Localización de las proteínas específicas del cemento radicular CEMP1 y CAP en células neoplásicas

Introducción: Las proteínas CEMP1 y CAP presentes en los cementoblastos y sus progenitores contribuyen a los procesos de mineralización en tejidos del ligamento periodontal, incluyendo la migración y la proliferación de fibroblastos gingivales; sin embargo su papel y relación con procesos neoplásicos no se han estudiado a profundidad. Para lograr un mejor entendimiento de la posible contribución de estas proteínas en los procesos tumorales, particularmente en las metástasis óseas, se investigó su expresión y localización en tejidos y líneas celulares de cáncer humano. Materiales y métodos: Trece casos de cáncer de próstata y mama que desarrollaron enfermedad metastásica ósea fueron analizados por medio de inmunohistoquímica; mientras que la expresión de las proteínas en dos líneas celulares de carcinoma de próstata (PC-3) y mama (MCF-7) se estudió por medio de ensayos de Western Blot. Resultados: Los tejidos de cáncer revelaron expresión citoplasmática y ocasionalmente nuclear de CAP en células tumorales y estructuras glandulares pequeñas, así como en el citoplasma de los fibroblastos estromales adyacentes al frente de invasión tumoral. En lo correspondiente a CEMP1, su expresión se localizó en el citoplasma de las células tumorales de 5 casos, pero no en el estroma. Ensayos de Wester Blot mostraron expresión de CEMP1 en las células PC-3 y MCF-7; y de CAP en las MCF-7. Conclusiones: Los resultados muestran que las proteínas de cemento radicular CEMP1 y CAP se expresan en tejidos neoplásicos y células neoplásicas, y que posiblemente contribuyen en ciertas condiciones patológicas como el cáncer metastásico en humanos.Palabras clave: CEMP1, CAP, metástasis óseas, TWIST, Runx2.Introduction: CEMP1 and CAP are recognized as cementum proteins, they appear to be limited to cementoblasts and their progenitors, and participate in the mineralization process of periodontal ligament tissues, including the proliferation and migration of periodontal ligament fibroblasts. However, their contribution in neoplastic processes had not been explored. In the present study, we investigated their protein expression and localization in cancer tissues and cells. Materials and Methods: CEMP1 and CAP expressions were analyzed immunohistochemically in 13 cancer cases with bone metastasis. In addition, Wester Blot essays were use to detect expression of the proteins in the prostate (PC-3) and mama (MCF-7) cancer cell lines. Results: CAP expression was detected in all tissues examined. Strong cytoplasmatic and rarely nuclear staining was found in small tumor nests, glandular structures and, in the stromal fibroblasts at the immediate vicinity of the tumor nests. CEMP1 was found in the cytoplasm of tumor cells in 5 cases, but its expression was negative in the stromal tissues. Also, cancer lines PC-3 and MCF-7 showed CEMP1 expression; however, CAP expression was observed only in MCF-7 cells. Conclusions: The results suggest that CEMP1 and CAP are present in tissues other that cementum and possibly contribute to pathological conditions such as metastatic cancer.Keywords: CEMP1, CAP, bone metastasis, TWIST, Runx2

Ciencia Odontológica 2.0

Libro que muestra avances de la Investigación Odontológica en MéxicoEs para los integrantes de la Red de Investigación en Estomatología (RIE) una enorme alegría presentar el segundo de una serie de 6 libros sobre casos clínicos, revisiones de la literatura e investigaciones. La RIE está integrada por cuerpos académicos de la UAEH, UAEM, UAC y UdeG

Localización de las proteínas específicas del cemento radicular CEMP1 y CAP en células neoplásicas

Introduction: CEMP1 and CAP are recognized as cementum proteins, they appear to be limited to cementoblasts and their progenitors, and participate in the mineralization process of periodontal ligament tissues, including the proliferation and migration of periodontal ligament fibroblasts. However, their contribution in neoplastic processes had not been explored. In the present study, we investigated their protein expression and localization in cancer tissues and cells. Materials and Methods: CEMP1 and CAP expressions were analyzed immunohistochemically in 13 cancer cases with bone metastasis. In addition, Wester Blot essays were use to detect expression of the proteins in the prostate (PC-3) and mama (MCF-7) cancer cell lines. Results: CAP expression was detected in all tissues examined. Strong cytoplasmatic and rarely nuclear staining was found in small tumor nests, glandular structures and, in the stromal fibroblasts at the immediate vicinity of the tumor nests. CEMP1 was found in the cytoplasm of tumor cells in 5 cases, but its expression was negative in the stromal tissues. Also, cancer lines PC-3 and MCF-7 showed CEMP1 expression; however, CAP expression was observed only in MCF-7 cells. Conclusions: The results suggest that CEMP1 and CAP are present in tissues other that cementum and possibly contribute to pathological conditions such as metastatic.Las proteínas CEMP1 y CAP presentes en los cementoblastos y sus progenitores contribuyen a los procesos de mineralización en tejidos del ligamento periodontal, incluyendo la migración y la proliferación de fibroblastos gingivales; sin embargo su papel y relación con procesos neoplásicos no se han estudiado a profundidad. Para lograr un mejor entendimiento de la posible contribución de estas proteínas en los procesos tumorales, particularmente en las metástasis óseas, se investigó su expresión y localización en tejidos y líneas celulares de cáncer humano. Materiales y métodos: Trece casos de cáncer de próstata y mama que desarrollaron enfermedad metastásica ósea fueron analizados por medio de inmunohistoquímica; mientras que la expresión de las proteínas en dos líneas celulares de carcinoma de próstata (PC-3) y mama (MCF-7) se estudió por medio de ensayos de Western Blot. Resultados: Los tejidos de cáncer revelaron expresión citoplasmática y ocasionalmente nuclear de CAP en células tumorales y estructuras glandulares pequeñas, así como en el citoplasma de los fibroblastos estromales adyacentes al frente de invasión tumoral. En lo correspondiente a CEMP1, su expresión se localizó en el citoplasma de las células tumorales de 5 casos, pero no en el estroma. Ensayos de Wester Blot mostraron expresión de CEMP1 en las células PC-3 y MCF-7; y de CAP en las MCF-7. Los resultados muestran que las proteínas de cemento radicular CEMP1 y CAP se expresan en tejidos neoplásicos y células neoplásicas, y que posiblemente contribuyen en ciertas condiciones patológicas como el cáncer metastásico en humanos

Bone regeneration in rat cranium critical-size defects induced by Cementum Protein 1 (CEMP1).

Gene therapy approaches to bone and periodontal tissue engineering are being widely explored. While localized delivery of osteogenic factors like BMPs is attractive for promotion of bone regeneration; method of delivery, dosage and side effects could limit this approach. A novel protein, Cementum Protein 1 (CEMP1), has recently been shown to promote regeneration of periodontal tissues. In order to address the possibility that CEMP1 can be used to regenerate other types of bone, experiments were designed to test the effect of hrCEMP1 in the repair/regeneration of a rat calvaria critical-size defect. Histological and microcomputed tomography (µCT) analyses of the calvaria defect sites treated with CEMP1 showed that after 16 weeks, hrCEMP1 is able to induce 97% regeneration of the defect. Furthermore, the density and characteristics of the new mineralized tissues were normal for bone. This study demonstrates that hrCEMP1 stimulates bone formation and regeneration and has therapeutic potential for the treatment of bone defects and regeneration of mineralized tissues

Cemp1-p3 Peptide Promotes the Transformation of Octacalcium Phosphate into Hydroxyapatite Crystals

Dental cementum contains unique molecules that regulate the mineralization process in vitro and in vivo, such as cementum protein 1 (CEMP1). This protein possesses amino acid sequence motifs like the human recombinant CEMP1 with biological activity. This novel cementum protein 1-derived peptide (CEMP1-p3, from the CEMP1’s N-terminal domain: (QPLPKGCAAVKAEVGIPAPH), consists of 20 amino acids. Hydroxyapatite (HA) crystals could be obtained through the combination of the amorphous precursor phase and macromolecules such as proteins and peptides. We used a simple method to synthesize peptide/hydroxyapatite nanocomposites using OCP and CEMP1-p3. The characterization of the crystals through scanning electron microscopy (SEM), powder X-ray diffraction (XRD), high--resolution transmission electron microscopy (HRTEM), and Raman spectroscopy revealed that CEMP1-p3 transformed OCP into hydroxyapatite (HA) under constant ionic strength and in a buffered solution. CEMP1-p3 binds and highly adsorbs to OCP and is a potent growth stimulator of OCP crystals. CEMP1-p3 fosters the transformation of OCP into HA crystals with crystalline planes (300) and (004) that correspond to the cell of hexagonal HA. Octacalcium phosphate crystals treated with CEMP1-p3 grown in simulated physiological buffer acquired hexagonal arrangement corresponding to HA. These findings provide new insights into the potential application of CEMP1-p3 on possible biomimetic approaches to generate materials for the repair and regeneration of mineralized tissues, or restorative materials in the orthopedic field

Carboxy-Terminal Cementum Protein 1-Derived Peptide 4 (cemp1-p4) Promotes Mineralization through wnt/β-catenin Signaling in Human Oral Mucosa Stem Cells

Human cementum protein 1 (CEMP1) is known to induce cementoblast and osteoblast differentiation and alkaline phosphatase (ALP) activity in human periodontal ligament-derived cells in vitro and promotes bone regeneration in vivo. CEMP1′s secondary structure analysis shows that it has a random-coiled structure and is considered an Intrinsic Disordered Protein (IDP). CEMP1′s short peptide sequences mimic the biological capabilities of CEMP1. However, the role and mechanisms of CEMP1′s C-terminal-derived synthetic peptide (CEMP1-p4) in the canonical Wnt/β-catenin signaling pathway are yet to be described. Here we report that CEMP1-p4 promotes proliferation and differentiation of Human Oral Mucosa Stem Cells (HOMSCs) by activating the Wnt/β-catenin pathway. CEMP1-p4 stimulation upregulated the expression of β-catenin and glycogen synthase kinase 3 beta (GSK-3B) and activated the transcription factors TCF1/7 and Lymphoid Enhancer binding Factor 1 (LEF1) at the mRNA and protein levels. We found translocation of β-catenin to the nucleus in CEMP1-p4-treated cultures. The peptide also penetrates the cell membrane and aggregates around the cell nucleus. Analysis of CEMP1-p4 secondary structure revealed that it has a random-coiled structure. Its biological activities included the induction to nucleate hydroxyapatite crystals. In CEMP1-p4-treated HOMSCs, ALP activity and calcium deposits increased. Expression of Osterix (OSX), Runt-related transcription factor 2 (RUNX2), Integrin binding sialoproptein (IBSP) and osteocalcin (OCN) were upregulated. Altogether, these data show that CEMP1-p4 plays a direct role in the differentiation of HOMSCs to a “mineralizing-like” phenotype by activating the β-catenin signaling cascade

Can Human Oral Mucosa Stem Cells Differentiate to Corneal Epithelia?

Human oral mucosa stem cells (hOMSCs) arise from the neural crest, they can self-renew, proliferate, and differentiate to several cell lines and could represent a good source for application in tissue engineering. Because of their anatomical location, hOMSCs are easy to isolate, have multilineage differentiation capacity and express embryonic stem cells markers such as—Sox2, Oct3/4 and Nanog. We have used SHEM (supplemented hormonal epithelial medium) media and cultured hOMSCs over human amniotic membrane and determined the cell’s capacity to differentiate to an epithelial-like phenotype and to express corneal specific epithelial markers—CK3, CK12, CK19, Pan-cadherin and E-cadherin. Our results showed that hOMSCs possess the capacity to attach to the amniotic membrane and express CK3, CK19, Pan-Cadherin and E-Cadherin without induction with SHEM media and expressed CK12 or changed the expression pattern of E-Cadherin to a punctual-like feature when treated with SHEM media. The results observed in this study show that hOMSCs possess the potential to differentiate toward epithelial cells. In conclusion, our results revealed that hOMSCs readily express markers for corneal determination and could provide the ophthalmology field with a therapeutic alternative for tissue engineering to achieve corneal replacement when compared with other techniques. Nevertheless, further studies are needed to develop a predictable therapeutic alternative for cornea replacement

Calcium Hydroxide Promotes Cementogenesis and Induces Cementoblastic Differentiation of Mesenchymal Periodontal Ligament Cells in a CEMP1-and ERK-Dependent Manner

Periodontal tissue engineering is a complex process requiring the regeneration of bone, cementum, and periodontal ligament (PDL). Since cementum regeneration is poorly understood, we used a dog model of dental pulpal necrosis and in vitro cellular wounding and mineralization assays to determine the mechanism of action of calcium hydroxide, Ca(OH)(2), in cementogenesis. Laser capture microdissection (LCM) followed by qRT-PCR were used to assay responses of periapical tissues to Ca(OH)(2) treatment. Additionally, viability, proliferation, migration, and mineralization responses of human mesenchymal PDL cells to Ca(OH)(2) were assayed. Finally, biochemical inhibitors and siRNA were used to investigate Ca(OH)(2)-mediated signaling in PDL cell differentiation. In vivo, Ca(OH)(2)-treated teeth formed a neocementum in a STRO-1- and cementum protein-1 (CEMP1)-positive cellular environment. LCM-harvested tissues adjacent to the neocementum exhibited higher mRNA levels for CEMP1, integrin-binding sialoprotein, and Runx2 than central PDL cells. In vitro, Ca(OH)(2) and CEMP1 promoted STRO-1-positive cell proliferation, migration, and wound closure. Ca(OH)(2) stimulated expression of the cementum-specific proteins CEMP1 and PTPLA/CAP in an ERK-dependent manner. Lastly, Ca(OH)(2) stimulated mineralization by CEMP1-positive cells. Blocking CEMP1 and ERK function abolished Ca(OH)(2)-induced mineralization, confirming a role for CEMP1 and ERK in the process. Ca(OH)(2) promotes cementogenesis and recruits STRO-1-positive mesenchymal PDL cells to undergo cementoblastic differentiation and mineralization via a CEMP1- and ERK-dependent pathway.NIH[NIH-RO1-DE13725]NIH[NIH-RO1-DE16671]PTPLA/CAPES Foundation[0668/07-9]State of Sao Paulo Research Foundation[FAPESP 06/51161-0