EGF Receptor Expression in Mineralized Tissues: An In Situ Hybridization and Immunocytochemical Investigation in Rat and Human Mandibles

There is extensive evidence that growth factors play a central part in the autocrine/paracrine regulation of cell growth and differentiation in mineralized tissues. In order to investigate involvement of the EGFr receptor (EGFr) in forming mineralized tissues, its expression was studied by in situ hybridization and immunocytochemistry in mandibles of growing rats, as well as in human embryos. In Hertwig's epithelial root sheath of rat molar, EGFr mRNAs appeared strongly expressed, while dental pulp and dental follicle showed weak labeling. The lingual epithelium of rat incisor showed strong labeling, which decreased after epithelial dislocation. Cells of the adjoining lingual dental pulp and dental follicle, as compared to epithelium, contained a low level of EGFr mRNAs. In contrast, a significant signal with antisense RNA probe was observed in bone. Sense RNA probes provided a regular background or no labeling. Undifferentiated cells located in the periosteum and endosteal spaces were labeled. EGFr mRNAs were also present in osteoblasts and in lesser amounts in some osteocytes. In rat and in human bone, both osteoblasts and osteocytes were positive on immunostaining. Similarly in the Hertwig's root sheath, EGFr immunostaining and in situ hybridization labeling were closely related. These data show that different patterns of EGFr expression in forming mineralized tissues are tissue-and stage-specific. However, in all these cells, the present in situ investigation supports the assumption that EGFr is involved in the early stages of cellular proliferation and differentiation. This report also suggests that EGFr may play a role in differentiated and mature cells of mineralized tissues.     

Models for the Study of Cementogenesis

Cementum is a mineralized tissue that acts to connect the periodontal ligament to the tooth root surface. Its composition is very much like bone, being comprised mainly of type I collagen, inorganic mineral and noncollagenous proteins, however the origin of the cells and factors necessary for cementum formation have yet to be elucidated.

Our laboratory has focused on the role that adhesion molecules, and their cell surface receptors, play in the formation of cementum and tooth root. In order to study this, we used a mouse molar as a model system. This system enabled us to study the formation of four distinct mineralized tissues; bone, cementum, dentin and enamel at various stages of their development. For these studies, we initiated experiments to examine potential cementoblast progenitor cells, in vitro. As a first step, we show that dental papilla and dental follicle cells, n vitro, obtained from molar tissues at day 21 of development, induce mineralized nodules, in vitro

In addition, we obtained tissues from mice where defects in root development may exist and determined bone sialoprotein (BSP) protein expression, a mineralized tissue specific adhesion molecule, in such tissues. As discussed here, we found that osteopetrotic (op/op) mice have delayed and/or defective root development and BSP does not localize in the dental tissues, at day 33 of development. In addition, dentin formation was defective and odontoblasts appeared immature, based on morphological examination. In contrast, the day 33 control molars demonstrated positive staining for BSP localized to root cementum, with normal formation of dentin.     

Changes in retinoblastoma gene expression during cervical cancer progression

The role of tumour suppressor genes in the development of human cancers has been studied extensively. In viral carcinogenesis, the inactivation of suppressor proteins such as retinoblastoma (pRb) and p53, and cellular oncogenes overexpression, such as c-myc, has been the subject of a number of investigations. In uterine-cervix carcinomas, where high-risk human papillomavirus (HPV) plays an important role, pRb and p53 are inactivated by E7 and E6 viral oncoproteins, respectively. However, little is known about the in situ expression of some of these proteins in pre-malignant and malignant cervical tissues. On the other hand, it has also been demonstrated that c-myc is involved in cervical carcinogenesis, and that pRb participates in the control of c-myc gene expression. By using immunostaining techniques, we investigated pRb immunodetection pattern in normal tissues, squamous intraepithelial lesions (SILs) and invasive carcinomas from the uterine cervix. Our data show low pRb detection in both normal cervical tissue and invasive lesions, but a higher expression in SILs. C-Myc protein was observed in most of the cellular nuclei of the invasive lesions, while in SILs was low. These findings indicate a heterogeneous pRb immunostaining during the different stages of cervical carcinogenesis, and suggest that this staining pattern could be a common feature implicated in the pathogenesis of uterine-cervix carcinoma.     

TGF-beta -- an excellent servant but a bad master

ABSTRACT: The transforming growth factor (TGF-beta) family of growth factors controls an immense number of cellular responses and figures prominently in development and homeostasis of most human tissues. Work over the past decades has revealed significant insight into the TGF-beta signal transduction network, such as activation of serine/threonine receptors through ligand binding, activation of SMAD proteins through phosphorylation, regulation of target genes expression in association with DNA-binding partners and regulation of SMAD activity and degradation. Disruption of the TGF-beta pathway has been implicated in many human diseases, including solid and hematopoietic tumors. As a potent inhibitor of cell proliferation, TGF-beta acts as a tumor suppressor; however in tumor cells, TGF-beta looses anti-proliferative response and become an oncogenic factor. This article reviews current understanding of TGF-beta signaling and different mechanisms that lead to its impairment in various solid tumors and hematological malignancies.     

尼古丁抑制人牙髓干细胞增殖和分化

目的探讨尼古丁的刺激对人牙髓干细胞增殖、分化的影响。方法培养人牙髓干细胞,流式细胞计量术鉴定细胞表面抗原;用不同浓度的尼古丁(10-4、10-3和10-2 mol/L)刺激牙髓干细胞,培养0、1、2、3和4 d后,CCK8法检测细胞增殖能力;茜素红染色法检测细胞分化过程中矿化结节形成,RT-qPCR和免疫印迹(Western blot)检测牙本质涎磷蛋白(DSPP)、碱性磷酸酶(ALP)、骨桥素(OPN)及细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)、p38(p-ERK、p-JNK、p-p38)等MAPK通路相关蛋白表达。结果培养第3、4天时,与对照组相比,尼古丁刺激时A值显著降低(P<0.05);与对照组相比,尼古丁刺激时矿化结节形成数、DSPP、ALP、OPN mRNA和蛋白、p-ERK、p-JNK、p-p38蛋白表达显著降低(P<0.05)。结论尼古丁抑制人牙髓干细胞增殖、成骨分化能力。     

Ing4 induces Cell Growth Inhibition in Human Lung Adenocarcinoma A549 Cells by Means of Wnt‐1/β‐Catenin Signaling Pathway

ING4, as a novel candidate tumor suppressor gene, has been implicated in several human malignances by tumor growth inhibition and apoptosis enhancement. The mechanism of ING4 remains largely unknown. The purpose of this study was to investigate the inhibitory tumor growth effects of ING4 on lung adenocarcinoma, and its mechanism, by ING4 cDNA transduction into A549 cells. Furthermore, the expression level of ING4 in lung adenocarcinoma tissues was examined. The expression of ING4 was markedly reduced in human lung adenocarcinoma tissues. Overexpression of ING4 can induce growth inhibition in A549 cells both in vitro and in vivo, and also induce up‐regulation of p27, down‐regulation of cyclinD1, SKP2, and Cox2, and inactivation of the Wnt‐1/β‐catenin pathway. Moreover, overexpression of ING4 can enhance the sensitivity of A549 cells to radiotherapy and chemotherapy. Thus, ING4 may play an inhibitory role on A549 cell proliferation and tumor growth in lung adenocarcinoma by up‐regulation or down‐regulation of cell proliferation‐regulating proteins such as p27, cyclinD1, SKP2, and Cox2 by means of inactivation of Wnt‐1/β‐catenin signaling. Anat Rec, 291:593–600, 2008. © 2008 Wiley‐Liss, Inc.     

The miRNA23b-regulated signaling network as a key to cancer development—implications for translational research and therapeutics

A growing body of evidence indicates that microRNA23b (miR23b) is pleiotropic—it plays important roles in regulating physiological functions of cells, in regulating differentiation of cells and in regulating cellular immune responses. Our review of the literature showed that dysregulation of miR23b expression is implicated in the disruption of these cellular mechanisms and development of diseases such as cancer. MiR23b dysregulation appears to do this by modulating the expression level of candidate gene products involved in a network of signaling pathways including TGF-beta and Notch pathways that govern malignant properties of cancer cells such as motility and invasiveness. More recently, miR23b regulation of gene expression has also been associated with cancer stem cells and chemoresistance. Our review covers miR23b’s role in immunity, endothelial function, differentiation, and cancer as well as its potential for translation into future cancer diagnostics and therapeutics.     

Thiamine pyrophosphatase activity of the prenatal mouse molar. A histochemical investigation

Thiamine pyrophosphatase (TPPase), an enzyme associated with the Golgi apparatus, has been implicated in the regulation of cellular oxidation as well as of transport across cell membranes. This enzyme has been localized in odontogenic tissues of the postnatal mouse and it was the intent of the present study to localize TPPase during prenatal odontogenesis. Mouse fetuses (CDI, Charles River) 14 through 19 days postconception were decapitated, the heads were frozen and mounted on the chuck of a cryostat. Frontal sections, 14 micrometers thick, were air-dried and incubated for TPPase activity. Subsequent to incubation the activity was visualized by immersion in 1% ammonium sulfide. The degree of enzyme activity varied not only with the chronological age of the fetus but also as a function of the tissue's metabolic state. Regions, such as the dental lamina, evidenced decreased TPPase activity with increasing age, while tissue layers such as the IEE displayed greater enzyme activity with increasing age.     

SWI/SNF chromatin remodelling complex and retroviral gene silencing

Because of the unique infectious cycle of retroviruses which involves the integration of the retroviral genome into the host chromosome, many cellular chromosomal proteins are used by the virus to maintain its gene expression. At the same time, cellular mechanisms for the surveillance and exclusion of non-self expression by such intragenomic parasites operate as an important host defence system in the cellular nuclei. Retroviruses have strategies for escaping from host defence systems, such as by maintaining or reactivating viral expression in specific host cell types. Understanding such epigenetical regulation would be essential for progress in retroviral virology. In this review, we emphasise the importance of the chromatin remodelling factor SWI/SNF complex as one of the key players in epigenetic regulation of host and viral gene expression. An understanding of these mechanisms will surely lead to new ideas on the pathogenicity of this virus, on the latent infection observed in many other viruses, and further forward the design of unique retroviral vectors for long-term transgene expression, providing strong tools for human gene therapy and regenerative medicine.     

Gene duplication and the evolution of vertebrate skeletal mineralization

The mineralized skeleton is a critical innovation that evolved early in vertebrate history. The tissues found in dermal skeletons of ancient vertebrates are similar to the dental tissues of modern vertebrates; both consist of a highly mineralized surface hard tissue, enamel or enameloid, more resilient body dentin, and basal bone. Many proteins regulating mineralization of these tissues are evolutionarily related and form the secretory calcium-binding phosphoprotein (SCPP) family. We hypothesize here the duplication histories of SCPP genes and their common ancestors, SPARC and SPARCL1. At around the same time that Paleozoic jawless vertebrates first evolved mineralized skeleton, SPARCL1 arose from SPARC by whole genome duplication. Then both before and after the split of ray-finned fish and lobe-finned fish, tandem gene duplication created two types of SCPP genes, each residing on the opposite side of SPARCL1. One type was subsequently used in surface tissue and the other in body tissue. In tetrapods, these two types of SCPP genes were separated by intrachromosomal rearrangement. While new SCPP genes arose by duplication, some old genes were eliminated from the genome. As a consequence, phenogenetic drift occurred: while mineralized skeleton is maintained by natural selection, the underlying genetic basis has changed.     

Disruption of the midkine gene (Mdk) resulted in altered expression of a calcium binding protein in the hippocampus of infant mice and their abnormal behaviour

BACKGROUND: Midkine (MK) is a growth factor implicated in the development and repair of various tissues, especially neural tissues. However, its in vivo function has not been clarified. RESULTS: Knockout mice lacking the MK gene (Mdk) showed no gross abnormalities. We closely analysed postnatal brain development in Mdk(-/-) mice using calcium binding proteins as markers to distinguish neuronal subpopulations. Intense and prolonged calretinin expression was found in the dentate gyrus granule cell layer of the hippocampus of infant Mdk(-/-) mice. In infant Mdk(+/+) mice, calretinin expression in the granule cell layer was weaker, and had disappeared by 4 weeks after birth, when calretinin expression still persisted in Mdk(-/-) mice. Furthermore, 4 weeks after birth, Mdk(-/-) mice showed a deficit in their working memory, as revealed by a Y-maze test, and had an increased anxiety, as demonstrated by the elevated plus-maze test. CONCLUSION: Midkine plays an important role in the regulation of postnatal development of the hippocampus.     

The amelogenin "enamel proteins" and cells in the periodontium

This overview will examine the multifunctional nature of a group of proteins known as the amelogenins. These secreted proteins were named in the 1960s because of their expected role during development of dental enamel [Eastoe JE. Adv Fluorine Res. 1965;21:5-17]. As gene expression assays became more sensitive, expression was also noted in tissues not involved with enamel formation leading to hypotheses concerning additional roles for these proteins. In vitro approaches led to the discovery that some of the amelogenins are able to regulate gene expression and to participate in cellular signaling. An extract containing predominately amelogenins has been used clinically in treatment of certain forms of pcriodontal disease with regenerative results noted originally in animal models, but later in human patients as well. Much literature has been devoted to the roles of amelogenins during mineral formation, and therefore this topic will be covered primarily in the Introduction. The goal of this review will be to focus on strategies that have been used to uncover roles of amelogenins related to gene expression and development apart from the roles in enamel mineral, and the possible functions that these proteins could have if delivered to normally nonexpressing tissues for therapeutic approaches.