Matrix Extracellular Phosphoglycoprotein Expression Research Articles

IMMUNOHISTOCHEMICAL DISTRIBUTION OF THE SIBLINGS IN AMELOBLASTOMA AND ODONTOGENIC KERATOCYST

Objective Ameloblastomas are benign but aggressive neoplasms of the jaw. The odontogenic keratocysts (OKC) are aggressive jaw cysts of odontogenic origin. A recent WHO classification designated OKC as a neoplasm but a current edition re-designated it as a cyst. The small integrin-binding ligand n-linked glycoproteins (SIBLINGs) are a family of molecules that some epithelial neoplasms use in furthering their progression. Members of the SIBLING family are bone sialoprotein (BSP), dentin matrix protein1 (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE), and osteopontin (OPN). The objective of this study was to compare the expression of the SIBLINGs in ameloblastomas and odontogenic keratocysts. Findings Using immunohistochemistry, with appropriate controls, 49 cases of ameloblastomas and 35 cases of OKCs were screened for the expression of all five SIBLINGs in a retrospective study on archived paraffin sections. Immunoreactivity was scored as positive if > 10% of tumor/cyst cells stained for a SIBLING and negative if Conclusion While all members of the SIBLINGs are expressed in ameloblastomas and OKCs, DSPP expression in OKCs is significantly higher than in ameloblsatomas, whereas, MEPE expression in OKCs is considerably lower than in ameloblastomas. The differences in the expression of DSPP and MEPE between ameloblastomas and OKCs may indicate differences in the degree of aggressive behaviors.

Effect of variations in dietary Pi intake on intestinal Pi transporters (NaPi-IIb, PiT-1, and PiT-2) and phosphate-regulating factors (PTH, FGF-23, and MEPE).

Hyperphosphatemia is a common condition in patients with chronic kidney disease (CKD) and can lead to bone disease, vascular calcification, and increased risks of cardiovascular disease and mortality. Inorganic phosphate (Pi) is absorbed in the intestine, an important step in the maintenance of homeostasis. In CKD, it is not clear to what extent Pi absorption is modulated by dietary Pi. Thus, we investigated 5/6 nephrectomized (Nx) Wistar rats to test whether acute variations in dietary Pi concentration over 2days would alter hormones involved in Pi metabolism, expression of sodium-phosphate cotransporters, apoptosis, and the expression of matrix extracellular phosphoglycoprotein (MEPE) in different segments of the small intestine. The animals were divided into groups receiving different levels of dietary phosphate: low (Nx/LPi), normal (Nx/NPi), and high (Nx/HPi). Serum phosphate, fractional excretion of phosphate, intact serum fibroblast growth factor 23 (FGF-23), and parathyroid hormone (PTH) were significantly higher and ionized calcium was significantly lower in the Nx/HPi group than in the Nx/LPi group. The expression levels of NaPi-IIb and PiT-1/2 were increased in the total jejunum mucosa of the Nx/LPi group compared with the Nx/HPi group. Modification of Pi concentration in the diet affected the apoptosis of enterocytes, particularly with Pi overload. MEPE expression was higher in the Nx/HPi group than in the Nx/NPi. These data reveal the importance of early control of Pi in uremia to prevent an increase in serum PTH and FGF-23. Uremia may be a determining factor that explains the expressional modulation of the cotransporters in the small intestine segments.

Regulatory roles of miRNA-758 and matrix extracellular phosphoglycoprotein in cervical cancer.

The present study aimed to examine the role and underlying mechanism of miRNA-758 (miR-758) expression in cancer tissues, blood and cervical exfoliated cells from patients with cervical cancer. A total of 49 patients with cervical cancer and 26 healthy people for cervical cancer screening were included in the present study. The patients with cervical cancer were treated with resection, and the tumor and adjacent tissues, blood and cervical exfoliated cells were collected. The expression levels of miR-758 and matrix extracellular phosphoglycoprotein (MEPE) mRNA in each sample were detected by reverse transcription-quantitative polymerase chain reaction. In addition, western blot analysis was used to detect the MEPE protein in tumor tissues, while ELISA was applied to detect the MEPE protein expression in the blood and cervical exfoliated cells. Compared with the normal control, MEPE mRNA expression was upregulated in cervical cancer tissues, blood and cervical exfoliated cells. At the protein level, MEPE was also upregulated significantly in patients with cervical cancer. miR-758 expression was decreased significantly in cervical cancer tissues, blood and cervical exfoliated cells (P<0.05), which was opposite to the trend observed for MEPE mRNA expression. Furthermore, MEPE expression was increased in the tumor tissue, blood and cervical exfoliated cells of cervical cancer patients, which was associated to the downregulated miR-758. Therefore, miR-758 may regulate the infiltration and invasion of cervical cancer by targeting MEPE.

Mechanical loading reduces inflammation-induced human osteocyte-to-osteoclast communication.

Multiple factors contribute to bone loss in inflammatory diseases such as rheumatoid arthritis (RA), but circulating inflammatory factors and immobilization play a crucial role. Mechanical loading prevents bone loss in the general population, but the effects of mechanical loading in patients with RA are less clear. Therefore, we aimed to investigate whether mechanical stimuli reverse the stimulatory effect of RA serum on osteocyte-to-osteoclast communication. Human primary osteocytes were pretreated with 10 % RA serum or healthy control serum for 7 days, followed by 1 h ± mechanical loading by pulsating fluid flow (PFF). Nitric oxide (NO) and prostaglandin E2 were measured in the medium. Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), interleukin-6 (IL-6), cyclooxygenase-2 (COX2), matrix-extracellular phosphoglycoprotein (MEPE), cysteine-rich protein 61 (CYR61), and SOST gene expression was quantified by qPCR. Osteoclast precursors were cultured with PFF-conditioned medium (PFF-CM) or static-conditioned medium (stat-CM), and osteoclast formation was assessed. RA serum alone did not affect IL-6, CYR61, COX2, MEPE, or SOST gene expression in osteocytes. However, RA serum enhanced the RANKL/OPG expression ratio by 3.4-fold, while PFF nullified this effect. PFF enhanced NO production to the same extent in control serum (2.6–3.5-fold) and RA serum-pretreated (2.7–3.6-fold) osteocytes. Stat-CM from RA serum-pretreated osteocytes enhanced osteoclastogenesis compared with stat-CM from control serum-pretreated osteocytes, while PFF nullified this effect. In conclusion, RA serum, containing inflammatory factors, did not alter the intrinsic capacity of osteocytes to sense mechanical stimuli, but upregulated osteocyte-to-osteoclast communication. Mechanical loading nullified this upregulation, suggesting that mechanical stimuli could contribute to the prevention of osteoporosis in inflammatory disease.

Increased Expression of Matrix Extracellular Phosphoglycoprotein (MEPE) in Cortical Bone of the Rat Tibia after Mechanical Loading: Identification by Oligonucleotide Microarray

Skeletal integrity in humans and animals is maintained by daily mechanical loading. It has been widely accepted that osteocytes function as mechanosensors. Many biochemical signaling molecules are involved in the response of osteocytes to mechanical stimulation. The aim of this study was to identify genes involved in the translation of mechanical stimuli into bone formation. The four-point bending model was used to induce a single period of mechanical loading on the right tibia, while the contra lateral left tibia served as control. Six hours after loading, the effects of mechanical loading on gene-expression were determined with microarray analysis. Protein expression of differentially regulated genes was evaluated with immunohistochemistry. Nine genes were found to exhibit a significant differential gene expression in LOAD compared to control. MEPE, Garnl1, V2R2B, and QFG-TN1 olfactory receptor were up-regulated, and creatine kinase (muscle form), fibrinogen-B beta-polypeptide, monoamine oxidase A, troponin-C and kinesin light chain-C were down-regulated. Validation with real-time RT-PCR analysis confirmed the up-regulation of MEPE and the down-regulation of creatine kinase (muscle form) and troponin-C in the loaded tibia. Immunohistochemistry showed that the increase of MEPE protein expression was already detectable six hours after mechanical loading. In conclusion, these genes probably play a role during translation of mechanical stimuli six hours after mechanical loading. The modulation of MEPE expression may indicate a connection between bone mineralization and bone formation after mechanical stimulation.

Sclerostin regulation of WISP1 and MMPS in chondrocytes is dependent on the LRP5/6 binding domain but not secretion from the cell

Sclerostin regulation of WISP1 and MMPS in chondrocytes is dependent on the LRP5/6 binding domain but not secretion from the cell

MEPE-Derived ASARM Peptide Inhibits Odontogenic Differentiation of Dental Pulp Stem Cells and Impairs Mineralization in Tooth Models of X-Linked Hypophosphatemia

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide − a substrate for PHEX and a strong inhibitor of mineralization − derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.

MEPE is a novel regulator of growth plate cartilage mineralization

Matrix extracellular phosphoglycoprotein (MEPE) belongs to the SIBLING protein family which play key roles in biomineralization. Although the growth plates of MEPE-overexpressing mice display severe morphological disruption, the expression and function of MEPE in growth plate matrix mineralization remains largely undefined. Here we show MEPE and its cleavage product, the acidic serine aspartate-rich MEPE-associated motif (ASARM) peptide, to be localised to the hypertrophic zone of the growth plate. We also demonstrate that the phosphorylated (p)ASARM peptide inhibits ATDC5 chondrocyte matrix mineralization. Stable MEPE-overexpressing ATDC5 cells also had significantly reduced matrix mineralization in comparison to the control cells. Interestingly, we show that the addition of the non-phosphorylated (np)ASARM peptide promoted mineralization in the ATDC5 cells. The peptides and the overexpression of MEPE did not affect the differentiation of the ATDC5 cells. For a more physiologically relevant model, we utilized the metatarsal organ culture model. We show the pASARM peptide to inhibit mineralization at two stages of development, as shown by histological and μCT analysis. Like in the ATDC5 cells, the peptides did not affect the differentiation of the metatarsals indicating that the effects seen on mineralization are direct, as is additionally confirmed by no change in alkaline phosphatase activity or mRNA expression. In the metatarsal organ cultures, the pASARM peptide also reduced endothelial cell markers and vascular endothelial growth factor mRNA expression. Taken together these results show MEPE to be an important regulator of growth plate chondrocyte matrix mineralization through its cleavage to an ASARM peptide.

Modulation of the Differentiation of Dental Pulp Stem Cells by Different Concentrations of β-Glycerophosphate

Dentinogenesis is a necessary prerequisite for dental tissue engineering. One of the steps for dentinogenesis is to obtain large quantities of highly purified odontoblasts. Therefore, we have undertaken an experiment applying different concentrations of β-glycerophosphate (β-GP) to induce the differentiation of dental pulp stem cells (DPSCs) in a long-term 28-day culture. In the meanwhile, we have studied the time- and maturation-dependent expression of matrix extracellular phosphoglycoprotein (MEPE) and that of the odontoblast-like marker-dentin sialoprotein (DSP), in order to investigate an optimized mineralized condition. Western blot results revealed that the expression of DSP became lower when accompanied by the increase of the β-GP concentration, and there was also an influence on MEPE expression when different concentrations of β-GP were applied. Meanwhile, the mineralized groups had an inhibitory function on the expression of MEPE as compared with the control group. Above all, all experimental groups successfully generated mineralized nodules by Alizarin Red S and the 5 mM β-GP group formed more mineralized nodules quantitated using the CPC extraction method. In conclusion, there is a significant modulation of the β-GP during the differentiation of the DPSCs. The degree of odontoblast differentiation is β-glycerophosphate concentration dependent. A low concentration of β-GP (5 mM) has been shown to be the optimal concentration for stimulating the maturation of the DPSCs. Moreover, MEPE accompanied with DSP clearly demonstrates the degree of the differentiation.

Matrix extracellular phosphoglycoprotein is expressed in causative tumors of oncogenic osteomalacia

Oncogenic osteomalacia (OOM), or tumor-induced osteomalacia, is a rare disease characterized by renal phosphate wasting and osteomalacia. It arises due to the secretion of fibroblast growth factor 23 (FGF-23) from causative tumors. Matrix extracellular phosphoglycoprotein (MEPE) is predominantly expressed in odontoblasts, osteoblasts, and osteocytes. Although the presence of MEPE mRNA has been reported in some OOM tumors, little is known about the prevalence of MEPE expression in OOM tumors. In this study, the expression of MEPE and FGF-23 in OOM tumors was investigated at the transcriptional and translational levels. Eleven causative OOM tumors were analyzed by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemistry for MEPE and FGF-23 expression. Hemangiopericytomas and giant cell tumors, pathological diagnoses that are common in cases of OOM, were obtained from non-osteomalacic patients and analyzed as controls. The gene expression level of FGF23 and MEPE in OOM tumors was 10(4)- and 10(5)-times higher, respectively, than in non-OOM tumors. Immunohistochemical staining revealed that FGF-23 protein was expressed in all OOM tumors, and MEPE was expressed in 10 out of 11 OOM tumors. Thus, MEPE expression was common in OOM tumors, similar to FGF-23. These results indicate that, in addition to the hypophosphatemic effects of FGF-23, MEPE or the MEPE-derived acidic serine aspartate-rich MEPE-associated motif peptide may contribute to decreased bone mineralization in OOM patients.

Inhibition of Osteoclastogenesis by Mechanically Loaded Osteocytes: Involvement of MEPE

In regions of high bone loading, the mechanoresponsive osteocytes inhibit osteoclastic bone resorption by producing signaling molecules. One possible candidate is matrix extracellular phosphoglycoprotein (MEPE) because acidic serine- and aspartate-rich MEPE-associated motif peptides upregulate osteoprotegerin (OPG) gene expression, a negative regulator of osteoclastogenesis. These peptides are cleaved from MEPE when relatively more MEPE than PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) is present. We investigated whether mechanical loading of osteocytes affects osteocyte-stimulated osteoclastogenesis by involvement of MEPE. MLO-Y4 osteocytes were mechanically loaded by 1-h pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) or kept under static control conditions. Recombinant MEPE (0.05, 0.5, or 5 μg/ml) was added to some static cultures. Mouse bone marrow cells were seeded on top of the osteocytes to determine osteoclastogenesis. Gene expression of MEPE, PHEX, receptor activator of nuclear factor kappa-B ligand (RANKL), and OPG by osteocytes was determined after PFF. Osteocytes supported osteoclast formation under static control conditions. Both PFF and recombinant MEPE inhibited osteocyte-stimulated osteoclastogenesis. PFF upregulated MEPE gene expression by 2.5-fold, but not PHEX expression. PFF decreased the RANKL/OPG ratio at 1-h PFF treatment. Our data suggest that mechanical loading induces changes in gene expression by osteocytes, which likely contributes to the inhibition of osteoclastogenesis after mechanical loading of bone. Because mechanical loading upregulated gene expression of MEPE but not PHEX, possibly resulting in the upregulation of OPG gene expression, we speculate that MEPE is a soluble factor involved in the inhibition of osteoclastogenesis by osteocytes.

Molecular Regulation of Matrix Extracellular Phosphoglycoprotein Expression by Bone Morphogenetic Protein-2

Matrix extracellular phosphoglycoprotein (MEPE) is mainly expressed in mineralizing tissues, and its C-terminal proteolytic cleavage product is an acidic-serine-asparate-rich-MEPE-associated motif (ASARM) that is a strong regulator of body phosphate metabolism and mineralization. There is sufficient data supporting a role for MEPE protein function in mineralization, however, little is known about the regulation of MEPE gene expression. As bone morphogenetic protein-2 (BMP-2) is one of the most important signals for calvarial mineralization and MEPE expression is higher in mineralized tissues, we attempted to uncover a regulatory circuit between BMP-2 and MEPE expression. Mepe expression is very low in proliferating MC3T3-E1 cells, but is dramatically increased in the mineralization stage and is strongly stimulated by treatment with BMP-2, even in proliferating cells. Overexpression and knock-down experiments of Smads, Dlx5, and Runx2 indicated that they are indispensable mediators of BMP-2-induced Mepe expression. In contrast, Msx2 showed strong inhibition of Mepe transcription. PHEX is an enzyme that prevents the release of the ASARM motif, a mineralization inhibitor, from the MEPE molecule. Thus, the MEPE/PHEX ratio may be a good indicator of mineralization progression because we found that the mRNA ratio and protein levels were low when osteoblasts were actively differentiating to the mineralization stage and the ratio was high when the cells reached the mineralization stage when it is assumed that osteocytes may protect themselves and make a space to survive from the mineralized matrix by releasing the ASARM motif. Collectively, MEPE expression is bone cell-specific and induced by the BMP-2 signaling pathway. In addition, the MEPE/PHEX ratio of the cell could be a very important barometer indicating the progression of tissue mineralization.

Expression of matrix extracellular phosphoglycoprotein in human dental pulp cells undergoing odontoblastic differentiation

To investigate the expression of matrix extracellular phosphorylated protein (MEPE) in human dental pulp cells (hDPC) undergoing odontogenic induction and explore the role of MEPE in odontoblast-like differentiation. hDPC were isolated by enzymatic digestion and preceded to odontogenic induction for 7, 14 and 21 days respectively. hDPC before induction served as controls. The expressions of MEPE, dentin sialophosphoprotein (DSPP)/dentin sialoprotein (DSP), bone sialoprotein (BSP) and collagen type I were determined by quantitative real-time RT-PCR and Western blotting. The mRNA levels of MEPE, DSPP, BSP and type I collagen were increased in a time-dependent manner as hDPC were induced along odontoblastic lineage. Statistical differences were detected for MEPE and BSP mRNA expressions in induced hDPC compared with control group (P < 0.001). For DSPP and type I collagen, the mRNA levels in the induced groups were (12 943.33 + or - 3805.73) and (250.55 + or - 31.86) respectively, which were significantly higher than those in control group on days 21 (P < 0.05). Western blotting also revealed the increased expressions of MEPE, DSP, BSP and type I collagen in the induced DPC. hDPC showed analogously temporal expressions of MEPE, DSPP/DSP, BSP and collagen type I while differentiating along odontoblast lineage. MEPE may play an important role in the odontogenic differentiation of hDPC and may be a potential marker of odontoblast-like differentiation.

Patterns of FGF-23, DMP1, and MEPE expression in patients with chronic kidney disease

Fibroblast growth factor 23 (FGF-23), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) are skeletal proteins involved in the regulation of phosphate homeostasis and bone metabolism. Circulating FGF-23 levels are increased in patients with chronic kidney disease (CKD); however, FGF-23 skeletal expression and its regulation by DMP1 and MEPE have yet to be evaluated. Thus, expression of these three proteins was characterized by immunohistochemistry in 32 pediatric and young adult patients with CKD stages 2–5. When compared to normal controls, bone FGF-23 and DMP1 expression were increased in all stages of CKD; significant differences in bone FGF-23 and DMP1 expression were not detected between pre-dialysis CKD and dialysis patients. Bone MEPE expression in CKD did not differ from controls. FGF-23 was expressed in osteocyte cell bodies located at the trabecular periphery. DMP1 was widely expressed in osteocyte cell bodies and dendrites throughout bone. MEPE was also expressed throughout bone, but only in osteocyte cell bodies. Bone FGF-23 expression correlated directly with plasma levels of the protein ( r = 0.43, p < 0.01) and with bone DMP1 expression ( r = 0.54, p < 0.01) and expression of both proteins were inversely related to osteoid accumulation. Bone MEPE expression was inversely related to bone volume. In conclusion, skeletal FGF-23 and DMP1 expression are increased in CKD and are related to skeletal mineralization. The patterns of expression of FGF-23, MEPE, and DMP1 differ markedly in trabecular bone, suggesting that individual osteocytes may have specialized functions. Increases in bone FGF-23 and DMP1 expression suggest that osteocyte function is altered early in the course of CKD.

Expression Analysis of Fibroblast Growth Factor-23, Matrix Extracellular Phosphoglycoprotein, Secreted Frizzled-Related Protein-4, and Fibroblast Growth Factor-7: Identification of Fibroblast Growth Factor-23 and Matrix Extracellular Phosphoglycoprotein as Major Factors Involved in Tumor-Induced Osteomalacia

To evaluate the expression of various phosphaturic factors in the tumor of a patient with tumor-induced osteomalacia (TIO) and to analyze serum levels of fibroblast growth factor (FGF)-23 in TIO and healthy subjects. We measured serum FGF-23 levels in 2 patients with TIO and 6 healthy volunteers. Expression of FGF-23, matrix extracellular phosphoglycoprotein (MEPE), FGF-7, and secreted frizzled-related protein-4 (sFRP-4) was analyzed in a hemangiopericytoma from a patient with TIO, in a hemangiopericytoma from a patient without TIO, and in various control cell lines. Serum FGF-23 levels were substantially higher in patients with TIO in comparison with those in healthy control subjects and normalized with successful resection of the tumor. Tissue expression analysis showed preferential expression of FGF-23 and MEPE in TIO-related hemangiopericytoma, whereas FGF-7 and sFRP-4 were widely expressed in all studied cell lines and tissues. These results support the use of FGF-23 measurements for the diagnosis and follow-up of patients with TIO. In addition, the specific expression of FGF-23 and MEPE in the TIO-associated tumor suggests an important role of these two phosphatonins in the pathogenesis of TIO. Because of the limited number of patients in our report, further studies are needed to clarify the role of different phosphatonins in the development of the clinical features of TIO.

Changes in Matrix Extracellular Phosphoglycoprotein Expression before and during in Vitro Osteogenic Differentiation of Human Dental Papilla Mesenchymal Cells

The purpose of this study is to characterise the expression of matrix extracellular phosphoglycoprotein (MEPE) in cultured mesenchymal cells isolated from human dental papilla (PaMCs) of impacted third molars either before or during differentiation of these cells into osteo/odontoblasts. PaMCs, like mesenchymal cells deriving from human dental pulp (DPMCs), resulted positive for a number of mesenchymal markers including CD146 and STRO-1. During the first week in culture they showed a faster proliferation rate than DPMCs, coupled to an earlier down-regulation of MEPE. Also when the cells were further cultured in osteogenic medium (containing beta-glycerophosphate, ascorbic acid and dexamethasone) for 40 days, MEPE down-regulation coupled to an increased expression of osteogenic markers, such as osteocalcin and alkaline phosphatase, occurred earlier in PaMCs than in DPMCs. Thus, our data, indicating that also in PaMCs MEPE expression is higher when cells proliferate, whereas it is downregulated as cells differentiated, are in favour of a role of MEPE as an early regulator of odontogenic differentiation. We also confirm the superior proliferative potential of PaMCs in comparison with DPMCs, coupled to a more rapid induction of osteogenic differentiation. Therefore, these cells represent an optimal source to be conveniently used for dental tissue engineering and tooth regeneration.

Immunolocalization of Sibling and RUNX2 Proteins During Vertical Distraction Osteogenesis in the Human Mandible

We tested the hypothesis that mechanical loading of human bone increases expression of the transcription factor RUNX2 and bone matrix proteins osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein-1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE). We examined this in tissue sections of atrophic mandibular bone taken from edentulous patients who had undergone distraction osteogenesis. In undistracted bone, weak to moderate staining for OPN and BSP was found in osteoblasts and bone matrix of immature woven bone. RUNX2 was also detectable in osteoblasts and in cells of the periosteum. In woven bone, but not in lamellar bone, a small number of osteocytes stained for all proteins tested. After distraction, staining intensity had increased in the existing old bone and staining was seen in more bone cells than before distraction. We also found a high expression of DMP1 and MEPE in many osteocytes embedded in woven bone and in some osteocytes of lamellar bone not seen before distraction. New bone trabeculae were forming in the fibrous tissue of the distraction gap containing all stages of intramembranous bone formation. Moderate to strong staining was seen for all five proteins tested in osteocytes located in woven bone of these trabeculae and for RUNX2, OPN, and BSP in osteoblasts lining the trabecular surfaces. We conclude that loading of atrophic human jawbone by distraction activates matrix synthesis of bone cells in and around existing bone. Increased staining of DMP1 and MEPE in osteocytes after loading is in line with the concept that these proteins may be involved in signaling the effector cells to adapt the bone structure to its mechanical demands.

SIBLING Expression Patterns in Duct Epithelia Reflect the Degree of Metabolic Activity

The SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family of secreted glycophosphoproteins includes bone sialoprotein (BSP), dentin matrix protein-1 (DMP1), dentin sialophosphoprotein (DSPP), osteopontin (OPN), and matrix extracellular phosphoglycoprotein (MEPE). For many years, they were thought in normal adults to essentially be limited to metabolically active mesenchymal cells that assembled the mineralized matrices of bones and teeth. Over the last decade they have also been upregulated in a variety of tumors. Three of these proteins (BSP, OPN, and DMP1) have been shown to interact with three matrix metalloproteinases (MMP-2, MMP-3, and MMP-9, respectively). Recently, all five SIBLINGs and their MMP partners when known were observed in specific elements of normal ductal epithelia in salivary gland and kidney. We have hypothesized that the SIBLINGs and their MMP partners may be expressed in ductal cells with high metabolic activity. In this paper, we show that all the SIBLINGs (except MEPE) and their MMP partners are expressed in the metabolically active epithelia of human eccrine sweat gland duct but not in the more passive ductal cells of the macaque (monkey) lacrimal gland. It is hypothesized that MEPE expression may be limited to cells involved in active phosphate transport. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Cross-talk between Wnt and Bone Morphogenetic Protein 2 (BMP-2) Signaling in Differentiation Pathway of C2C12 Myoblasts

Loss of function of the Wnt co-receptor, lipoprotein receptor-related protein 5, decreases bone formation, and a point mutation in this gene results in high bone mass, indicating the importance of this signaling pathway in bone formation. However, the exact mechanism is currently unknown. We examined a potential role for Wnt signaling and functional cross-talk of bone morphogenetic protein 2 (BMP-2) in osteoblast differentiation. To assess the contribution of Wnt, we generated C2C12 cells over-expressing Wnt3a or Wnt5a and treated these with BMP-2. We showed that expression of matrix extracellular phosphoglycoprotein was induced by BMP-2 in Wnt3a over-expressing C2C12 cells but not in Wnt5a over-expressing C2C12 cells. Over-expression of Wnt3a blocked BMP-2-induced inhibition of myotube formation in C2C12 cells when switched to low mitogen medium. In these cultures, expression of inhibitor of DNA binding/differentiation (Id) 1, a helix-loop-helix protein induced by BMP-2, decreased in stable Wnt3a- but not in Wnt5a-expressing cells. This suppression is mediated by a GC-rich region of the BMP-2-responsive element of the Id1 gene promoter, and interaction between Smad1/4 and beta-catenin is crucial for Wnt-mediated suppression of the BMP-2 response in C2C12 cells. Over-expression of the inhibitor of canonical Wnt signaling, Dickkopf, inhibits this suppression. In contrast, BMP-2 or Smad1/4 up-regulated Wnt3a or activated beta-catenin-induced lymphoid-enhancing factor 1/T cell factor-dependent transcriptional activity. These findings identify functional cross-talk of Id1 expression between Wnt and BMP signaling and demonstrate a novel mechanism for Wnt regulation of the BMP-2 response, linking Id1 expression to Wnt/beta-catenin signaling.

MEPE is downregulated as dental pulp stem cells differentiate

Previous studies on dental pulp cell culture have described heterogenous mixtures of cells that differentiate into odontoblasts and form mineralized dentin. The aim of this study was to characterize the matrix extracellular phosphoglycoprotein (MEPE) expression by dental pulp stem cells (DPSC), related to cell differentiation. DPSC differentiation to form mineralized nodules was characterized by Alizarin red staining and micro-Raman spectroscopy. Osteogenesis SuperArray analysis was used to broadly screen for osteogenesis-related genes altered by DPSC differentiation. Relative levels of expression of MEPE and DSP were determined by semiquantitative RT-PCR and Western blot. Mineral analysis showed that as DPSC differentiated, they formed a carbonated hydroxyapatite mineral. Differentiation was initially marked by upregulation by Runx2, TGFbeta-related genes, EGFR and genes involved in collagen metabolism. ALP activity first increased, as DPSCs reached confluence but later decreased when cells further differentiated three weeks after confluence. MEPE was the only marker that was downregulated as DPSCs differentiated. DPSC differentiation can be characterized by downregulation of MEPE as other markers of DPSC differentiation, such as DSP, are upregulated. Expression of MEPE related to DSP and can be used to monitor DPSC as they are used for studies of odontoblast differentiation, tissue engineering or vital pulp therapy. The downregulation of MEPE as DPSC differentiate, suggests that MEPE is an inhibitor of mineralization.