however , mgp has other properties that may relate to its biological role . it is synthesized in a wide variety of tissues during embryonic life [ 15 ] .
mgp is synthesized in embryonic kidney tubules and mgp protein and vitronectin colocalize at focal sites .
mgp is highly expressed in cancers of the ovary , testes , kidney , prostate , and glioblastomas but its function in neoplastic cells is unknown [ 1215 ] .
mgp is a migration - promoting protein for glioblastoma , suggesting that it can promote the cancer spreading .
vitronectin binding suggested a mechanism to anchor mgp in the extracellular matrix where it may bind to bmps and/or calcium to prevent calcification .
mgp has at least 2 functional domains , a vitronectin extracellular matrix binding domain in the c - terminal and a calcification inhibitory domain in the n - terminal half of the protein .
mutations in human mgp that prevent synthesis of functional mgp suffer from keutel syndrome , with excessive calcification of cartilage and pulmonary artery stenosis [ 5 , 18 , 19 ] .
excessive calcification occurs if mgp is absent , but the mechanism of calcification inhibition is unknown . a postulated mechanism of calcification inhibition is regulation of bone morphogenetic protein-2 ( bmp-2 ) , a cytokine / morphogen in the transforming growth factor beta superfamily [ 10 , 2023 ] .
a role in transport of calcium - phosphate complexes has also been suggested , because mgp binds calcium phosphate crystals and forms a serum complex with fetuin - a and calcium phosphate [ 24 , 25 ] .
calcification inhibition by mgp is connected to the gla residues contained in the n - terminal half of mgp which are involved in binding of bmp-2 , hydroxyapatite , and fetuin - a calcium phosphate complexes [ 22 , 2426 ] .
the gla domain includes the vitamin k - dependent , gamma - carboxyglutamic acids ( gla ) , and all 3 phosphoserines [ 2 , 27 , 28 ] .
the functional role of gla and the gla domain is supported by calcification defects that occur after treatment with warfarin , an inhibitor of gla formation [ 29 , 30 ] .
the present studies show mgp binds fibronectin and augments cell adhesion and spreading on fibronectin .
mgp localizes with fibronectin in many embryonic tissues , possibly augmenting cell matrix interaction in development .
one aspect of mgp function is mediated by binding fibronectin and increasing cell interactions with fibronectin .
matrix gla protein was purified as described from bovine bone , with the final step being reversed phase purification on a c18 column . after purification on hplc ,
mgp is a relatively insoluble protein with an approximate water solubility of 10  g / ml ; purified mgp was kept at 20c in a solution of approximately 40% acetonitrile and 0.06% trifluoroacetic acid in water .
a typical purification peak contained 0.2 to 0.4  mg / ml . the final concentration of mgp was achieved by dilution into aqueous buffer at 10  g / ml or lower .
concentration of mgp was estimated by absorbance at 280  nm , using an extinction coefficient of 1.0  mg / ml = 1.0 absorbance units .
rabbit antibodies to bovine mgp and the purified igg fraction were produced as described using pierce immunopure columns [ 6 , 31 ] .
the mgp peptide corresponding to amino acids 6177 in the secreted human protein nh2-eryamvygynaaynryf - cooh was synthesized by the molecular resource center of the university of tennessee , hsc .
bovine serum albumin , human plasma fibronectin , and its proteolytic fragments , including the iii1-c polypeptide ( anastellin ) , guinea pig liver tissue transglutaminase ( ttg ) , human fibrinogen , gelatin , pooled normal rabbit serum , were purchased from sigma .
the reducible crosslinking chemical dithiobis ( succinimidyl proprionate ) ( dsp ) and the immunopure igg purification kit were purchased from pierce .
protein binding was assessed by the ability of filter immobilized target proteins to bind to iodinated mgp in an overlay buffer .
the methods for assessing mgp binding by nitrocellulose transferred proteins from sds polyacrylamide gels ( sds - page ) and electroblotting were described previously [ 6 , 3133 ] .
briefly , proteins were run in an sds - page and then transferred to shleicher and schuell bas - nc reinforced nitrocellulose membranes by electroblotting on a hoefer electroblot apparatus . in some experiments proteins were transferred to membranes by dot - blotting on a bio - rad bio - dot apparatus as described in .
the membranes are incubated in 1% bovine serum albumin ( bsa ) , 10  mm phosphate , 140  mm nacl , and ph 7.4 ( bsa - pbs ) overnight at 4c .
the filter overlay is performed in bsa - pbs with 0.1% tween 20 buffers containing radiolabeled mgp for 4 hours at ambient temperature and then washed in incubation buffer .
the membrane is exposed to kodak biomax ms film in a kodak biomax transcreen - he intensifying screen and developed on an automatic film developer .
fibronectin ( fn ) , fibrinogen ( fg ) , and vitronectin ( vn ) were mixed with tissue transglutaminase ( ttg ) and iodinated mgp , respectively , in buffer containing 0.05  m tris , 2.5  mm calcium , and 1  mm dithiothreitol ( tcd buffer ) at 37 degrees .
final concentrations of 0.18  mg / ml fn , 0.18  mg / ml fg , or 0.18  mg / ml vn were incubated with 0.0125 
mg / ml ttg and trace amounts of i - mgp ( 2.5  ng / ml ) containing 30,000  dpm . to determine the effect of antibodies to mgp , 31  g / ml rabbit anti - mgp igg was included during incubation ( see figure 2(c ) ) .
after incubation , samples were processed immediately for sds page in sample buffer containing 2% sds and 2% 2-mercaptoethanol treated at 100 degrees for 2  min .
the electrophoretic gels were processed by wrapping in plastic film and exposure to kodak biomax ms film with he enhancing screen for the appropriate time at 80c . after exposure
0.5  mg / ml fibronectin iii1-c polypeptide , 0.5  mg / ml fn , 0.5  mg / ml vn , or 0.5  mg / ml ovalbumin were incubated with trace amounts of -i - mgp ( 2.5  ng / ml , 30,000  dpm ) at ambient temperature for 2 hours in 5  mm phosphate , 75  mm nacl , 2.5  mm edta , and 0.05  mg / ml gelatin ph 7.4 . after 2 hours , either 1  mg / ml dsp or an equal volume of dmso for controls
the reaction was quenched for 15  min at ambient temperature with tris ph 8 to react with free dsp the frozen at 20 degrees for sds gel electrophoresis or gel filtration chromatography . for sds - page , samples are diluted with 2% sds sample buffer ph 6.8 with or without 2% 2-mercaptoethanol and boiled for 2  min . for gel filtration chromatography
, samples are diluted with 4  m guanidine hcl , 0.1  m tris , 0.1% tween 20 , and 0.5  mg / ml gelatin ph 8.0 .
some samples were reduced with 2% 2-mercaptoethanol for 10  min at ambient temperature to break dsp crosslinks before dilution with buffer .
gel filtration chromatography was performed on an ap ( pharmacia - lkb ) sephacryl hr s200 packed in a bio - rad column ( 1.6  19  cm ) equilibrated with 4  m guanidine , 0.1  m tris , and 0.1% tween 20 , ph 8.0 .
constant volume fractions ( 30 drops / fraction ) were collected and counted on a packard auto - gamma counter to determine the elution position of radioactive mgp .
controls were mgp and ovalbumin incubated with dsp , mgp alone , proteins incubated without dsp crosslinking , and dsp treated samples reduced with mercaptoethanol to reduce and break crosslinks . to determine whether mgp affected cell attachment to fibronectin , hela cells were used as an immortal cancer derived cell line that is popular as a model of cell behaviors such as attachment , spreading , and integrin action [ 3437 ] .
initial studies demonstrated they had a dose - dependent cell attachment response to increasing concentrations of the matrix proteins .
short term attachment studies within 1 hour after cell release from feeder plates were performed .
hela cells are grown in liebovitz l-15 medium supplemented with 10% fetal bovine serum ( fbs ) with gentamicin and amphotericin b added at the recommended concentrations .
8095% confluent cells are harvested by release with 5  mm edta in calcium - free pbs ph 7.4 , washed with 10% fbs l-15 and resuspended to 250,000  cells / ml in serum - free l-15 .
the cell suspension is used immediately for cell attachment assays below . in a typical cell attachment assay
, 96-well plates are coated with the indicated concentrations of fibronectin in 50  mm sodium bicarbonate buffer ph 9.6 ( coat buffer ) .
for control with no fibronectin the well is treated with coat buffer alone . after incubating overnight at 4c
covered with parafilm to prevent evaporation , fibronectin solutions are removed and replaced with the indicated amount of mgp in coat buffer or coat buffer alone as control . after incubating at 37c in a humidified atmosphere for 3 hours , the second mgp or control solutions are removed and replaced with blocking solution made with 1% bsa in pbs ph 7.4 which has been heat inactivated at 65c for 30  min .
after 1.5 hours at 37c in a humidified atmosphere , the wells are emptied and 25,000 hela cells added in 0.1  ml of serum - free l-15 medium using an 8-well multiwell pipet .
vitronectin mediated cell attachment experiments were performed with the same procedure , except that vitronectin replaces fibronectin in the plate coating .
the wells are immediately washed twice with serum - free l-15 medium and then fixed with 96% ethanol for 10  min and then stained with 0.1% crystal violet dye in distilled water for 30  min at ambient temperature .
the dye is removed and the wells washed gently with 0.375  ml of distilled water 3 times .
after wells have dried completely , 0.1  ml / well of 0.2% triton x100 is added and incubated at ambient temperature overnight covered with parafilm to prevent evaporation .
the plates are read at 595  nm on a molecular devices spectramax 2500 multiwell plate reader .
data is entered as replicates ( usually 6 but a minimum of 4 replicates for each data point ) into prism 2.0 for analysis , nonlinear regression plotting , and statistical analysis . in some experiments
the first fibronectin coat is replaced by a first mgp coat and then followed by a second coat of fibronectin to ascertain the effect of switching the order of addition of fibronectin and mgp . in some experiments after an initial coat of fibronectin the remaining nonspecific protein binding sites on the plate are blocked with bsa - pbs before addition of mgp to see if mgp effects required nonspecific protein binding sites on wells . in other experiments the effect of rabbit anti - mgp antibody on mgp enhancement of fibronectin - cell binding was assessed by adding 31  g / ml ( 0.0312 a280/ml ) of anti - mgp igg in the second coat step containing mgp .
hela cells are grown and harvested nonenzymatically as described above and , after washing once with l-15 supplemented with 10% fbs , cells are resuspended in serum - free l-15 medium to 15000  cells / ml .
9000 hela cells/0.6  ml are added to each chamber of lab - tek 4 chamber glass slides .
lab - tek chamber slides are previously coated with 0.0 , 0.2 , or 0.4  g / ml of fibronectin in coat buffer overnight at 4c and then a second coat of 3  g / ml mgp for 3 hours at 37c and then blocked with heat inactivated bsa - pbs for 1.5  h at 37c as described above .
cells attach for 2 hours and then are rinsed gently twice with serum - free l-15 medium .
freshly made 1% paraformaldehyde in pbs is added to fix cells for 10  min at ambient temperature , washed pbs for 10  min , and then treated with 0.1% triton x-100 in pbs for 5  min to permeabilize cells .
the bsa - pbs is then replaced with 1  :  40 dilution of phalloidin alexafluor488 in heat inactivated bsa - pbs for 20  min .
after one wash with heat inactivated bsa - pbs the chambers are washed twice with pbs .
the slides are placed in a 10  cm petri dish immersed in pbs and then imaged using 10x or 40x water immersion objectives on a zeiss axiophot fluorescence microscope equipped with a digital camera .
1416 random microscopic field images taken on the 10x objective were analyzed , with a minimum of 100  cells analyzed for each condition .
the procedures in nih image are as follows : an introduction and tutorial were used to determine cell areas .
briefly , low power images of cells taken with the 10x objective were modified by autocontrast and grayscale conversion in photoshop .
the images were opened in nih image , scaled to fit the window , and modified with the lutz tool to turn cells red then under analyze , analyze particle .
after examination to manually remove clumped / adherent cells from analysis , the results are copied into excel as area / cell .
the cell spreading analysis is similar to that used in a previous publication measuring cell attachment and spreading on titanium surfaces .
paraffin embedded sections of formalin fixed day 19 embryos were obtained as described previously [ 4 , 6 ] .
the sections were deparaffinized with xylene , rehydrated , and boiled for 2  min in 0.01  m sodium citrate , ph 6 for antigen retrieval as described previously .
sections were blocked with buffer containing normal horse serum at 1.5% then incubated with rabbit anti - mgp at 20  g / ml or mouse antifibronectin monoclonal antibody ( clone ist-3 sigma ) at 1  :  5000 dilution .
after an overnight 4c incubation and washing , biotinylated horse antiuniversal igg ( vector ) is added .
images were collected on a zeiss axiophot microscope equipped with a pc and 6.3x and 40x objectives .
as seen in figure 1(a ) , overlay of dot - blotted proteins with radioactive mgp demonstrates that mgp binds to fibronectin and fibronectin fragment iii1-c ( anastellin ) .
mgp did not bind ovalbumin or other fragments of fibronectin with the exception of the 110 kilodalton fibronectin fragment that weakly binds to mgp .
mgp binds to the sds - page band of fibronectin iii1-c ( figure 1(b ) ) .
we previously reported that mgp binds to vitronectin and fibronectin but not laminin , type ii collagen , osteocalcin , tissue transglutaminase , chondroitin sulfate glycosaminoglycan , biglycan , beta - casein , ovalbumin , or bovine serum albumin .
figure 1(c ) shows that mgp61 - 77 also binds to the first type iii fibronectin domain .
ttg was incubated in mixtures of i - mgp with fibronectin , fibrinogen , or vitronectin , respectively .
the products were analyzed by autoradiographs of samples run on sds - page to identify shifts in radioactive mgp ( figure 2(a ) ) .
radioiodinated mgp was incorporated into higher fibronectin ( fn ) and fibrinogen ( fg ) bands .
there was no mgp incorporated into higher molecular weight bands for mgp incubated with fn or fg the absence of ttg .
transglutaminase shifted a small amount of mgp to higher molecular weight but not to the size or extent found with either fibrinogen or fibronectin .
the band at the bottom of the figure 2(a ) is free radioiodinated mgp .  
figure 2(b ) shows the coomassie blue stained gel exposed to film in figure 2(a ) .
ttg converted only a small fraction of the fibronectin ( fn ) to multimers as there is little change in lanes with and without ttg .
ttg was more efficient at crosslinking fibrinogen ( fg ) to multimers which can be as additional bands when ttg is present but not in samples without ttg ( figure 2(b ) ) .
note that the coomassie blue stained band in all lanes is bsa , a component of the buffer used to separate radioiodinated mgp from unincorporated iodine during iodination .
mgp in the ttg crosslinked fibronectin lane comigrated at the stacking gel surface or the top of the running gel .
the presence of anti - mgp polyclonal antibodies blocked the ability of tissue transglutaminase to incorporate mgp into a fibronectin multimer ( figure 2(c ) ) . increasing
the concentration of ttg increased the amount of mgp shifted to higher molecular weight with fibronectin .
transglutaminase could incorporate mgp into larger complexes of fibronectin but not with vitronectin , ovalbumin , or bovine serum albumin . 

chemical crosslinking could confirm that the mgp and iii1-c are in close proximity in solution .
i - mgp and iii1-c , vitronectin , or ovalbumin was incubated under associative conditions then reacted with dsp , a disulfide containing homobifunctional crosslinking agent .
the prediction is that interacting proteins would become covalently crosslinked in the presence of a crosslinking agent .
if dsp is added to a mixture of fibronectin iii1-c and i - mgp , a higher molecular weight crosslinked species is observed on autoradiographs after sds - page ( figure 3(a ) , iiic , 2me , and + dsp lane ) . if samples are reduced with 2-mercaptoethanol ( 2me ) , the dsp crosslink is reduced and larger bands disappear ( figure 3(a ) iiic , + 2me , and + dsp lane ) .
no additional higher molecular weight bands are present for iiic in the absence of dsp crosslinker ( iiic , 2me , and dsp lane ) .
the lack of higher molecular weight species is due to disruption of protein interactions in 2% sds buffer heated to 100c .
mgp and either vitronectin or fibronectin was crosslinked by dsp although not to the same extent as seen with iii1-c ( figure 3(a ) , vn and fn , 2me , and + dsp lanes ) .
it is possible that the increased crosslinking of iii1-c and mgp results from aggregation of the bound complex that increases crosslinking efficiency .
ovalbumin is a negative control for binding ; mgp and ovalbumin were not crosslinked by dsp .
if the products of dsp crosslinked i - mgp and fibronectin iii1-c are denatured in 4  m guanidine containing buffer then analyzed by sephacryl s200 gel filtration , an elution profile with mgp shifted to a peak near the excluded volume , ve , is the result ( figure 3(b ) , filled circles ) .
if the crosslinked sample is reduced with mercaptoethanol prior to column loading , the elution profile is similar to controls , for example , i - mgp alone ( figure 3(b ) , open circles ) .
the i mgp peak normally elutes around fraction 37 with a minor peak at fraction 28 ( figure 3(b ) , open triangles ) .
control reactions with i - mgp with dsp , i - mgp + iii1-c in the absence of dsp were identical in gel filtration elution profile with mgp alone or the reduced crosslink ( open diamonds or squares , resp . ) .
a four - fold decreased amount of iii1-c in the mixture with labeled mgp and dsp resulted in a smaller s200 column ve peak ( data not shown ) .
the data show that iii1-c and the crosslinking agent were necessary to produce the shift in the mgp peak and are consistent with a close interaction between fibronectin iii1-c and mgp .
figure 4(a ) shows that cell attachment increases with increasing fibronectin until a maximum is reached at fibronectin coating concentrations between 0.8 and 3.2  g / ml ( solid line with closed circles ) .
mgp enhanced cell attachment to fibronectin ( open circles with the dashed line , figure 4(a ) ) .
the cell attachment curve for equal amounts of fibronectin is shifted to the left if mgp is added at 3  g / ml . the result is analogous to increasing the apparent affinity of cells for equivalent amounts of fibronectin ( figure 4(a ) ) .
in addition , mgp did not increase cell attachment beyond the maximum observed for fibronectin alone .
the lack of inherent cell attachment activity for mgp is in agreement with the absence of cell binding activity previously reported .
in contrast to mgp augmenting cell attachment to fibronectin , mgp did not augment attachment of cells to vitronectin .  
there was a small inhibitory effect of mgp on cell binding to vitronectin , but in some experiments there was no significant effect .
under the same conditions and cell line , mgp consistently augments cell attachment on fibronectin but not on vitronectin .
antibody to mgp inhibited the ability of mgp to augment cell attachment ( figure 4(b ) ) .
anti - mgp rabbit igg abolished the ability of mgp to enhance cell attachment to fibronectin ( mgp compared to mgp + antimgp , p  .05 ) , but an equal amount of nonspecific rabbit igg did not .
the presence of igg at the concentrations used did not affect cell attachment to fibronectin , because anti - mgp igg or control igg added in the absence of mgp was identical to the control fibronectin alone ( control , figure 4(b ) ) .
increasing amounts of mgp dose dependently enhanced cell attachment to fibronectin ( figure 4(c ) ) .
a dose response curve of increasing mgp ( 0.0 , 0.8 , 3.0 , and 6.0  g / ml ) reveals a change in apparent affinity with an increasing shift of the binding curve to the left indicating increased cell attachment at each concentration of fibronectin . again , maximum binding of cells to fibronectin was not affected ( figure 4(c ) ) .
there was no inherent mgp cell attachment activity , because cells did not attach even at the highest amounts of mgp ( 6.0  g / ml ) if fibronectin was not present .
cells attach and spread on fibronectin as integrins and other cell adhesive proteins form focal adhesions which connect to the actin cytoskeleton . to determine whether mgp affects cell spreading on fibronectin , the average area of cells attached to fibronectin alone or fibronectin plus mgp was determined by calculation of average cell area from over 100  cells as described in experimental procedures .
mgp induced significantly greater cell spreading at each fibronectin concentration ( figure 5(a ) , fn versus fn + mgp , p  .0001 indicated by asterisk ) .
there were too few cells attached in the mgp alone wells to measure spreading ( mgp does not have cell adhesive activity on its own ) .
the appearance of selected microscopic fields of cells adherent on 0.4  g / ml fibronectin coated surfaces is shown is in figures 5(b ) and 5(c ) .
the appearance of selected microscopic fields of cells on the same concentration of fibronectin plus mgp is shown in figures 5(d ) and 5(e ) .
it was previously shown that mgp in the rat embryonic kidney localizes in the developing ureter and the collecting tubules .
fibronectin is widely distributed in the rat embryonic kidney , including in the ureter and collecting tubules ( figures 6(a ) , 6(b ) , and 6(c ) ) .
localization of mgp in a serial section reveals that fibronectin is present in some of the same collecting tubules and ureter epithelium as mgp ( figures 6(d ) , 6(e ) , and 6(f ) ) .
arrows in figures 6(b ) , 6(c ) , 6(e ) , and 6(f ) indicate similar structures in serial sections with both fibronectin and mgp .
fibronectin has a wider distribution within the developing kidney , being present in other tubules with little or no mgp , in connective tissue areas , and in forming glomeruli ( figures 6(a)6(c ) ) . developing glomeruli has little or no mgp at this stage of development ( figure 6(d ) ) .
this confirms the results from a previous publication that showed that mgp binds to fibronectin , vitronectin , and fibrinogen but not tissue transglutaminase , type ii collagen , fibromodulin , osteocalcin , osteonectin , heparin , decorin , casein , or ovalbumin .
the current study shows that mgp binds to a region of fibronectin made up by its first type iii repeat called iii1-c or anastellin .
mgp did not bind the 30 , 45 kilodalton fragments of fibronectin that do not contain a type iii repeat [ 40 , 41 ] .
mgp showed some affinity for a 110 kilodalton fragment of fibronectin that is comprised of many type iii repeats [ 40 , 41 ] .
a region of mgp comprised of amino acids 6177 was proposed as an extracellular matrix binding region .
the current study shows that mgp6177 peptide binds to fibronectin and also to the fibronectin iii1-c fragment .
for example , human mgp is originally 84 amino acids long but has 7 amino acids removed from the c - terminus , so that the mature protein is 77 amino acids long and ends with the peptide sequence identical to the mgp6177 peptide used for this study .
while bovine mgp was used in this study , the results imply that the human mgp will bind to extracellular matrix proteins via a sequence at its carboxyl terminal .
the proteolytic processing of the mgp c - terminal to remove amino acids 7884 may relate to activating a protein interaction site .
the crosslinked multimerized fibronectin formed by incubation with ttg is analogous to the insoluble matrix fibronectin present in many tissues .
the close association of mgp and fibronectin suggested that mgp may become incorporated into fibronectin crosslinked multimers .
indeed , addition of mgp during crosslinking of fibronectin by ttg resulted in its incorporation into a larger multimer form of fibronectin .
the i mgp remained with the fibronectin multimer after boiling in 2% sds that dissociates the mgp and fibronectin interaction .
it is possible that transglutaminase crosslinks mgp to fibronectin , although further studies are necessary to prove crosslinking occurs and determine which amino acids of mgp and fibronectin are involved .
the shift of smaller amounts of mgp to higher molecular weight by ttg suggested it may become crosslinked to itself or to ttg , but we were unable to directly establish that mgp is a substrate of ttg .
to prove mgp was a substrate for ttg we attempted incorporation of radiolabeled putrescine as described .
the association of mgp with crosslinked fibronectin confirms a close association of mgp with fibronectin .
although mgp binds to vitronectin , ttg did not incorporate mgp into a high molecular weight component with vitronectin .
the results suggest that mgp may become a component of crosslinked fibronectin and fibrinogen in vivo .
this has been established by overlay assays and by dsp crosslinking of mgp to the fibronectin iii1-c polypeptide .
overlay assays show that the c - terminal mgp 6177 amino acid peptide can bind to fibronectin and to the iii1-c polypeptide .
a noncarboxylated form of mgp ( ucmgp or glu - mgp ) accumulates in calcified atherosclerotic plaque [ 22 , 30 ] .
further studies will be necessary to determine whether glu - mgp and mgp share binding for fibronectin and the iii1-c domain .
this is not simply an additive effect of providing more adhesive protein , because mgp by itself has no adhesive activity .
the lack of inherent cell adhesion activity suggests that mgp acts by altering the ability of cells to bind fibronectin .
mgp binding of fibronectin seems to be the key , because the enhanced cell attachment was observed in bsa - coated surfaces in which mgp - fibronectin binding was the only way that the enhancement could occur .
mgp enhanced cell attachment to fibronectin but not to vitronectin , another extracellular matrix protein bound by mgp .
these studies explored the mechanism of mgp binding to fibronectin and the effect of exogenous fibronectin and mgp on cell attachment and spreading .
hela cells are a well characterized cell line that is a popular model for cell attachment .
hela cells express the integrins for fibronectin and vitronectin binding [ 3437 ] . the goal was to characterize the effect of mgp for fibronectin binding and cell - fibronectin interactions in a short , 1 hour period when
cellular synthesis and secretion of fibronectin , mgp , and other attachment proteins could be minimized .
there has been a positive correlation of mgp expression with tumor progression and poor prognosis in glioma [ 15 , 16 , 45 ] , but a negative correlation of mgp expression with tumor progression and metastasis in renal and prostate carcinoma   and decreased mgp has been found in colon carcinoma .
future studies should determine whether mgp has the effect in multiple normal and neoplastic cell types .
other studies could determine the effect of mgp for migration and invasion assays in hela compared to other cancer cell types , but longer term studies need to consider the ability of cells to synthesize fibronectin , mgp , integrins , or metalloproteinases that affect migration and invasion .
the expression of mgp mrna is very high in the embryonic rat kidney [ 1 , 4 ] .
we demonstrated that mgp protein is also synthesized during this time and that localization of mgp mrna was strongest in the developing ureter and collecting tubules on embryonic days 1719 .
immunolocalization studies showed mgp localized in embryonic ureters and collecting tubules also contained abundant fibronectin .
calcification inhibition and bmp-2 binding requires posttranslational carboxylation to produce gla in a vitamin k - dependent step [ 22 , 26 , 29 ] .
the phosphorylation of 3 serine residues at the amino terminal end of mgp is necessary for its secretion to inhibit deposition of hydroxyapatite in the extracellular matrix [ 2 , 47 ] .
the amino terminal 154 amino acids of mgp contains the phosphoserines and gla necessary for calcification inhibition , whereas a c - terminal peptide mgp6177 has been shown previously to bind vitronectin and in the current studies to bind fibronectin [ 6 , 26 ] .
it is not clear whether the binding of fibronectin or modulating the cell response to fibronectin is related to the calcification inhibitory activity of mgp .
it is more likely that this mgp property is related to migration - promoting activity that is demonstrated for glioblastoma cells .
it is possible that mgp is a multifunctional protein that can act to modify cell - matrix interactions during embryonic life or in cancer cells and also as a calcification inhibitor in selected adult tissues .
this could help explain why many tissues that contain mgp synthesizing cells do not become calcified in mgp knockout animals and in keutel syndrome , a human disease characterized by loss of mgp function .
mgp binds via a 6177 amino acid sequence present at the physiologic c - terminus .
mgp can become part of transglutaminase crosslinked multimers of fibronectin , suggesting it may be a component of fibronectin matrices .
cells attach and spread better on fibronectin and mgp coated surfaces than to fibronectin alone , even though mgp itself has no cell attachment activity . in tissues , mgp localizes near fibronectin , suggesting that interactions between the proteins are possible in vivo .
the ability of mgp to alter cell interactions with fibronectin is a potential reason for cancer cells and certain embryonic cells to overexpress mgp .
 
 background . matrix gla protein ( mgp ) is a vitamin k - dependent , extracellular matrix protein . 
 mgp is a calcification inhibitor of arteries and cartilage . 
 however mgp is synthesized in many tissues and is especially enriched in embryonic tissues and in cancer cells . 
 the presence of mgp in those instances does not correlate well with the calcification inhibitory role . 
 this study explores a potential mechanism for mgp to bind to matrix proteins and alter cell matrix interactions . methods . to determine whether mgp influences cell behavior through interaction with fibronectin , we studied mgp binding to fibronectin 
 , the effect of mgp on fibronectin mediated cell attachment and spreading and immunolocalized mgp and fibronectin . 
 
 results . 
 first , mgp binds to fibronectin . 
 the binding site for mgp is in a specific fibronectin fragment , called iii1-c or anastellin . 
 the binding site for fibronectin is in a mgp c - terminal peptide comprising amino acids 6177 . 
 second , mgp enhances cell attachment and cell spreading on fibronectin . 
 mgp alone does not promote cell adhesion . 
 third , mgp is present in fibronectin - rich regions of tissue sections . conclusions . 
 mgp binds to fibronectin . 
 the presence of mgp increased cell - fibronectin interactions . 
 
1. Introduction
2. Materials and Methods 
3. Results
4. Discussion
5. Conclusions