Abstract
Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius, separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5'rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non-osteocytic teleost fish, but only in calcified cartilage. In addition, our results also indicate that the presence of MGP mRNA in heart tissue is due, at least in fish, to the expression of the MGP gene in only two specific cell types, smooth muscle and endothelial cells, whereas no expression was found in the striated muscle fibers of the ventricle. In light of these results and recent information on expression of MGP gene in these same cell types in mammalian aorta, it is likely that the levels of MGP mRNA previously detected in Xenopus, birds, and mammalian heart tissue may be restricted to regions rich in smooth muscle and endothelial cells. Our results also emphasize the need to re-evaluate which cell types are involved in MGP gene expression in other soft tissues and bring further evidence that fish are a valuable model system to study MGP gene expression and regulation.
Highlights
MATRIX GLA PROTEIN (MGP) is a secreted vitamin K– dependent protein previously found to accumulate within the organic matrix of mammalian bone from which it was originally purified.[1]
We describe the purification of MGP from a teleost fish, A.regius, and deduce its complete amino acid sequence from the cloned cDNA
The tissue distribution of MGP mRNA and sites of protein accumulation were determined by a combination of Northern and in situ hybridization analysis as well as immunohistochemistry using specific antibodies developed against the mature MGP protein
Summary
MATRIX GLA PROTEIN (MGP) is a secreted vitamin K– dependent protein previously found to accumulate within the organic matrix of mammalian bone from which it was originally purified.[1]. MGP was purified from mammalian calcified cartilage,(4) and more recently, the accumulation of the protein was detected at sites of arterial calcification.[5,6,7,8,9] In addition, expression of the MGP gene was found to occur in vitro in different cell lines,(6,10–12) and in vivo in cartilage[13] and in various soft tissues.[3,13,14]. The identification of mammalian-like multinucleated osteoclasts and the occurrence of efficient bone resorption functioning as a calcium homeostasisregulating mechanism is controversial in marine fishes, in those with a non-osteocytic (often called acellular) bone type.[18,19,20] In these marine fish, and in contrast with fresh water fish or higher vertebrates, osteoblasts do not usually become trapped as osteocytes in the mineralized matrix of vertebra but recede to the periphery of the bone matrix as mineralization proceeds. Chondrocytes, on the other hand, are usually found within hyaline cartilage that does not mineralize, or are inserted into an Alcian blue stainable extracellular matrix indicative of the presence of cartilage-specific mucopolysaccharides[21] and at the borders of cartilaginous regions undergoing calcification.[19]
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