Lung development, repair and cancer: A study on the role of MMP20 gene in adenocarcinoma.

Multiple matrix metalloproteinases have significant roles in tissue organization during lung development, and repair. Imbalance of proteinases may lead to chronic inflammation, changes in tissue structure, and are also highly associated to cancer development. The role of MMP20 is not well studied in lung organogenesis, however, it was previously shown to be present at high level in lung adenocarcinoma. The current study aimed to identify the functional properties of MMP20 on cell proliferation and motility in a lung adenocarcinoma in vitro cell model, and relate the interaction of MMP20 with other molecular signalling pathways in the lung cells after gaining tumoral properties. In this study, two different single guide RNA (sgRNAs) that specifically targeted on MMP20 sites were transfected into human lung adenocarcinoma A549 cells by using CRISPR-Cas method. Following that, the changes of PI3-K, survivin, and MAP-K mRNA gene expression were determined by Real-Time Polymerase Chain Reaction (RT-PCR). The occurrence of cell death was also examined by Acridine Orange/Propidium Iodide double staining. Meanwhile, the motility of the transfected cells was evaluated by wound healing assay. All the data were compared with non-transfected cells as a control group. Our results demonstrated that the transfection of the individual sgRNAs significantly disrupted the proliferation of the A549 cell line through suppression in the gene expression of PI3-K, survivin, and MAP-K. When compared to non-transfected cells, both experimental cell groups showed reduction in the migration rate, as reflected by the wider gaps in the wound healing assay. The current study provided preliminary evidence that MMP20 could have regulatory role on stemness and proliferative genes in the lung tissues and affect the cell motility. It also supports the notion that targeting MMP20 could be a potential treatment mode for halting cancer progression.

The possible influence of genetic aetiological factors on molar-incisor hypomineralisation.

OBJECTIVE: The present study searched for evidence of possible associations between some genetic factors that could affect the development of molar-incisor hypomineralisation (MIH). METHODS: In 113 patients who were surgically treated at an Otorhinolaryngology and Cervicofacial Surgery Clinic (ORL) during early childhood, human leukocyte antigen (HLA) DQ2 and DQ8 haplotypes and single nucleotide polymorphisms (SNP) of eight amelogenesis-related genes were searched in genomic DNA. Genotypes were determined by high resolution melting (HRM), TaqMan genotyping assays, and Sanger sequencing. Association between MIH and the HLA DQ2 and DQ8 alleles was tested using a univariate logistic regression. The significance of genetic variants was analysed using the Cochran-Armitage tests for trend and the Fisher exact tests. RESULTS: We identified MIH in 22 (19.5 %) of the 113 children. Among the evaluated genetic variants, SNP rs2245803 in the MMP20 gene in a homozygous form in a recessive model was associated with MIH development (OR, 2.796; 95 %CI, 1.075 - 4.783; p = 0.0496) with the genotype distribution of TT(3), TG(6) or GG(13) in children with MIH and distribution of TT(18), TG(42) or GG(31) in children without MIH. CONCLUSIONS: While the aetiology of MIH remains unclear, our findings suggest that variants of genes associated with amelogenesis may play important roles in susceptibility to MIH.

Effects of DSPP and MMP20 Silencing on Adhesion, Metastasis, Angiogenesis, and Epithelial-Mesenchymal Transition Proteins in Oral Squamous Cell Carcinoma Cells.

Recent reports highlight the potential tumorigenic role of Dentin Sialophosphoprotein (DSPP) and its cognate partner Matrix Metalloproteinase 20 (MMP-20) in Oral Squamous Cell Carcinomas (OSCCs). However, the function/mechanism of these roles is yet to be fully established. The present study aimed to investigate the effects of DSPP and MMP20 silencing on specific proteins involved in oral cancer cell adhesion, angiogenesis, metastasis, and epithelial-mesenchymal transition (EMT). Stable lines of DSPP/MMP20 silenced OSCC cell line (OSC2), previously established via lentiviral-mediated shRNA transduction, were analyzed for the effects of DSPP, MMP20, and combined DSPP-MMP20 silencing on MMP2, MMP9, integrins αvß3 and αvß6, VEGF, Kallikerin- 4,-5,-8,-10, E-cadherin, N-cadherin, Vimentin, met, src, snail, and Twist by Western blot. Results show a significant decrease (p < 0.05) in the expression of MMP2, MMP9, integrin αvß3, αvß6, VEGF, Kallikerins -4, -5, -8, -10, N-cadherin, vimentin met, src, snail and twist following DSPP and MMP20 silencing, individually and in combination. On the other hand, the expression of E-cadherin was found to be significantly increased (p < 0.05). These results suggest that the tumorigenic effect of DSPP and MMP20 on OSC2 cells is mediated via the upregulation of the genes involved in invasion, metastasis, angiogenesis, and epithelial-mesenchymal transition (EMT).

DNA sequencing reveals AMELX, ODAM and MMP20 variations in dental fluorosis.

OBJECTIVE: Dental fluorosis (DF) is a dental development disorder caused by chronic fluoride overconsumption. There are differences in the susceptibility to and severity of DF in studied populations. The objective of the present study was to determine if single-nucleotide variations (SNVs) in the genes Amelogenin (AMELX), Odontogenic Ameloblast Associated (ODAM) and Matrix Metalloproteinase 20 (MMP20) are associated with DF by evaluating the relationship between variations in these genes and the degree of DF severity. SUBJECTS AND METHODS: Schoolchildren from two regions of Durango State and Mexico City, Mexico, were studied. The DF phenotype was determined using the Thylstrup and Fejerskov (TF) index. DNA was obtained from the buccal mucosa of each participant, and the presence of the variations rs946252 in AMELX, rs1514392 in ODAM and rs1784418 in MMP20 was determined by bidirectional DNA sequencing. RESULTS: A total of 180 DNA samples from 30 schoolchildren from 2 areas of Durango State were sequenced and analyzed. Differences in the severity of DF were found between the study areas (p = 0.006). SNVs in theMMP20 gene were present in 76.9 % of the participants in the high fluoride concentration and lower DF severity area. CONCLUSION: AMELX and ODAM variations was not different between the two populations with respect to DF severity; however, the presence of rs1784418 differed between phenotypes with regard to susceptibility to DF. Therefore, MMP20 might be related to the various phenotypes of DF and may serve as a protective marker.

Integrated Multiomics Approach to Identify Genetic Underpinnings of Heart Failure and Its Echocardiographic Precursors: Framingham Heart Study.

BACKGROUND: Heart failure (HF) may arise from alterations in metabolic, structural, and signaling pathways, but its genetic architecture is incompletely understood. To elucidate potential genetic contributors to cardiac remodeling and HF, we integrated genome-wide single-nucleotide polymorphisms, gene expression, and DNA methylation using a transomics analytical approach. METHODS: We used robust rank aggregation (where the position of a certain gene in a rank order list [based on statistical significance level] is tested against a randomly shuffled rank order list) to derive an integrative transomic score for each annotated gene associated with a HF trait. RESULTS: We evaluated ≤8372 FHS (Framingham Heart Study) participants (54% women; mean age, 55±17 years). Of these, 62 (0.7%) and 35 (0.4%) had prevalent HF with reduced ejection fraction and HF with preserved left ventricular ejection fraction, respectively. During a mean follow-up of 8.5 years (minimum-maximum, 0.005-18.6 years), 223 (2.7%) and 234 (2.8%) individuals developed incident HF with reduced ejection fraction and HF with reduced ejection fraction, respectively. Top genes included MMP20 and MTSS1 (promotes actin assembly at intercellular junctions) for left ventricular systolic function; ITGA9 (receptor for VCAM1 [vascular cell protein 1]) and C5 for left ventricular remodeling; NUP210 (expressed during myogenic differentiation) and ANK1 (cytoskeletal protein) for diastolic function; TSPAN16 and RAB11FIP3 (involved in regulation of actin cytoskeleton) for prevalent HF with reduced ejection fraction; ANKRD13D and TRIM69 for incident HF with reduced ejection fraction; HPCAL1 and PTTG1IP for prevalent HF with reduced ejection fraction; and ZNF146 (close to the COX7A1 enzyme) and ZFP3 (close to SLC52A1-the riboflavin transporter) for incident HF with reduced ejection fraction. We tested the HF-related top single-nucleotide polymorphisms in the UK biobank, where rs77059055 in TPM1 (minor allele frequency, 0.023; odds ratio, 0.83; P=0.002) remained statistically significant upon Bonferroni correction. CONCLUSIONS: Our integrative transomics approach offers insights into potential molecular and genetic contributors to HF and its precursors. Although several of our candidate genes have been implicated in HF in animal models, independent replication is warranted.

Survey of dentin sialophosphoprotein and its cognate matrix metalloproteinase-20 in human cancers.

BACKGROUND: Matrix metalloproteinases-20 (MMP20) expression is widely regarded as tooth specific, with expression limited to dental hard tissues. Recently, we reported MMP20 expression and interaction with dentin sialophosphoprotein (DSPP), a member of the Small Integrin Binding Ligand N-linked Glycoproteins (SIBLINGs), in human oral squamous cell carcinoma (OSCC) and dysplastic oral premalignant lesions (OPLs), suggesting a role for MMP20-DSPP interaction in oral carcinogenesis. METHODS: This study aimed to survey the expression of MMP20 and its cognate DSPP partner in the breast, colon, prostate, thyroid, and cervical neoplasms. Using commercially available tissue microarrays (TMAs) and cell lines, we performed immunohistochemistry, immunofluorescence, proximity ligation assay, and western blot experiments to determine the expressions of MMP20 and DSPP in the breast, colon, prostate, thyroid, cervical neoplasms, and their normal counterparts. RESULTS: Significantly high expression levels of MMP20 and DSPP were observed in the malignant breast, colon, prostate, thyroid, and cervical neoplasms compared with their benign and normal counterparts. Furthermore, MMP20 levels increased with advanced stages of colon and thyroid cancers. DSPP expression increased significantly with tumor stage in all cancers examined. CONCLUSIONS: The co-localization and potential MMP20-DSPP interaction previously reported in oral cancers are present in other cancers. These results suggest MMP20-DSPP pairing as a potential marker of disease activity in some epithelial cancers with diagnostic and prognostic implications.

[High expression of MMP20 may be a potential prognostic factor for poor outcomes of patients with endometrial carcinoma].

OBJECTIVE: To explore the expression of MMP20 in endometrial carcinoma and its clinical significance. METHODS: Bioinformatics analysis was used to explore the correlation of MMP20 expression with the prognosis of the patients with endometrial carcinoma. We collected 21 pairs of fresh endometrial carcinoma and adjacent endometrial tissues to detect the expression of MMP20 mRNA using real-time PCR. We also examined MMP20 protein expressions in 134 paraffin-embedded endometrial carcinoma tissues and 34 paraffin-embedded endometrial tissues using immunohistochemistry. The correlation of MMP20 expression with the clinicopathological features and prognosis of the patients were analyzed. RESULTS: Based on TCGA database analysis, the patients with endometrial carcinoma showing an increased MMP20 mRNA expression had a significantly poorer prognosis than those with a low MMP20 mRNA expression (P=0.00065). Real-time PCR analysis and immunohistochemistry confirmed the up-regulated expressions of MMP20 at both the mRNA (P=0.0062) and protein (P=0.005) levels in endometrial carcinoma tissues compared to the adjacent endometrial tissues. An elevated MMP20 protein expression was positively associated with FIGO stage of endometrial carcinoma (P=0.014) and inversely correlated with the survival time of the patients (P=0.019). The Cox regression model analysis showed that an increased MMP20 expression was an independent predictor of a poor prognosis of the patients with endometrial carcinoma (P=0.067). CONCLUSIONS: High expression of MMP20 is a potential prognostic factor for poor outcomes of patients with endometrial carcinoma.

Interferon γ suppresses dentin sialophosphoprotein in oral squamous cell carcinoma cells resulting in antitumor effects, via modulation of the endoplasmic reticulum response.

The expression of proinflammatory cytokines in various malignant neoplasms is widely considered to represent the host immune response to tumor development. The role of interferon (IFN)γ in head and neck squamous cell carcinoma, and its association with endoplasmic reticulum (ER) stress pathways, remains a subject of ongoing investigation. Dentin sialophosphoprotein (DSPP), which is a member of the small integrin­binding N­linked glycoproteins family, has been implicated in malignant transformation and invasion of oral squamous cell carcinoma (OSCC). Recent studies have established matrix metalloproteinase (MMP)20 as the cognate MMP partner of DSPP. The present study examined the effects of IFNγ treatment on DSPP and MMP20 expression, ER stress, the unfolded protein response (UPR), and calcium (Ca) homeostasis regulatory mechanisms in OSCC cells. The OSC2 OSCC cell line was treated with IFNγ at specific time­points. At each time­point, the mRNA expression levels of DSPP and MMP20, and those of ER­stress­, UPR­ and Ca homeostasis­associated proteins [78­kDa glucose­regulated protein (GRP78), sarco/endoplasmic reticulum Ca2+­ATPase (SERCA2b), inositol 1,4,5­trisphosphate receptor (IP3r), protein kinase R­like ER kinase (PERK) and inositol­requiring enzyme 1 (IRE1)], were assessed by reverse transcription­quantitative polymerase chain reaction. The protein expression levels of B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein (Bax), proliferating cell nuclear antigen (PCNA) and cytochrome c were analyzed by western blotting. Cell viability, apoptosis and migration were evaluated by MTT, Annexin V­fluorescein isothiocyanate flow cytometry and wound­healing assays, respectively. IFNγ treatment significantly downregulated the mRNA expression levels of the major ER stress regulator GRP78 and, to a lesser extent, the UPR­associated molecule IRE1; however, IFNγ had no significant effect on PERK. With regards to ER Ca homeostasis molecules, treatment with IFNγ downregulated the mRNA expression levels of SERCA2b and upregulated those of IP3r. Furthermore, DSPP and MMP20 mRNA expression levels were significantly reduced following IFNγ treatment. Notably, treatment with IFNγ hampered OSC2 migration, reduced cell viability and PCNA protein expression, enhanced apoptosis, downregulated Bcl­2, and upregulated Bax and cytochrome c. Overall, IFNγ inhibited OSCC cell viability and migration, and increased apoptosis, possibly by regulating ER stress and UPR mechanisms. In addition, IFNγ­induced DSPP and MMP20 downregulation may correspond with alteration in ER Ca homeostasis.

DSPP-MMP20 gene silencing downregulates cancer stem cell markers in human oral cancer cells.

BACKGROUND: Recent findings indicate that dentin sialophosphoprotein (DSPP) and matrix metalloproteinase (MMP) 20 interact in oral squamous cell carcinoma (OSCC). The objective of this study was to determine the effects of DSPP/MMP20 gene silencing on oral cancer stem cell (OCSC) markers. METHODS: The expression of well-established OCSC markers: ABCG2; ALDH1; CD133; CD44; BMI1; LGR4, and Podoplanin in DSPP/MMP20-silenced OSCC cell line, OSC2, and controls were assayed by western blot (WB), and flow cytometry techniques. The sensitivity of OSC2 cells to cisplatin following DSPP/MMP20 silencing was also determined. RESULTS: DSPP/MMP20 silencing resulted in downregulation of OCSC markers, more profoundly ABCG2 (84%) and CD44 (81%), following double silencing. Furthermore, while treatment of parent (pre-silenced) OSC2 cells with cisplatin resulted in upregulation of OCSC markers, DSPP/MMP20-silenced OSC2 cells similarly treated resulted in profound downregulation of OCSC markers (72 to 94% at 50 µM of cisplatin), and a marked reduction in the proportion of ABCG2 and ALDH1 positive cells (~ 1%). CONCLUSIONS: We conclude that the downregulation of OCSC markers may signal a reduction in OCSC population following MMP20/DSPP silencing in OSCC cells, while also increasing their sensitivity to cisplatin. Thus, our findings suggest a potential role for DSPP and MMP20 in sustaining OCSC population in OSCCs, possibly, through mechanism(s) that alter OCSC sensitivity to treatment with chemotherapeutic agents such as cisplatin.

NaF Reduces KLK4 Gene Expression by Decreasing Foxo1 in LS8 Cells.

Decreased expression and increased phosphorylation of Forkhead box o1 (Foxo1) in ameloblasts were observed both in vivo and in vitro when treated by fluoride. The present study aims to investigate the possible relationship between Foxo1 and enamel matrix proteinases, matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), in NaF-treated ameloblasts. Ameloblast-like cells (LS8 cells) were exposed to NaF at selected concentration (0/2 mM) for 24 h. Gene overexpression and silencing experiments were used to up- and down-regulate Foxo1 expression. The expression levels of Foxo1, MMP20, and KLK4 were detected by quantitative real-time PCR and western blot. Dual luciferase reporter assay was performed to evaluate the regulation of Foxo1 on the transcriptional activity of KLK4 promoter. The results showed that KLK4 expression was decreased in LS8 cells treated by NaF, while MMP20 expression was not changed. Foxo1 activation led to significantly up-regulation of KLK4 in LS8 cells under NaF condition. Knockout of Foxo1 markedly decreased klk4 expression in mRNA level, and intensified inhibition occurred in LS8 cells when combined with NaF treatment. However, the variation trend of MMP20 was not clear. Dual luciferase reporter assay showed that Foxo1 activation enhanced the transcriptional activity of KLK4 promoter. These findings suggest that the decrease of Foxo1 expression induced by high fluoride was a cause for low KLK4 expression.

The dynamics of TGF-ß in dental pulp, odontoblasts and dentin.

Transforming growth factor-beta (TGF-ß) is critical for cell proliferation and differentiation in dental pulp. Here, we show the dynamic mechanisms of TGF-ß in porcine dental pulp, odontoblasts and dentin. The mRNA of latent TGF-ß1 and TGF-ß3 is predominantly expressed in odontoblasts, whereas the mRNA expression level of latent TGF-ß2 is high in dental pulp. TGF-ß1 is a major isoform of TGF-ß, and latent TGF-ß1, synthesized in dental pulp, is primarily activated by matrix metalloproteinase 11 (MMP11). Activated TGF-ß1 enhances the mRNA expression levels of MMP20 and full-length dentin sialophosphoprotein (DSPP) in dental pulp cells, coinciding with the induction of odontoblast differentiation. Latent TGF-ß1 synthesized in odontoblasts is primarily activated by MMP2 and MMP20 in both odontoblasts and dentin. The activity level of TGF-ß1 was reduced in the dentin of MMP20 null mice, although the amount of latent TGF-ß1 expression did not change between wild-type and MMP20 null mice. TGF-ß1 activity was reduced with the degradation of DSPP-derived proteins that occurs with ageing. We propose that to exert its multiple biological functions, TGF-ß1 is involved in a complicated dynamic interaction with matrix metalloproteinases (MMPs) and/or DSPP-derived proteins present in dental pulp, odontoblasts and dentin.

Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis.

Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY LNA miRNA microarray identify miR-185-5p as a miRNA target commonly induced by all three Runx2 mutants. Real-time quantitative PCR was applied to determine the expression of miR-185-5p and Dlx2 in samples. Dual-luciferase reporter assays were conducted to confirm Dlx2 as a legitimate target of miR-185-5p. The suppressive effect of miR-185-5p on amelogenesis and osteogenesis of miR-185-5p was evaluated by RT-PCR and western blot examination of Amelx, Enam, Klk4, and Mmp20 gene and protein expression, and by Alizarin Red stain. We found that mutant Runx2 suppressed amelogenesis and osteogenesis. miR-185-5p, induced by Runx2, suppressed amelogenesis and osteogenesis. Furthermore, we identified Dlx2 as direct target of miR-185-5p. Consistently, Dlx2 expression was inversely correlated with miR-185-5p levels. This study highlights the molecular etiology and significance of miR-185-5p in CCD, and suggests that targeting miR-185-5p may represent a new therapeutic strategy in prevention or intervention of CCD.

Common variants in MMP20 at 11q22.2 predispose to 11q deletion and neuroblastoma risk.

MYCN amplification and 11q deletion are two inversely correlated prognostic factors of poor outcome in neuroblastoma. Here we identify common variants at 11q22.2 within MMP20 that associate with neuroblastoma cases harboring 11q deletion (rs10895322), using GWAS in 113 European-American cases and 5109 ancestry-matched controls. The association is replicated in 44 independent cases and 1902 controls. Our study yields novel insights into the genetic underpinnings of neuroblastoma, demonstrating that the inherited common variants reported contribute to the origin of intra-tumor genetic heterogeneity in neuroblastoma.Chromosomal abnormalities such as 11q deletion are associated with poor prognosis in neuroblastoma. Here, the authors perform a genome-wide association study and identify an association between a variant within a Matrix metalloproteinase (MMP) gene member, MMP20, and 11q-deletion subtype neuroblastoma.

Expression of enamel proteins in rough endoplasmic reticulum and golgi complex in human dental germs / Expresión de proteínas del esmalte en retículo endoplásmico rugoso y complexo golgiensis en gérmenes dentales humanos

Tooth enamel is the hardest tissue in the body. The organic matrix configuration is provided by the main proteins amelogenin, ameloblastin and enamelysin (MMP20), an enzyme that helps to shape the matrix. The aim of this study was to determine by histochemistry the expression of amelogenin and enamelysin through the rough endoplasmic reticulum in the late stages of amelogenesis, and its expression in the Complexus golgiensis (Golgi complex / Golgi apparatus) in the early stages in human fetuses. In early stages a colocalization of both proteins inside the Golgi apparatus was found, being more evident the relationship between Golgi and amelogenin (99.92 %). In the late stage, a colocalization of both proteins and rugged endoplasmic reticulum was found. With enamelysin being more evident in relation with rough endoplasmic reticulum (99.95 %). Our findings demonstrated the presence of amelogenin and enamelysin in odontoblast and ameloblast. However, the presence of these two proteins in odontoblast remains unknown.

El esmalte dental es el tejido más duro del cuerpo. La configuración de la matriz orgánica es proporcionada por las proteínas principales amelogenina, ameloblastina y enamelisina (MMP20), una enzima que ayuda a dar forma a la matriz. El objetivo de este estudio fue determinar mediante histoquímica la expresión de amelogenina y enamelisina a través del retículo endoplasmático rugoso en las últimas etapas de la amelogénesis , y su expresión en el Complexo golgiensis en las primeras etapas de formación en fetos humanos. En las primeras etapas se observó colocalización de ambas proteínas en el interior del Complexo golgiensis, siendo más evidente la relación entre Golgi y amelogenina (99,92 %). En la última etapa, se identificó una colocalización de ambas proteínas y retículo endoplásmico rugoso. Resulto más evidente la enamelisina en relación con el retículo endoplasmático rugoso (99,95 %). Nuestros resultados demostraron la presencia de amelogenina y enamelisina en odontoblastos y ameloblastos, sin embargo se desconoce la presencia de estas dos proteínas en odontoblastos.

Postradiation Matrix Metalloproteinase-20 Expression and Its Impact on Dental Micromorphology and Radiation-Related Caries.

Recent evidence suggests that head-and-neck radiotherapy (HNRT) increases active forms of matrix metalloproteinase-20 (MMP-20) in human tooth crowns, degrading the dentin-enamel junction (DEJ) and leading to enamel delamination, which is a pivotal step in the formation of radiation-related caries (RRC). Additional participation of enzymatic degradation of organic matrix components in caries progression was attributed to MMP-20 in dentin. Therefore, the current study tested the hypothesis that MMP-20 is overexpressed in the DEJ, dentin-pulp complex components, and carious dentin of post-HNRT patients, leading to detectable micromorphological changes to the enamel and dentin. Thirty-six teeth were studied, including 19 post-HNRT specimens and 17 nonirradiated controls. Optical light microscopy was used to investigate the micromorphological components of the DEJ, dentin-pulp complex components, and carious dentin. The samples were divided into 2 subgroups: nondemineralized ground sections (n = 20) and demineralized histological sections (n = 16). In addition, immunohistochemical analysis using the immunoperoxidase technique was conducted to semiquantitatively assess MMP-20 expression in the DEJ, dentin-pulp complex components, and carious dentin. No apparent damage to the DEJ microstructure or other dentin-pulp complex components was observed and no statistically significant differences were detected in MMP-20 expression (p > 0.05) between the irradiated and control groups. This study rejected the hypothesis that MMP-20 is overexpressed in the DEJ, dentin-pulp complex components, and carious dentin of post-HNRT patients, leading to detectable micromorphological changes. Hence, direct effects of radiation may not be regarded as an independent factor to explain aggressive clinical patterns of RRC.

EDTA soluble chemical components and the conditioned medium from mobilized dental pulp stem cells contain an inductive microenvironment, promoting cell proliferation, migration, and odontoblastic differentiation.

BACKGROUND: The critical challenge in tissue engineering is to establish an optimal combination of stem cells, signaling morphogenetic molecules, and extracellular matrix scaffold/microenvironment. The extracellular matrix components of teeth may be reconstituted as an inductive microenvironment in an ectopic tooth transplantation bioassay. Thus, the isolation and identification of the chemical components of the inductive microenvironment in pulp/dentin regeneration will accelerate progress towards the goal of tissue engineering of the tooth. METHODS: The teeth demineralized in 0.6 M hydrochloric acid were sequentially extracted by 4.0 M guanidine hydrochloride (GdnHCl), pH 7.4, and 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 7.4. The extracted teeth were transplanted into an ectopic site in severe combined immunodeficiency (SCID) mice with mobilized dental pulp stem cells (MDPSCs). The unextracted tooth served as a positive control. Furthermore, the soluble components for the inductive microenvironment, the GdnHCl extracts, or the EDTA extracts together with or without MDPSC conditioned medium (CM) were reconstituted systematically with autoclaved teeth in which the chemical components were completely inactivated and only the physical microenvironment was preserved. Their pulp/dentin regenerative potential and angiogenic potential were compared 28 days after ectopic tooth transplantation by histomorphometry and real-time RT-PCR analysis. RESULTS: Expression of an odontoblastic marker, enamelysin, and a pulp marker, thyrotropin-releasing hormone degrading enzyme (TRH-DE), was lower, and expression of a periodontal cell marker, anti-asporin/periodontal ligament-associated protein 1 (PLAP-1), was higher in the transplant of the EDTA-extracted teeth compared with the GdnHCl-extracted teeth. The autoclaved teeth reconstituted with the GdnHCl extracts or the EDTA extracts have weak regenerative potential and minimal angiogenic potential, and the CM significantly increased this potential. Combinatorial effects of the EDTA extracts and the CM on pulp/dentin regeneration were demonstrated in vivo, consistent with their in-vitro effects on enhanced proliferation, migration, and odontoblastic differentiation. CONCLUSIONS: The EDTA-extracted teeth demonstrated significantly lower pulp/dentin regenerative potential compared with the GdnHCl-extracted teeth. The EDTA soluble chemical components when reconstituted with the physical structure of autoclaved teeth serve as an inductive microenvironment for pulp/dentin regeneration, promoting cell proliferation, migration, and odontoblastic differentiation.

Expression Pattern of Matrix Metalloproteinase 20 (MMP20) in Human Tumors.

BACKGROUND/AIM: Matrix metalloproteinase 20 (MMP20) is a member of the family of matrix metalloproteinases. Under normal conditions the expression of MMP20 is restricted to ameloblasts and odontoblasts. In order to identify a possible expression of MMP20 under pathological conditions, we investigated three major human tumor entities, i.e. colon, breast and lung tumors, on the mRNA and protein level. MATERIALS AND METHODS: Real-time RT-PCR and immunocytochemical analyses of established human tumor cell lines were employed for our study; immunohistochemical analysis was performed on both primary tumors and normal control tissues. RESULTS: MMP20 was identified on both the mRNA and the protein level in breast MCF-7, colon HT-29, and lung A549 cell lines. MMP20 was also detected in primary tumor tissue by immunohistochemistry. CONCLUSION: MMP20 is a new potential candidate for tumor diagnosis or therapy.

Abrogation of epithelial BMP2 and BMP4 causes Amelogenesis Imperfecta by reducing MMP20 and KLK4 expression.

Amelogenesis Imperfecta (AI) can be caused by the deficiencies of enamel matrix proteins, molecules responsible for the transportation and secretion of enamel matrix components, and proteases processing enamel matrix proteins. In the present study, we discovered the double deletion of bone morphogenetic protein 2 (Bmp2) and bone morphogenetic protein 4 (Bmp4) in the dental epithelium by K14-cre resulted in hypoplastic enamel and reduced density in X-ray radiography as well as shortened enamel rods under scanning electron microscopy. Such enamel phenotype was consistent with the diagnosis of hypoplastic amelogenesis imperfecta. Histological and molecular analyses revealed that the removal of matrix proteins in the mutant enamel was drastically delayed, which was coincided with the greatly reduced expression of matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4). Although the expression of multiple enamel matrix proteins was down-regulated in the mutant ameloblasts, the cleavage of ameloblastin was drastically impaired. Therefore, we attributed the AI primarily to the reduction of MMP20 and KLK4. Further investigation found that BMP/Smad4 signaling pathway was down-regulated in the K14-cre;Bmp2(f/f);Bmp4(f/f)ameloblasts, suggesting that the reduced MMP20 and KLK4 expression may be due to the attenuated epithelial BMP/Smad4 signaling.

Medium modification with bone morphogenetic protein 2 addition for odontogenic differentiation.

The aim of this study was to evaluate whether medium modification improves the odontogenic differentiation of human dental pulp stem cells (DPSC) in vitro and in vivo. DPSC isolated from human impacted third molar teeth were analysed for clusters of differentiation with flow cytometry. Odontogenic differentiation was stimulated by medium modification with the addition of bone morphogenetic protein 2 (BMP2). The expression of dentin sialophosphoprotein, dentin matrix protein 1, enamelysin/matrix metalloproteinase 20 and the phosphate-regulating gene with homologies to endopeptidases on the X chromosome of the cells were analysed with RT-PCR at 7, 14 and 21 days. Then, DPSC were transplanted on the back of immunocompromised mice via a hydroxyapatite tricalcium phosphate scaffold, and the structure of the formed tissue was investigated. The cells were identified as mesenchymal stem cells with a 98.3% CD73 and CD90 double-positive cell rate. The increase in mineralization capacity and expression of human enamel-dentin specific transcripts proportional to the culture period were determined after differentiation. Six weeks after transplantation, an osteo-dentin matrix was formed in the group in which odontogenic differentiation was stimulated, and the odontogenic characteristics of the matrix were confirmed by histological examination and RT-PCR analysis. Odontogenic differentiation of the isolated and characterized human DPSC was improved with medium modification by the addition of BMP2 in vitro and in vivo. The defined medium and applied technique have a potential use for forming reparative dentin in the future, but the effects of the method should be investigated in long-term studies.

High-Level Expression and Prognostic Significance of Matrix Metalloprotease-19 and Matrix Metalloprotease-20 in Human Pancreatic Ductal Adenocarcinoma.

OBJECTIVE: Matrix metalloproteinase (MMP)-19 and MMP-20 are important members of the MMP family, and their roles in tumor survivorship and progression are continually reported. This work aimed to determine the expression and prognostic significance of MMP-19 and MMP-20 in pancreatic ductal adenocarcinoma (PDAC). METHODS: Immunohistochemistry was used to investigate the levels of MMP-19 and MMP-20 expression in carcinoma tissues and paracancerous tissues from 102 PDAC patients. RESULTS: The MMP-19 and MMP-20 were, respectively, expressed in 71.6% (73/102) and 70.6% (72/102) of carcinoma tissues, and the expression was positively correlated (r = 0.643, P < 0.001). High-level expression of MMP-19 and MMP-20 was strongly correlated with aggressive clinicopathological characteristics. Kaplan-Meier analysis showed that high-level expression of MMP-19 and MMP-20 was significantly associated with decreased event-free survival (P < 0.001) and overall survival (P < 0.001). Multivariate analysis showed that high-level expression of MMP-19 could act as an independent predictive biomarker for poor event-free survival and overall survival. CONCLUSIONS: Levels of MMP-19 and MMP-20 expression are significantly increased in PDAC. High-level expression of MMP-19 and MMP-20 is closely correlated to progression and prognosis of PDAC, and these may be considered as promising markers for unfavorable prognoses.