Matrix Gla Protein mRNA Levels Research Articles

Sodium thiosulfate protects human aortic smooth muscle cells from osteoblastic transdifferentiation via high-level phosphate

Vascular calcification is recognized as a common complication in some patients, such as chronic renal failure. The purpose of this study was to investigate the role of sodium thiosulfate (STS) for the transdifferentiation of human aortic vascular smooth muscle cells into osteoblast-like cells induced by high-level phosphate. All human aortic vascular smooth muscle cells were divided into STS group 1 (treatment with STS) and STS group 2 (culture in a medium containing a high level of phosphate). STS group 1 included a normal group, a high-level phosphate group, and other subgroups based on treatment with different concentrations of STS. Cells of STS group 2 were cultured in a medium containing a high level of phosphate for 72 hours, and then divided into a high-phosphate control group and other subgroups based on treatment with different concentrations of STS. The mRNA and protein expressions of bone morphogenetic protein-2 (BMP-2), core binding factor α-1 (Cbfα-1), and matrix Gla protein (MGP) were detected. Meanwhile, calcium concentration and alkaline phosphatase (ALP) activation were measured. In STS group 1, the mRNA levels of BMP-2 and Cbfα-1 were elevated significantly in the high-level phosphate group compared with the normal group (p < 0.05). However, both gene expressions were attenuated in the STS-treated groups (vs. normal group, p < 0.05). MGP mRNA levels were reduced in the high-level phosphate group (vs. normal group, p < 0.05). In the STS-treated groups, mRNA expression of MGP was elevated compared to the high-level phosphate group (p < 0.05). In STS group 2, expression of MGP was enhanced significantly (vs. high-phosphate control group, p < 0.05) with both BMP-2 and Cbfα-1 reducing in the STS-treated groups (vs. high-phosphate-control group, p < 0.05). STS attenuates calcium concentration and ALP activation. It can reverse osteoblast differentiation of vascular smooth muscle cells and modulate the expressions of calcification-related factors.

Lanthanum Prevents High Phosphate-Induced Vascular Calcification by Preserving Vascular Smooth Muscle Lineage Markers

Vascular calcification (VC) represents a major cardiovascular risk factor in chronic kidney disease patients. High phosphate (Pi) levels are strongly associated with VC in this population. Therefore, Pi binders are commonly used to control high Pi levels. The aim of this work was to study the mechanism of action of lanthanum chloride (LaCl3) on the progression of Pi-induced VC through its direct effect on vascular smooth muscle cells (VSMCs) in vitro. High Pi induced VSCM Ca deposition. We evaluated the action of LaCl3, compared to gadolinium chloride (GdCl3), and found different effects on the modulation of VSMC lineage markers, such as α-actin and SM22α. In fact, only LaCl3 preserved the expression of both VSMC lineage markers compared to high Pi-treated cells. Interestingly, both LaCl3 and GdCl3 reduced the high Pi-induced elevations of bone morphogenic protein 2 mRNA expression, with no reduction of the high core binding factor-alpha 1 mRNA levels observed in calcified VSMCs. Furthermore, we also found that only LaCl3 completely prevented the matrix GLA protein mRNA levels and osteonectin protein expression elevations induced by high Pi compared to GdCl3. Finally, LaCl3, in contrast to GdCl3, prevented the high Pi-induced downregulation of Axl, a membrane tyrosine kinase receptor involved in apoptosis. Thus, our results suggest that LaCl3 prevents VC by preserving VSMC lineage markers and by decreasing high Pi-induced osteoblastic differentiation.

Upregulation of cardiac matrix Gla protein expression in response to hypertrophic stimuli

Matrix Gla protein (MGP) expression is increased in cardiac hypertrophy, but the precise mechanisms regulating its expression are unknown. Here we characterized the effect of pressure overload and myocardial infarction in vivo as well as mechanical stretch and hypertrophic agonists in vitro on MGP expression. When angiotensin II (Ang II) was administered by osmotic minipumps, left ventricular (LV) MGP mRNA levels increased significantly from 6 h to 2weeks, whereas intravenous arginine8-vasopressin increased LV MGP mRNA levels within 4 h. During post-infarction remodeling process, MGP mRNA levels were elevated at 24 h (1.3-fold, p<0.05) and the maximal increase was observed at 4 weeks (2.8-fold, p<0.01). Ang II increased MGP mRNA levels 20% (p<0.05) in neonatal rat cardiac myocytes and 40% (p<0.05) in cardiac fibroblasts, whereas endothelin-1 decreased MGP mRNA levels 30% (p<0.01) in myocytes and had no effect in fibroblasts. Cyclic mechanical stretch resulted in reduction of MGP gene expression in both cardiac myocytes and fibroblasts. These results demonstrate that MGP is rapidly upregulated in response to cardiac overload well before the development of LV hypertrophy and post-infarction remodeling process. Our results also suggest that Ang II may be involved in mediating load-induced activation of MGP expression.

Excess magnesium inhibits excess calcium-induced matrix-mineralization and production of matrix gla protein (MGP) by ATDC5 cells

We found that excessive extracellular Ca 2+ and/or Mg 2+ affected the process of matrix mineralization and glycosaminoglycan (GAG) production by cells of the prechondrogenic cell line, ATDC5. Excess Ca 2+ induced both matrix mineralization and GAG production in the cells. On the other hand, excess Mg 2+ reduced this Ca 2+-mediated rise in both mineralization and GAG production in them. Next we measured the mRNA levels of cartilage-associated genes such as calcium-sensing receptor (CaSR), matrix gla protein (MGP), bone gla protein (BGP), and Runt-related transcription factor 2 (Runx2) in ATDC5 cells. Excess Ca 2+ increased the MGP, BGP, and CaSR mRNA levels, and excess Mg 2+ reduced the Ca 2+-induced increase in the MGP mRNA level in the cells. The changes in the MGP mRNA level paralleled those in the MGP protein level. These data show that Ca 2+ and Mg 2+ regulated the matrix mineralization positively and negatively, respectively, in ATDC5 cells and suggest that excess Mg 2+ might inhibit the excess Ca 2+-promoted mineralization mediated by MGP induction in chondrocytes.

Relationship between immunological rejection and matrix GLA protein in cryopreserved vascular allografts

Introduction Cryopreserved tissue allografts used for cardiovascular diseases become calcified as a late complication after transplantation, probably caused by immunological rejection. Recent attention has been focused on the inhibitory effect of matrix Gla protein (MGP) on ectopic vascular calcification, but the behavior of MGP in cryopreserved allografts is uncertain. In this study we examined the relationship between immunological rejection and MGP in cryopreserved rat aortic grafts after transplantation. Methods Cryopreserved rat aortae were isografted or allografted intraperitoneally. Fresh isografts were also tested. The grafts were retrieved 9 days after transplantation and the intragraft MGP mRNA was measured by a real-time quantitative PCR method. The effect of daily administration of FK506 on MGP mRNA levels in cryopreserved isografts and allografts after transplantation was also evaluated. Results There was no significant difference in intragraft MGP mRNA levels between fresh and cryopreserved isografts 9 days after transplantation. MGP expression levels in cryopreserved allografts were significantly lower as compared to those in cryopreserved isografts ( P < .01). Daily administration of FK506 enhanced intragraft MGP mRNA (ninefold) in cryopreserved allografts ( P < .01), but not in cryopreserved isografts. Conclusions Immunological rejection is likely to inhibit MGP expression in cryopreserved vascular allografts, resulting in late-onset calcification.

Spatial-specific TGF-β1 adenoviral expression determines morphogenetic phenotypes in embryonic mouse lung

The precise spatial-temporal role that expression and activation of transforming growth factor (TGF)-β plays in mammalian organ morphogenesis remains incompletely understood. Using replication deficient adenoviral vectors containing engineered TGF-β1 cDNAs, we studied the spatial effects of locally over-expressing either latent or mutated, constitutively active TGF-β1 protein during embryonic mouse lung branching morphogenesis in culture. Transfer of exogenous genes into lung epithelium was achieved by intra-tracheal microinjection of recombinant adenovirus, while submerging lungs in virus resulted in gene transfer into the pleura and subjacent mesenchymal cells, as revealed by cytochemical staining for β-galactosidase. Only lungs transfected with active, but not latent TGF-β1 gene, showed elevated levels of active TGF-β. Epithelial over-expression of active, but not latent TGF-β1, via intra-tracheal micro-injection inhibited lung branching morphogenesis by 36%. In contrast, lungs submerged with either active or latent TGF-β1 recombinant virus did not demonstrate an inhibitory effect upon branching. Pulmonary gene regulation was assayed by competitive polymerase chain reaction coupled with reverse transcription. Direct respiratory tract micro-injection of adenovirus over-expressing active TGF-β1 resulted in a dose-dependent inhibition of epithelial surfactant protein (SP)-C and SP-B mRNA levels by up to 76% and 70%, respectively, while in contrast, fibronectin and matrix Gla protein (MGP) mRNA levels remained stable. However, lungs that had been submerged in adenovirus expressing active TGF-β1 demonstrated a concentration-dependent induction of both fibronectin and MGP mRNA levels up to 4.3- and 4.7-fold respectively in the presence of 1 X 10 11 pfu/ml active TGF-β1 virus. On the other hand, lungs treated with adenovirus expressing latent TGF-β1 either by micro-injection or submerging failed to demonstrate any regulatory effect either upon epithelial or mesenchymal gene expression. We conclude that adenovector-mediated overexpression of activated TGF-β1 in specific spatial compartments results respectively in either inhibition of branching morphogenesis and epithelium-specific gene expression, or in induction of matrix gene expression without affecting morphogenesis or epithelium-specific gene expression, depending on the route of administration. Also, the lack of effect of latent TGF-β1 over-expression strongly suggests that TGF-β activation per se provides an important locus of fine regulation ofthe spatial effects of TGF-β signaling during embryonic lung branching morphogenesis.

Matrix Gla protein gene expression is induced by transforming growth factor-beta in embryonic lung culture.

Matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein with a wide tissue distribution. The current study was designed to investigate the possible regulation of MGP by exogenous transforming growth factor-beta (TGF-beta) during lung development. Using reverse transcription coupled competitive polymerase chain reaction methodology, we determined that exogenous TGF-beta 1 increases MGP mRNA levels in embryonic mouse lung culture in a concentration-dependent manner. MGP mRNA levels were elevated by 5.0-fold at 50 ng/ml TGF-beta 1 in E11 embryonic mouse lungs explanted for 4 days in serumless culture. MGP mRNA stimulation by TGF-beta 1 was a time-dependent event: MGP message increment was initially detected after 1 day in culture, and MGP mRNA levels continued to increase up to 4 days in the presence of TGF-beta 1. In addition, embryonic lungs in serumless medium without exogenously added TGF-beta 1 showed an increase, although to a lesser extent, in pulmonary MGP mRNA level during culture, indicating that MGP expression is also developmentally regulated. The present results indicate that MGP gene expression can be stimulated by exogenous TGF-beta 1 during early embryonic mouse lung branching morphogenesis in culture.

Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins, osteocalcin, and matrix Gla protein in vitro

The role of the vitamin K dependent proteins, osteocalcin which is bone specific and matrix Gla protein (MGP) found in many tissues, has been studied by inhibition of synthesis of their characteristic amino acid, gamma-carboxyglutamic acid (Gla) with the anticoagulant sodium warfarin. The effect of sodium warfarin on expression of these proteins, and other phenotypic markers of bone and cartilage during cellular differentiation and development of tissue extracellular matrix, was examined in several model systems. Parameters assayed include cell growth (reflected by histone gene expression) and collagen types I and II, osteopontin, alkaline phosphatase, and mineralization. Studies were carried out in calvarial bone organ cultures, normal diploid rat osteoblast and chondrocyte cultures, and rat osteosarcoma cell lines ROS 17/2.8 and 25/1. In normal diploid cells, warfarin consistently stimulated cell proliferation (twofold). In osteoblast cultures, MGP mRNA levels were generally increased (three to tenfold). Notably, MGP mRNA levels were not affected in chondrocyte cultures, either with chronic or acute warfarin treatments. Osteocalcin mRNA levels and synthesis were decreased up to 50% in ROS 17/2.8 cells and in chronically treated (1 and 5 micrograms/ml sodium warfarin) rat osteoblast cultures after 22 days. Early stages of osteoblast phenotype development from the proliferation period to initial tissue formation (nodules) appeared unaffected; while after day 14, further growth and mineralization of the nodule areas were significantly decreased in warfarin-treated cultures. In summary, warfarin has opposing effects on the expression of two vitamin K dependent proteins, MGP and osteocalcin, in osteoblast cultures and MGP is regulated differently between cartilage and bone as reflected by cellular mRNA levels. Additionally, warfarin effects expression of nonvitamin K dependent proteins which may reflect the influence of warfarin on endoplasmic reticulum associated enzymes.

Expression of the matrix Gla protein in urogenital malignancies.

Matrix Gla protein (MGP), is a vitamin-K-dependent protein which is synthesized in a variety of tissues such as lung, heart, kidney, cartilage and bone. The function of MGP in these tissues is unclear. We have previously reported elevated MGP mRNA levels in a breast-cancer cell line, 600PEI, as compared to normal breast epithelium. Here we describe high MGP expression in primary renal-cell carcinomas, prostate carcinomas and testicular germ-cell tumors, as determined by Northern analysis. MGP was over-expressed in 21 out of 28 patients with renal-cell carcinoma, and in 16 out of 29 patients with testicular germ-cell tumors, as compared to matched normal tissues. For the renal-cell carcinomas, a statistically significant inverse correlation was observed between the level of MGP expression and tumor size, lymph-node metastasis and tumor grade. MGP was also highly expressed in 13 primary prostatic carcinomas as compared to prostate cell lines derived from metastatic tumors, and to lymph-node metastasis. Our findings indicate that the loss of MGP expression may be associated wih tumor progression and metastasis.

Developmental expression and hormonal regulation of the rat matrix GLA protein (MGP) gene in chondrogenesis and osteogenesis

Matrix Gla protein (MGP), a vitamin K dependent protein, has recently been identified in many tissues. However, it is accumulated only in bone and cartilage suggesting that the expression of MGP may be related to the development and/or maintenance of the phenotypic properties of these tissues. We systematically evaluated MGP mRNA expression as a function of bone and cartilage development and also as regulated by vitamin D during growth and cellular differentiation. Three experimental models of cartilage and bone development were employed: an in vivo model for endochondral bone formation, as well as in primary cells of normal diploid rat chondrocyte and osteoblast cultures. MGP was expressed at the highest level during cartilage formation and calcification in vivo during endochondral bone formation. In chondrocyte cultures, MGP mRNA was present throughout the culture period but increased only after 3 weeks concomitantly with type I collagen mRNA. In osteoblast cultures, MGP mRNA was expressed during the proliferative period and exhibited increased expression during the period of matrix development. In contrast to osteocalcin (bone Gla protein), this increase was not dependent on mineralization but was related to the extent of differentiation associated with and potentially induced by extracellular matrix formation. During the proliferative period, type I collagen mRNA peaked and thereafter declined, while type I collagen protein steadily accumulated in the extracellular matrix. Constant MGP levels were maintained in the mineralization period of osteoblast differentiation in vitro which is consistent with the constant levels found during the osteogenic period of the in vivo system. MGP mRNA levels in both osteoblasts and chondrocytes in culture were significantly elevated by 1,25-(OH)2D3 (10(-8) M, 48 h) throughout the time course of cellular growth and differentiation. Interestingly, when MGP mRNA transcripts from vitamin D treated and untreated chondrocytes and osteoblasts were analyzed by high resolution Northern blot analysis, we observed two distinct species of MGP mRNA in the vitamin D treated chondrocyte cultures while all other cultures examined exhibited only a single MGP mRNA transcript. Primer extension analysis indicated a single transcription start site in both osteoblasts and chondrocytes with or without vitamin D treatment, suggesting that the lower molecular weight MGP message in vitamin D treated chondrocytes may be related to a modification in post-transcriptional processing. In conclusion, these results show that the selective accumulation of MGP in bone and cartilage tissues in vitro may be related to the development and/or maintenance of a collagenous matrix as reflected by increases in MGP mRNA during these periods.(ABSTRACT TRUNCATED AT 400 WORDS)

Lung, heart, and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla protein. Implications for the possible functions of matrix Gla protein and for the tissue distribution of the gamma-carboxylase.

We have used cDNA probes for two small vitamin K-dependent bone matrix proteins, bone Gla protein (BGP) and matrix Gla protein (MGP), to evaluate the possibility that either of these proteins might be synthesized by the various soft tissues previously shown to have gamma-carboxylase activity. BGP mRNA was found in bone but not in any of the soft tissues tested, a result which reinforces the view that plasma BGP is a specific marker for bone metabolism. In contrast, MGP mRNA was found in all rat tissues examined. Lung and heart have 10-fold higher levels of MGP mRNA than bone, and kidney has a 5-fold higher level. Despite the high levels of MGP mRNA in heart and kidney, these tissues contain 40-500-fold lower concentrations of MGP protein than bone. Immunofluorescence was used to identify cells that contain MGP in kidney, lung, heart, and spleen. In each tissue, MGP was found in discrete tissue-specific cell types. In most of the soft tissues tested, MGP is the first well characterized substrate for the vitamin K-dependent carboxylase found to be synthesized. The exceptionally broad tissue distribution for MGP synthesis demonstrates that the function of MGP is not specific to connective tissues, and the low levels of MGP antigen in soft tissues with high MGP mRNA levels indicate that MGP is unlikely to act solely by virtue of its accumulation in an extracellular matrix.