular system of rats and humans and that it is clearly associated with calcification pathologies in these tissues, being highly accumulated at sites of ectopic mineral deposits. Furthermore, the extensive number of Gla residues (16 Gla residues in sturgeon and, by comparison, 15 in all mammals) and the absence of other identifiable functional domains, together with our in vivo and in vitro evidence for a high mineral binding affinity, strongly suggest that GRP might be a potent physiological modulator of soft tissue calcification, acting by directly influence mineral formation and or recruitment, and an important new player in the complexity of phenotypes involving connective tissue mineralization, whose mechanisms and regulatory pathways remain to be fully understood. This study was approved by the Faro Hospital and Lisbon Central Hospital ethics committee. We included in our study patients with stage 5 chronic kidney disease who underwent surgery for arteriovenous fistula creation. A sample of the radial artery wall was collected at the time of surgery from each patient. Calcified and noncalcified carotid samples were also collected at autopsy. Skin biopsies were taken under local anesthesia from the affected skin of PXE, dermatomyositis with calcinosis, and scleroderma with calcinosis (lateral neck or axilla) patients. All patients exhibited clinical signs and were diagnosed for the corresponding pathology. Control skin biopsies were obtained from forearm regions of volunteer healthy subjects. Human blood was collected from volunteer healthy subjects by venipuncture at Faro Hospital. Informed consent was obtained from all participants. Pig ears were obtained from the local slaughterhouse, immediately frozen for transport, and kept at −80° until further processing. Rat skin and blood samples were obtained from Mus musculus specimens maintained at the University of Algarve animal facilities. Total RNA was extracted from rat adult tissues (including bony, cartilaginous, and major soft tissues) as described.19Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar One microgram of total RNA was treated with RQ1 RNase-free DNase (Promega, Madison, WI) and reverse-transcribed at 37°C with MMLV-RT (Invitrogen, San Diego, CA) using specific reverse primers RnGRP1R (5′-CACTCAAAAACAAGACAAAGCAAACATCCG-3′), RnGAPDH_RT1R (5′-GAAGACGCCAGTAGACTCCACGACAT-3′) and RnHPRTI_RT1R (5′-CACAAGGGAAGTGACAATCTACCTGACG-3′). Quantitative real-time polymerase chain reaction (qPCR) was performed with an iCycler iQ apparatus (Bio-Rad, Amadora, Portugal), using primer sets RnGRP1F (5′-TCCTTCCTACCTCTACAACCGCCAAAA-3′)/RnGRP1R to amplify rat GRP, RnGAPDH_RT1F (5′-CGGCAAGTTCAACGGCACAGTCAAG-3′)/RnGAPDH_RT1R to amplify rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and RnHPRTI_RT1F (5′-AAATGTCTGTTGCTGCGTCCCTTTTGAT-3′)/RnHPRTI_RT1R to amplify rat HPRTI. PCR reactions, set up in duplicates, were performed as previously described18Viegas CS Simes DC Laizé V Williamson MK Price PA Cancela ML Gla-rich Protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates.J Biol Chem. 2008; 283: 36655-36664Crossref PubMed Scopus (93) Google Scholar using Absolute QPCR SYBR green fluorescein mix (ABgene, Epsom, UK). Fluorescence was measured at the end of each extension cycle in the FAM-490 channel and melting profiles of each reaction were performed to check for unspecific product amplification. Levels of gene expression were calculated using the comparative method (ΔΔCt) and normalized using gene expression levels of GAPDH or HPRTI housekeeping genes. Gene expression in lung was set to 1 and used as reference for relative expression in other tissues. qPCR was performed in quadruplicates and a normalized SD was calculated. Samples were collected as previously described18Viegas CS Simes DC Laizé V Williamson MK Price PA Cancela ML Gla-rich Protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates.J Biol Chem. 2008; 283: 36655-36664Crossref PubMed Scopus (93) Google Scholar and included either in paraffin or in Historesin Plus (Leica Microsystems, Lisbon, Portugal), according to the manufacturer's instructions. Mineral deposits were detected with silver nitrate (Sigma-Aldrich, Taufkirchen, Germany) by the von Kossa method, and physiological structures were identified by counterstaining with hematoxylin and eosin or toluidine blue.20Ortiz-Delgado JB Simes DC Viegas CS Schaff BJ Sarasquete C Cancela ML Cloning of matrix Gla protein in a marine cartilaginous fish. Prionace glauca: preferential protein accumulation in skeletal and vascular systems.Histochem Cell Biol. 2006; 126: 89-101Crossref PubMed Scopus (17) Google Scholar A 417-bp fragment of rat GRP cDNA (spanning from nucleotide 417 to the 3′ end) cloned in pCRII-TOPO was either linearized with ApaI and transcribed with SP6 RNA polymerase to generate an antisense riboprobe or linearized with KpnI and transcribed with T7 RNA polymerase to generate a sense riboprobe. A 364-bp fragment of the human GRP cDNA (spanning from nucleotide 459 to 822 according to EST sequences retrieved from GenBank sequence data base and previously identified as GRP),18Viegas CS Simes DC Laizé V Williamson MK Price PA Cancela ML Gla-rich Protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates.J Biol Chem. 2008; 283: 36655-36664Crossref PubMed Scopus (93) Google Scholar amplified by PCR with HsGRPis2F (5′-CATCCTATCTCTACAACCGCCACC-3′) and HsGRPis1R (5′-TTCAGCGTTTTTATTTGTAAGCCATA-3′) primers and genomic DNA, and cloned in pCRII-TOPO, was either linearized with KpnI and transcribed with T7 RNA polymerase to generate an antisense riboprobe or linearized with ApaI and transcribed with SP6 RNA polymerase to generate a sense riboprobe. Probes were then labeled with digoxigenin using RNA labeling kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. RNA in situ hybridization was performed on paraffin sections of rat and human tissues with digoxigenin-labeled antisense riboprobes, as previously described.18Viegas CS Simes DC Laizé V Williamson MK Price PA Cancela ML Gla-rich Protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates.J Biol Chem. 2008; 283: 36655-36664Crossref PubMed Scopus (93) Google Scholar Briefly, sections were digested with 40 μg/ml proteinase K (Sigma) in 1X phosphate-buffered saline containing 0.1% Tween 20 (Sigma) for 30 minutes and then hybridized at 68°C overnight in a humidified chamber. After hybridization, sections were washed and the signal revealed with the alkaline phosphatase-coupled antidigoxigenin-AP antibody (Roche) and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate solution (Sigma). Negative controls for GRP mRNA detection were performed with sense probes. Affinity-purified rabbit polyclonal antibody against GRP was obtained from SDI- Strategic Diagnostics (Newark, DE), using for immunization a synthetic amino acid peptide corresponding to the C terminus of rat GRP (RQWHYDGLYPSYLYNRQNI), synthesized by NeoMPS, Inc. (San Diego, CA), purified to 95% purity, and conjugated to keyhole limpet hemocyanin. A cysteine residue was introduced at the N terminus of the peptide for binding to keyhole limpet hemocyanin. The affinity purified antiserum was termed CTerm-GRP. Pig and rat skin (dermis and epidermis) and human blood vessels were cleaned from fat tissue, lyophilized, and reduced to fine powder. From the calculated weight a 10-fold excess (w/v) of 4 mol/L guanidine HCl (Sigma-Aldrich) extraction solution was added with vigorous stirring at 4°C for 24 hours, and the extracted material was separated by centrifugation for 10 minutes at 10000 × g. A portion of these crude guanidine extract was dialyzed against 50 mmol/HCI, using 3500 molecular weight tubing (Spectra-Por 3, Spectrum, Gardena, CA) with four changes of medium over 2 days and analyzed for the presence of GRP by Western blotting using the purified CTerm-GRP antibody. Rat GRP was further isolated from the crude guanidine HCl extract by reverse-phase high performance liquid chromatography as previously described.18Viegas CS Simes DC Laizé V Williamson MK Price PA Cancela ML Gla-rich Protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates.J Biol Chem. 2008; 283: 36655-36664Crossref PubMed Scopus (93) Google Scholar Resulting fractions were analyzed for the presence of GRP and Gla-containing proteins by dot blot using the CTerm-GRP and M3B (American Diagnostica, Stamford, CT) antibodies, respectively. For further isolation, pig guanidine HCl extract was incubated with 0.1% hydroxyapatite (Calbiochem, San Diego, CA) for 24 hours at 4°C, with constant rotation. After incubation, hydroxyapatite was separated from the crude extract by centrifugation for 10 minutes at 10000 × g. Hydroxyapatite was further cleaned by washing twice with 6 mol/L guanidine HCl for 1 hour and twice with distillated water for 30 minutes at room temperature with constant stirring. The resulting hydroxyapatite powder was demineralized using a 10-fold excess of 10% formic acid for 4 hours at 4°C with vigorous stirring, as described for bone demineralization18Viegas CS Simes DC Laizé V Williamson MK Price PA Cancela ML Gla-rich Protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates.J Biol Chem. 2008; 283: 36655-36664Crossref PubMed Scopus (93) Google Scholar, 21Simes DC Williamson MK Delgado JBO Viegas CSB Price PA Cancela ML Purification of Matrix Gla protein (MGP) from a marine teleost fish. Argyrosomus regius: calcified cartilage and not bone as the primary site of MGP accumulation in fish.J Bone Min Res. 2003; 18: 244-259Crossref PubMed Scopus (39) Google Scholar and further dialyzed against 50 mmol/L HCl as described above. Aliquots of the formic acid dialyzed extract were analyzed by Western blotting using the purified CTerm-GRP antibody. Aliquots of total protein were fractionated into a 4 to 12% gradient polyacrylamide precast gel containing 0.1% sodium dodecyl sulfate (NuPage, Invitrogen), and protein profile revealed by staining the gel as described.18Viegas CS Simes DC Laizé V Williamson MK Price PA Cancela ML Gla-rich Protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates.J Biol Chem. 2008; 283: 36655-36664Crossref PubMed Scopus (93) Google Scholar, 21Simes DC Williamson MK Delgado JBO Viegas CSB Price PA Cancela ML Purification of Matrix Gla protein (MGP) from a marine teleost fish. Argyrosomus regius: calcified cartilage and not bone as the primary site of MGP accumulation in fish.J Bone Min Res. 2003; 18: 244-259Crossref PubMed Scopus (39) Google Scholar Transfer onto nitrocellulose membranes (Amersham Biosciences, Carnaxide, Portugal) and protein immunodetection was performed as described previously.21Simes DC Williamson MK Delgado JBO Viegas CSB Price PA Cancela ML Purification of Matrix Gla protein (MGP) from a marine teleost fish. Argyrosomus regius: calcified cartilage and not bone as the primary site of MGP accumulation in fish.J Bone Min Res. 2003; 18: 244-259Crossref PubMed Scopus (39) Google Scholar, 22Simes DC Williamson MK Schaff BJ Gavaia PJ Ingleton PM Price PA Cancela ML Characterization of osteocalcin (BGP) and matrix Gla protein (MGP) fish specific antibodies: validation for immunodetection studies in lower vertebrates.Calcif Tissue Int. 2004; 74: 170-180Crossref PubMed Scopus (19) Google Scholar GRP protein was detected by incubating blots overnight with 5 μg/ml anti-CTerm-GRP antibody in 5% (w/v) nonfat dried milk powder in Tris-buffered saline/Tween 20 (15 mmol/L NaCl, 10 mmol/L Tris-HCl buffer, pH 8, 0.05% Tween 20) as primary antibody and alkaline phosphatase-labeled goat anti-rabbit IgG antibody (Sigma-Aldrich) diluted 1:30,000 in Tris-buffered saline/Tween 20, as secondary antibody. Visualization of immunoreactive bands was achieved using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate solution (Calbiochem) as described.21Simes DC Williamson MK Delgado JBO Viegas CSB Price PA Cancela ML Purification of Matrix Gla protein (MGP) from a marine teleost fish. Argyrosomus regius: calcified cartilage and not bone as the primary site of MGP accumulation in fish.J Bone Min Res. 2003; 18: 244-259Crossref PubMed Scopus (39) Google Scholar, 22Simes DC Williamson MK Schaff BJ Gavaia PJ Ingleton PM Price PA Cancela ML Characterization of osteocalcin (BGP) and matrix Gla protein (MGP) fish specific antibodies: validation for immunodetection studies in lower vertebrates.Calcif Tissue Int. 2004; 74: 170-180Crossref PubMed Scopus (19) Google Scholar Samples of total protein were applied onto a nitrocellulose membrane (Invitrogen) as previously described.22Simes DC Williamson MK Schaff BJ Gavaia PJ Ingleton PM Price PA Cancela ML Characterization of osteocalcin (BGP) and matrix Gla protein (MGP) fish specific antibodies: validation for immunodetection studies in lower vertebrates.Calcif Tissue Int. 2004; 74: 170-180Crossref PubMed Scopus (19) Google Scholar GRP detection was performed using the purified CTerm-GRP antibody as primary antibody and alkaline phosphatase-labeled goat anti-rabbit IgG (Sigma-Aldrich), as secondary antibody. Gla-containing proteins were detected using 5 μg/ml of the purified monoclonal M3B antibody (American Diagnostica) as primary antibody and alkaline phosphatase-labeled goat anti-mouse IgG as secondary antibody. Immunohistochemical staining experiments were performed on paraffin and Historesin Plus-embedded tissue sections as described.20Ortiz-Delgado JB Simes DC Viegas CS Schaff BJ Sarasquete C Cancela ML Cloning of matrix Gla protein in a marine cartilaginous fish. Prionace glauca: preferential protein accumulation in skeletal and vascular systems.Histochem Cell Biol. 2006; 126: 89-101Crossref PubMed Scopus (17) Google Scholar, 21Simes DC Williamson MK Delgado JBO Viegas CSB Price PA Cancela ML Purification of Matrix Gla protein (MGP) from a marine teleost fish. Argyrosomus regius: calcified cartilage and not bone as the primary site of MGP accumulation in fish.J Bone Min Res. 2003; 18: 244-259Crossref PubMed Scopus (39) Google Scholar Briefly, the endogenous peroxidase activity was blocked with 3% H2O2 in Coons buffer (CBT: 0.1 mol/L Veronal, 0.15 mol/L NaCl, 0.1% Triton X-100) for 15 minutes. Nonspecific antibody binding was blocked with 0.5% (w/v) bovine serum albumin directly after peroxidase activity blocking, or after treatment with chondroitinase ABC (Sigma-Aldrich) (0.1 U/ml) for 1 hour at 37°C. Incubation with the purified polyclonal CTerm-GRP or the M3B antibodies (5 μg/ml and 5 μg/ml, respectively, diluted in CBT), as primary antibodies, was performed overnight in a humidified chamber at room temperature. Peroxidase activity was detected using as secondary antibodies the peroxidase-conjugated goat anti-rabbit and anti-mouse IgG, respectively (Sigma- Aldrich), and 0.025% 3,3-diaminobenzidine (Sigma- Aldrich) as described.20Ortiz-Delgado JB Simes DC Viegas CS Schaff BJ Sarasquete C Cancela ML Cloning of matrix Gla protein in a marine cartilaginous fish. Prionace glauca: preferential protein accumulation in skeletal and vascular systems.Histochem Cell Biol. 2006; 126: 89-101Crossref PubMed Scopus (17) Google Scholar, 21Simes DC Williamson MK Delgado JBO Viegas CSB Price PA Cancela ML Purification of Matrix Gla protein (MGP) from a marine teleost fish. Argyrosomus regius: calcified cartilage and not bone as the primary site of MGP accumulation in fish.J Bone Min Res. 2003; 18: 244-259Crossref PubMed Scopus (39) Google Scholar Negative controls consisted in the substitution of the primary antibody with both normal rabbit serum and CBT. Counterstaining was performed with hematoxylin and eosin or toluidine blue. Human and rat sera were prepared as described,23Cranenburg EC Vermeer C Koos R Boumans ML Hackeng TM Bouwman FG Kwaijtaal M Brandenburg VM Ketteler M Schurgers LJ The circulating inactive form of matrix Gla Protein (ucMGP) as a biomarker for cardiovascular calcification.J Vasc Res. 2008; 45: 427-436Crossref PubMed Scopus (148) Google Scholar, 24Price PA Nguyen TM Williamson MK Biochemical characterization of the serum fetuin-mineral complex.J Biol Chem. 2003; 278: 22153-22160Crossref PubMed Scopus (106) Google Scholar quick-frozen in 1-ml aliquots on dry ice, and stored at −80°C until use. Depletion of the high abundant proteins was performed for each 1 ml of human and rat serum aliquots using the ProteoMiner kit (Bio-Rad), according to the manufacturer's instructions. Five microliters of each of the eluted fractions was applied onto a nitrocellulose membrane (Invitrogen). GRP detection was performed with the purified CTerm-GRP antibody as described in dot blotting. We have recently shown that GRP in sturgeon is expressed exclusively by cartilage-associated cells, while in rat it was also expressed by bone cells. However, our in silico analysis further suggested that GRP could be present in other nonskeletal tissues.18Viegas CS Simes DC Laizé V Williamson MK Price PA Cancela ML Gla-rich Protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates.J Biol Chem. 2008; 283: 36655-36664Crossref PubMed Scopus (93) Google Scholar To continue these studies and establish the pattern of GRP tissue distribution, we have determined its spatial levels of expression in adult rat skeletal and soft tissues. Expression of GRP was detected by real-time PCR in all 16 tissues analyzed, the most significant levels being observed in cartilage- and bone-containing tissues such as skull, inner ear, tail, outer ear, and nose (see Supplemental Figure S1 at http://ajp.amjpathol.org). However, the presence of higher levels of expression in outer ear and nose, a result confirmed when using GAPDH to normalize GRP gene expression (results not shown), could not be easily explained. To further elucidate this question and clearly establish the identity of GRP-expressing cells, we performed in situ hybridization in sections from all rat tissues expressing GRP. Interestingly, in sections of outer ear (Figure 1) and nose (results not shown) GRP was found to be highly expressed i
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