Matrix Gla Protein Expression Research Articles

Matrix Gla Protein Metabolism in Vascular Smooth Muscle and Role in Uremic Vascular Calcification

Matrix Gla protein (MGP) is an inhibitor of vascular calcification but its mechanism of action and pathogenic role are unclear. This was examined in cultured rat aortas and in a model of vascular calcification in rats with renal failure. Both carboxylated (GlaMGP) and uncarboxylated (GluMGP) forms were present in aorta and disappeared during culture with warfarin. MGP was also released into the medium and removed by ultracentrifugation, and similarly affected by warfarin. In a high-phosphate medium, warfarin increased aortic calcification but only in the absence of pyrophosphate, another endogenous inhibitor of vascular calcification. Although GlaMGP binds and inactivates bone morphogenic protein (BMP)-2, a proposed mediator of vascular calcification through up-regulation of the osteogenic transcription factor runx2, neither warfarin, BMP-2, nor the BMP-2 antagonist noggin altered runx2 mRNA content in aortas, and noggin did not prevent warfarin-induced calcification. Aortic content of MGP mRNA was increased 5-fold in renal failure but did not differ between calcified and noncalcified aortas. Immunoblots showed increased GlaMGP in noncalcified (5-fold) and calcified (20-fold) aortas from rats with renal failure, with similar increases in GluMGP. We conclude that rat aortic smooth muscle produces both GlaMGP and GluMGP in tissue-bound and soluble, presumably vesicular, forms. MGP inhibits calcification independent of BMP-2-driven osteogenesis and only in the absence of pyrophosphate, consistent with direct inhibition of hydroxyapatite formation. Synthesis of MGP is increased in renal failure and deficiency of GlaMGP is not a primary cause of medial calcification in this condition.

Effect of Calcium and the Calcimimetic AMG 641 on Matrix-Gla Protein in Vascular Smooth Muscle Cells

Vascular calcification (VC) is frequently observed in patients with chronic renal failure and appears to be an active process involving transdifferentiation of vascular smooth muscle cells (VSMCs) to osteoblast-like cells. Reports of VC prevention in uremic rodents by calcimimetics coupled with identification of the calcium-sensing receptor (CaSR) in VSMCs led us to hypothesize that CaSR activation in arterial cells and VSMCs may elicit expression of an endogenous inhibitor of VC. Toward this end, we determined the effects of calcium and the calcimimetic AMG 641 on arterial wall and isolated VSMC expression of matrix-Gla protein (MGP). Bovine VSMCs were incubated with increasing calcium chloride or AMG 641 concentrations, while in vivo experiments were carried out on healthy and uremic rats. Both AMG 641 and hypercalcemia induced MGP expression in the arterial wall in healthy and uremic rats. The results obtained in vitro supported those from in vivo experiments. In conclusion, selective CaSR activation, either by extracellular calcium or AMG 641, increased MGP expression in vivo in the arterial wall and in vitro in bovine VSMCs. This local upregulation of MGP expression provides one potential mechanism by which calcimimetics prevent VC.

Phosphate-dependent stimulation of MGP and OPN expression in osteoblasts via the ERK1/2 pathway is modulated by calcium

Inorganic phosphate (Pi) acts as a signaling molecule in bone-forming cells, affecting cell functions and gene expression. Particularly, Pi stimulates the expression of mineralization-associated genes such as matrix gla protein (MGP) and osteopontin (OPN) through the ERK1/2 pathway. With respect to the presence of elevated extracellular calcium and Pi levels during bone remodeling, we questioned whether calcium might play a role in the Pi-dependent effects in osteoblasts. We first showed by Western blot and real-time PCR that the concomitant presence of 10mM Pi and 1.8mM calcium is required to stimulate ERK1/2 phosphorylation and MGP/OPN genes expression. The mechanisms involved in the cellular effects of calcium in the presence of Pi were subsequently examined. Firstly, the use of the calcium-sensing receptor (CaSR) agonist gadolinium and the G-protein inhibitor pertussis toxin enabled us to determine that a CaSR mechanism is not involved in the Pi and calcium mediated cellular effects. By transmission electron microscopy, we next demonstrated that adding 10mM Pi to the culture medium containing 1.8mM calcium led to the formation calcium phosphate precipitates (CaPp). Moreover, treatment of osteoblasts with exogenous pre-synthesized CaPp stimulated ERK1/2 phosphorylation and MGP/OPN genes expression. In spite of high extracellular calcium and Pi concentrations, this stimulation was blunted in the presence of phosphocitrate, an inhibitor of crystal formation. Finally, we showed that despite that CaPp are not endocytosed, their effect on ERK1/2 phosphorylation and MGP/OPN genes expression were dependent on lipid rafts integrity. In summary, we showed that calcium is required for Pi-dependent ERK1/2 phosphorylation and regulation of mineralization-associated genes in osteoblasts and that its effect could originate from extracellular-related effects of CaPp that are dependent on the integrity of lipid rafts.

Estrogen Inhibits Vascular Calcification via Vascular RANKL System

Rationale: Arterial calcification and osteoporosis are associated in postmenopausal women. RANK (the receptor activator of nuclear factor κB), RANKL (RANK ligand), and osteoprotegerin are key proteins in bone metabolism and have been found at the site of aortic calcification. The role of these proteins in vasculature, as well as the contribution of estrogen to vascular calcification, is poorly understood. Objective: To clarify the mechanism of RANKL system to vascular calcification in the context of estrogen deficiency. Methods and Results: RANKL induced the calcification inducer bone morphogenetic protein-2 by human aortic endothelial cells (HAECs) and decreased the calcification inhibitor matrix Gla protein (MGP) in human aortic smooth muscle cells (HASMCs), as quantified by real-time PCR and Western blot analysis. RANKL also induced bone-related gene mRNA expression and calcium deposition (Alizarin red staining) followed by the osteogenic differentiation of HASMCs. Estrogen inhibited RANKL signaling in HAECs and HASMCs mainly through estrogen receptor α. Apolipoprotein E–deficient mice fed with Western high-fat diet for 3 months presented atherosclerotic calcification (Oil red and Alizarin red staining) and osteoporosis (microcomputed tomographic analysis) after ovariectomy and increased expression of RANKL, RANK, and osteopontin in atherosclerotic lesion, as detected by in situ hybridization. Estrogen replacement inhibited osteoporosis and the bone morphogenetic protein osteogenic pathway in aorta by decreasing phosphorylation of smad-1/5/8 and increasing MGP mRNA expression. Conclusions: RANKL contributes to vascular calcification by regulating bone morphogenetic protein-2 and MGP expression, as well as bone-related proteins, and is counteracted by estrogen in a receptor-dependent manner.

Matrix Gla Protein Expression in NRK-52E Cells Exposed to Oxalate and Calcium Oxalate Monohydrate Crystals

Purpose: Matrix Gla protein (MGP), a potent calcification inhibitor in extracellular matrix, is a molecular determinant regulating vascular calcification. Here, we characterized MGP expression in cultured renal tubular epithelial cells exposed to oxalate and calcium oxalate monohydrate (COM) crystals. Methods:NRK-52E cells were cultured and exposed to medium containing oxalate or COM for 1, 3, 6, 12 and 24 h. The levels of MGP mRNA expression were quantified using real-time PCR (RT-PCR), and the levels of protein expression were characterized by Western blot. Results: MGP was expressed rapidly and intensively following exposure to COM and was time-dependent with exposure to oxalate. Conclusion: Our results have given a profile of MGP expression in renal tubular epithelial cells exposed to oxalate and crystals and provided support for an association between MGP and kidney stones. Further investigations on the mechanism of the MGP in the process of crystal-cell interaction may be important in the future.

Discovery and verification of matrix gla protein, a TNM staging and prognosis-related biomarker for gastric cancer

To analyze microarray datasets deposited in the public database and to identify TNM associated genes in gastric cancers. Microarray datasets of gastric cancer were selected from GEO database. Differentially expressed genes related to TNM staging were evaluated by significant analysis of the microarray using MultiExperiment Viewer (MEV) platform. Candidate gene expressions in gastric cancer tissues and cell lines were verified by reverse transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR, Western blot and immunohistochemistry. GES4007 dataset was re-analyzed leading to the identification of 14 genes associated with TNM staging. Over-expression of matrix gla protein (MGP) was confirmed in gastric cancer cell lines and tumor tissues by quantitative RT-PCR, Western blot and immunohistochemistry. Increased MGP expression was found in 22 of 54 cases of (40.7%) gastric cancer specimens compared to the normal gastric tissues. The up-regulation of MGP mRNA expression closely correlated with TNM stage (P = 0.001) and prognosis (P = 0.006). Public databases of microarray studies are the valuable resources for data mining. MGP has been identified and confirmed as a novel biomarker for the TNM stage and prognosis of gastric cancer.

Inhibition of Bone Morphogenetic Proteins Protects Against Atherosclerosis and Vascular Calcification

The bone morphogenetic proteins (BMPs), a family of morphogens, have been implicated as mediators of calcification and inflammation in the vascular wall. To investigate the effect of altered expression of matrix Gla protein (MGP), an inhibitor of BMP, on vascular disease. We used MGP transgenic or MGP-deficient mice bred to apolipoprotein E mice, a model of atherosclerosis. MGP overexpression reduced vascular BMP activity, atherosclerotic lesion size, intimal and medial calcification, and inflammation. It also reduced expression of the activin-like kinase receptor 1 and the vascular endothelial growth factor, part of a BMP-activated pathway that regulates angiogenesis and may enhance lesion formation and calcification. Conversely, MGP deficiency increased BMP activity, which may explain the diffuse calcification of vascular medial cells in MGP deficient aortas and the increase in expression of activin-like kinase receptor 1 and vascular endothelial growth factor. Unexpectedly, atherosclerotic lesion formation was decreased in MGP-deficient mice, which may be explained by a dramatic reduction in expression of endothelial adhesion molecules limiting monocyte infiltration of the artery wall. Our results indicate that BMP signaling is a key regulator of vascular disease, requiring careful control to maintain normal vascular homeostasis.

Inhibition of bone morphogenetic protein protects against atherosclerosis and vascular calcification

The bone morphogenetic proteins (BMP), a family of morphogens, have been implicated in atherogenesis as mediators of inflammation and calcification. We investigated the role of altered expression of Matrix Gla Protein (MGP), an inhibitor of BMP, on the development of atherosclerotic lesions and vascular calcification using MGP transgenic or MGP deficient mice bred to apolipoprotein E (Apoe−/−) mice, a model of atherosclerosis. MPG overexpression reduced vascular BMP signaling, which resulted in reduced lesion size, and intimal and medial calcification. It was associated with reduced expression of the activin‐like kinase receptor (ALK)1 and the vascular endothelial growth factor (VEGF), part of a BMP‐activated pathway that also regulates angiogenesis. MGP‐deficiency, on the other hand, strongly enhanced vascular BMP signaling and expression of ALK1 and VEGF. Despite the high propensity of the Apoe−/− mice to develop lesions, lesion formation was minimal in the setting of MGP deficiency, which causes chondroosteogenic differentiation and calcification, suggesting that the abnormal cell differentiation did not allow for lesion formation. Together, our results indicate that vascular BMP signaling is a key regulator in the development of vascular disease, requiring careful control to maintain normal vascular homeostasis.Research support: NIH/NHLBI, AHA.

Dual transcriptional regulation by runx2 of matrix Gla protein in Xenopus laevis

Matrix Gla protein (MGP) is an extracellular mineral-binding protein expressed in several tissues but it only accumulates in bone and calcified cartilage under physiological conditions. Available evidence indicates that it acts as a physiological inhibitor of mineralization. Runx2 is a transcription factor essential for bone formation in mammals, affecting osteoblast and chondrocyte differentiation by regulating key genes crucial for bone and cartilage development. Being an important cartilage-associated gene, MGP is a potential target for Runx2, and thus we have investigated the possible functional interactions between them. In A6 cells, Runx2 was found to modulate MGP transcription and deletion analysis of MGP distal and proximal promoter-luciferase constructs identified cis-regulatory regions. Interestingly, we have also identified a runx2-binding site that mediates transcriptional repression of XlMGP. Mutation of this element, located between − 54 and + 33 bp, results in 18-fold up-regulation of transcription. Furthermore, and in addition to the previously reported Xlrunx2 types I and II, we have identified three transcripts encoding novel, truncated Xlrunx2 isoforms. Although only type I and type II could transactivate XlMGP, the truncated isoforms identified in this study, which result from alternative splicing, could be involved in negative regulation of MGP expression, as described for other RUNX2 truncated isoforms acting in other target genes. In vivo microinjection of XlMGP promoter constructs and runx2 mRNA confirmed that those promoters are targets for this transcription factor. These data also indicate that MGP is under dual regulation by runx2 through the use of various isoforms and context-dependent formation of transcriptional complexes.

Phosphate-Dependent Regulation of MGP in Osteoblasts: Role of ERK1/2 and Fra-1

Inorganic phosphate (Pi) and the matrix Gla protein (MGP) are key regulators of bone formation. We have recently shown that Pi upregulates MGP in growth plate chondrocytes, which may represent a negative feedback loop for the control of mineralization. Osteoblasts from Fra-1-deleted mice express low levels of MGP, whereas the expression of MGP is elevated in Fra-1 transgenic osteoblasts, suggesting a role for Fra-1 in MGP expression and bone formation. In this study, we aimed at deciphering the relationships between Pi and MGP in osteoblasts to determine the molecular mechanisms involved in the Pi-dependent regulation of MGP. In MC3T3-E1 cells and primary calvaria-derived osteoblasts, Pi increased MGP and Fra-1 expression at both the mRNA and protein levels. We also found that Pi enhanced the phosphorylation of ERK1/2. U0126 (MEK1/2 inhibitor) suppressed Pi-stimulated MGP and Fra-1 expression, indicating that ERK1/2 is required for Pi-dependent regulation of MGP and Fra-1. In addition, using in vitro DNA binding and chromatin immunoprecipitation assays, we showed that Fra-1 interacts with the MGP promoter in response to Pi in MC3T3-E1 cells. Finally, we found that in fra-1 knockdown MC3T3-E1 osteoblasts, the level of MGP expression is no more significantly upregulated by Pi. We further showed that primary osteoblasts from Fra-1-deficient mice failed to exhibit a Pi-dependent stimulation of MGP expression. These data show, for the first time, that Pi regulates MGP expression in osteoblasts through the ERK1/2-Fra-1 pathway.

Variations in gene and protein expression in human nucleus pulposus in comparison with annulus fibrosus and cartilage cells: potential associations with aging and degeneration

Regardless of recent progress in the elucidation of intervertebral disc (IVD) degeneration, the basic molecular characteristics that define a healthy human IVD are largely unknown. Although work in different animal species revealed distinct molecules that might be used as characteristic markers for IVD or specifically nucleus pulposus (NP) cells, the validity of these markers for characterization of human IVD cells remains unknown. Eleven potential marker molecules were characterized with respect to their occurrence in human IVD cells. Gene expression levels of NP were compared with annulus fibrosus (AF) and articular cartilage (AC) cells, and potential correlations with aging were assessed. Higher mRNA levels of cytokeratin-19 (KRT19) and of neural cell adhesion molecule-1 were noted in NP compared to AF and AC cells. Compared to NP cytokeratin-18 expression was lower in AC, and alpha-2-macroglobulin and desmocollin-2 lower in AF. Cartilage oligomeric matrix protein (COMP) and glypican-3 expression was higher in AF, while COMP, matrix gla protein (MGP) and pleiotrophin expression was higher in AC cells. Furthermore, an age-related decrease in KRT19 and increase in MGP expression were observed in NP cells. The age-dependent expression pattern of KRT19 was confirmed by immunohistochemistry, showing the most prominent KRT19 immunoreaction in the notochordal-like cells in juvenile NP, whereas MGP immunoreactivity was not restricted to NP cells and was found in all age groups. The gene expression of KRT19 has the potential to characterize human NP cells, whereas MGP cannot serve as a characteristic marker. KRT19 protein expression was only detected in NP cells of donors younger than 54 years.

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

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

Replicative senescence of vascular smooth muscle cells enhances the calcification through initiating the osteoblastic transition

Medial artery calcification, which does not accompany lipid or cholesterol deposit, preferentially occurs in elderly population, but its underlying mechanisms remain unclear. In the present study, we investigated the potential role of senescent vascular smooth muscle cells (VSMCs) in the formation of senescence-associated medial calcification. Replicative senescence was induced by the extended passages (until passages 11-13) in human primary VSMCs, and cells in early passage (passage 6) were used as control young cells. VSMC calcification was markedly enhanced in the senescent cells compared with that in the control young cells. We identified that genes highly expressed in osteoblasts, such as alkaline phosphatase (ALP) and type I collagen, were significantly upregulated in the senescent VSMCs, suggesting their osteoblastic transition during the senescence. Knockdown of either ALP or type I collagen significantly reduced the calcification in the senescent VSMCs. Of note, runt-related transcription factor-2 (RUNX-2), a core transcriptional factor that initiates the osteoblastic differentiation, was also upregulated in the senescent VSMCs. Knockdown of RUNX-2 significantly reduced the ALP expression and calcification in the senescent VSMCs, suggesting that RUNX-2 is involved in the senescence-mediated osteoblastic transition. Furthermore, immunohistochemistry of aorta from the klotho(-/-) aging mouse model demonstrated in vivo emergence of osteoblast-like cells expressing RUNX-2 exclusively in the calcified media. We also found that statin and Rho-kinase inhibitor effectively reduced the VSMC calcification by inhibiting P(i)-induced apoptosis and potentially enhancing matrix Gla protein expression in the senescent VSMCs. These findings strongly suggest an important role of senescent VSMCs in the pathophysiology of senescence-associated medial calcification, and the inhibition of osteoblastic transition could be a new therapeutic approach for the prevention of senescence-associated medial calcification.

Prognostic value of matrix Gla protein in breast cancer

To assess the prognostic value of matrix Gla protein (MGP) expression in cases of breast cancer, 9 samples from patients diagnosed with breast cancer who were followed up for more than 10 years were microdissected and then analyzed using Affymetrix U133 Plus 2.0 Arrays. Genes that exhibited significant differences in expression between patients with a good prognosis and those with a poor prognosis were identified. The MGP gene was among the genes up-regulated in cases where the prognosis was poor, indicating that the mRNA levels of MGP are a potential prognostic indicator of breast cancer. However, immunohistostaining of breast tissue microarrays (n=207) did not reveal a correlation between the protein expression of MGP and overall survival, neither was there a correlation between the protein expression of MGP and ER status or bone metastasis. In breast cancer cases, the mRNA level of MGP may be a marker indicating poor prognosis; however, protein expression determined by immunohistostaining is not.

Sp proteins and Runx2 mediate regulation of matrix gla protein (MGP) expression by parathyroid hormone

As part of its catabolic action in bone, parathyroid hormone (PTH) inhibits extracellular matrix mineralization. We previously showed that PTH dose-dependently induces matrix gla protein (MGP) expression in osteoblasts and this induction is at least partially responsible for PTH-mediated inhibition of mineralization. Recently, we identified PKA and ERK/MAPK as the key signaling pathways involved in PTH regulation of MGP expression. The goal of this study was to further characterize the mechanism by which PTH stimulates expression of MGP. Deletion analysis of the murine Mgp gene promoter identified a PTH-responsive region between -173 bp and-49 bp. Using gel-mobility shift assays we found that Sp1/Sp3, and Runx2 bind to distinct sites within this region. Mutation of either the Sp or the Runx2 site reduced MGP induction by PTH, while mutation of both sites completely abolished PTH responsiveness. Overexpression of Runx2 or Sp1 activated the Mgp reporter, while Sp3 was a dose-dependent repressor of Sp1 and PTH-induced MGP expression. Collectively, these data show that PTH regulates MGP gene transcription in osteoblasts through altered activities of Sp and Runx2 transcription factors.

Immunohistochemical localization and mRNA expression of matrix Gla protein and fetuin-A in bone biopsies of hemodialysis patients

Matrix Gla protein (MGP) and fetuin-A are inhibitors of arterial calcifications. In blood of rats, calcium-phosphate-fetuin-MGP complexes, produced in bone, have been identified. Indeed, an association between bone resorption, release of such complexes, and arterial calcifications has been reported. We have investigated the synthesis and localization of fetuin-A and MGP in bone of hemodialysis patients and the possible contribution of bone cells in arterial calcifications. Bone biopsies from 11 hemodialysis patients were used for histology, in situ hybridization of fetuin-A and MGP messenger RNA (mRNA), immunohistochemistry of fetuin-A, and total, carboxylated, and non-carboxylated MGP proteins. Patients showed various types of renal osteodystrophy, or normal bone. MGP was synthesized and expressed (total and carboxylated) by osteoblasts, osteocytes, and most osteoclasts, while fetuin-A by osteoblasts and osteocytes. Fetuin-A and carboxylated MGP proteins were positive in the calcified matrix, while total MGP was negative. Osteoid seams were negative to fetuin-A, lightly positive to carboxylated MGP, and occasionally positive to total MGP. Undercarboxylated MGP was mostly undetectable. In adult humans, fetuin-A is produced also by osteoblasts, and not only by hepatocytes, as previously believed. MGP, essentially carboxylated, is synthesized by osteoblasts and most osteoclasts. Increased bone turnover can be an important contributor to arterial calcifications.

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.

A calcimimetic (R-568), but not calcitriol, prevents vascular remodeling in uremia

Renal insufficiency increases cardiovascular risk, accelerates atherogenesis, and causes vascular wall remodeling. Here we evaluated the effect of the calcimimetic R-568 and non-hypercalcemic doses of calcitriol on vascular structure. Subtotal nephrectomy was produced in Sprague-Dawley rats followed by treatment with R-568, calcitriol, or vehicle for 12 weeks. The aortic wall was significantly thicker in vehicle-treated uremic rats than in those with a sham-operation but R-568-treated uremic rats had a lower value. In contrast, calcitriol increased wall thickness in both the sham-operated and uremic groups. The calcification score, measured by von Kossa staining, and the number of proliferating cells in the intima and media were significantly higher in the calcitriol-treated uremic group. The expression of the calcium sensing receptor was higher in the intima of sham-operated and uremic rats treated with R-568 compared to animals treated with vehicle or calcitriol, while the expression of the vitamin D receptor was upregulated by both calcitriol and R-568. Our study shows that in uremic rats, calcitriol increased while R-568 attenuated media calcification and proliferation of vascular smooth muscle and endothelial cells.

CCN family 2/connective tissue growth factor (CCN2/CTGF) stimulates proliferation and differentiation of auricular chondrocytes

CCN family 2/connective tissue growth factor (CCN2/CTGF) is an atypical growth factor for growth plate chondrocytes. It plays an important role in their proliferation and differentiation in vitro, but does not stimulate hypertrophy or calcification of articular chondrocytes. We herein report for the first time that CCN2/CTGF promotes growth and differentiation of auricular chondrocytes and maintains their molecular phenotype in vitro and in vivo. Auricular chondrocytes were isolated from rabbit auricular cartilage by trypsin-collagenase treatment, and treated with human recombinant CCN2/CTGF or infected with adenovirus harboring the ccn2/ctgf gene. Cell proliferation was measured by [(3)H] thymidine incorporation and MTS assay, and changes in gene expression of auricular chondrocyte markers were monitored by real-time polymerase chain reaction, Northern hybridization, and histological analysis. For in vivo studies, auricular chondrocytes were cultured as pellets and implanted subcutaneously after treatment of recombinant human CCN2/CTGF. Ectopically formed cartilage was subjected to histological analysis. Cell death was monitored by in situ TUNEL analysis. CCN2/CTGF stimulated proliferation, differentiation and synthesis of elastin and proteoglycans of rabbit primary auricular chondrocytes in a dose-dependent manner. CCN2/CTGF caused a 2.5-fold increase in the expression of elastin in comparison to the control, resulting in enhanced deposition of elastin fibers in a monolayer culture of auricular chondrocytes. Mineralization was not induced; in contrast, CCN2/CTGF stimulated expression of matrix gla protein which is known to impair mineralization. Furthermore, pretreatment of pellets of auricular chondrocytes with CCN2/CTGF and subcutaneous implantation significantly enhanced the growth of ectopic auricular cartilage pieces expressing phenotypic markers of auricular chondrocytes including type II and X collagen. Notably, chondrocyte apoptosis was impaired by CCN2/CTGF. These findings show that CCN2/CTGF may be a suitable agent for promoting differentiation and growth of auricular chondrocytes, while preventing mineralization and apoptosis, and suggests that CCN2/CTGF may be useful for the repair or reconstruction of elastic cartilage.

ERK1/2 and Fra-1 mediate phosphate-dependent stimulation of matrix Gla protein expression in osteoblasts

ERK1/2 and Fra-1 mediate phosphate-dependent stimulation of matrix Gla protein expression in osteoblasts