Membrane Mucin Muc4 promotes blood cell association with tumor cells and mediates efficient metastasis in a mouse model of breast cancer.

Mucin-4 (Muc4) is a large cell surface glycoprotein implicated in the protection and lubrication of epithelial structures. Previous studies suggest that aberrantly expressed Muc4 can influence the adhesiveness, proliferation, viability and invasiveness of cultured tumor cells, as well as the growth rate and metastatic efficiency of xenografted tumors. Although it has been suggested that one of the major mechanisms by which Muc4 potentiates tumor progression is via its engagement of the ErbB2/HER2 receptor tyrosine kinase, other mechanisms exist and remain to be delineated. Moreover, the requirement for endogenous Muc4 for tumor growth progression has not been previously explored in the context of gene ablation. To assess the contribution of endogenous Muc4 to mammary tumor growth properties, we first created a genetically engineered mouse line lacking functional Muc4 (Muc4ko), and then crossed these animals with the NDL (Neu DeLetion mutant) model of ErbB2-induced mammary tumorigenesis. We observed that Muc4ko animals are fertile and develop normally, and adult mice exhibit no overt tissue abnormalities. In tumor studies, we observed that although some markers of tumor growth such as vascularity and cyclin D1 expression are suppressed, primary mammary tumors from Muc4ko/NDL female mice exhibit similar latencies and growth rates as Muc4wt/NDL animals. However, the presence of lung metastases is markedly suppressed in Muc4ko/NDL mice. Interestingly, histological analysis of lung lesions from Muc4ko/NDL mice revealed a reduced association of disseminated cells with platelets and white blood cells. Moreover, isolated cells derived from Muc4ko/NDL tumors interact with fewer blood cells when injected directly into the vasculature or diluted into blood from wild type mice. We further observed that blood cells more efficiently promote the viability of non-adherent Muc4wt/NDL cells than Muc4ko/NDL cells. Together, our observations suggest that Muc4 may facilitate metastasis by promoting the association of circulating tumor cells with blood cells to augment tumor cell survival in circulation.

Suppression of planar cell polarity signaling and migration in glioblastoma by Nrdp1-mediated Dvl polyubiquitination.

The lethality of the aggressive brain tumor glioblastoma multiforme (GBM) results in part from its strong propensity to invade surrounding normal brain tissue. Although oncogenic drivers such as epidermal growth factor receptor activation and Phosphatase and Tensin homolog inactivation are thought to promote the motility and invasiveness of GBM cells via phosphatidylinostitol 3-kinase activation, other unexplored mechanisms may also contribute to malignancy. Here we demonstrate that several components of the planar cell polarity (PCP) arm of non-canonical Wnt signaling including VANGL1, VANGL2 and FZD7 are transcriptionally upregulated in glioma and correlate with poorer patient outcome. Knockdown of the core PCP pathway component VANGL1 suppresses the motility of GBM cell lines, pointing to an important mechanistic role for this pathway in glioblastoma malignancy. We further observe that restoration of Nrdp1, a RING finger type E3 ubiquitin ligase whose suppression in GBM also correlates with poor prognosis, reduces GBM cell migration and invasiveness by suppressing PCP signaling. Our observations indicate that Nrdp1 physically interacts with the Vangl1 and Vangl2 proteins to mediate the K63-linked polyubiquitination of the Dishevelled, Egl-10 and Pleckstrin (DEP) domain of the Wnt pathway protein Dishevelled (Dvl). Ubiquitination hinders Dvl binding to phosphatidic acid, an interaction necessary for efficient Dvl recruitment to the plasma membrane upon Wnt stimulation of Fzd receptor and for the propagation of downstream signals. We conclude that the PCP pathway contributes significantly to the motility and hence the invasiveness of GBM cells, and that Nrdp1 acts as a negative regulator of PCP signaling by inhibiting Dvl through a novel polyubiquitination mechanism. We propose that the upregulation of core PCP components, together with the loss of the key negative regulator Nrdp1, act coordinately to promote GBM invasiveness and malignancy.

LRIG1 opposes epithelial-to-mesenchymal transition and inhibits invasion of basal-like breast cancer cells.

LRIG1 (leucine-rich repeat and immunoglobulin-like domain containing), a member of the LRIG family of transmembrane leucine-rich repeat-containing proteins, is a negative regulator of receptor tyrosine kinase signaling and a tumor suppressor. LRIG1 expression is broadly decreased in human cancer and in breast cancer and low expression of LRIG1 has been linked to decreased relapse-free survival. Recently, low expression of LRIG1 was revealed to be an independent risk factor for breast cancer metastasis and death. These findings suggest that LRIG1 may oppose breast cancer cell motility and invasion, cellular processes that are fundamental to metastasis. However, very little is known of LRIG1 function in this regard. In this study, we demonstrate that LRIG1 is downregulated during epithelial-to-mesenchymal transition (EMT) of human mammary epithelial cells, suggesting that LRIG1 expression may represent a barrier to EMT. Indeed, depletion of endogenous LRIG1 in human mammary epithelial cells expands the stem cell population, augments mammosphere formation and accelerates EMT. Conversely, expression of LRIG1 in highly invasive Basal B breast cancer cells provokes a mesenchymal-to-epithelial transition accompanied by a dramatic suppression of tumorsphere formation and a striking loss of invasive growth in three-dimensional culture. LRIG1 expression perturbs multiple signaling pathways and represses markers and effectors of the mesenchymal state. Furthermore, LRIG1 expression in MDA-MB-231 breast cancer cells significantly slows their growth as tumors, providing the first in vivo evidence that LRIG1 functions as a growth suppressor in breast cancer.

Amelogenin splice isoforms stimulate chondrogenic differentiation of ATDC5 cells.

OBJECTIVE: Amelogenins are the most abundant matrix proteins in enamel. Among the amelogenin isoforms, full-length amelogenin (M180) and leucine-rich amelogenin peptide (LRAP) are expressed in various tissues and are implicated as signalling molecules in mesenchymal cells. Here, we examined the effects of M180 and LRAP on a chondrogenic cell line, ATDC5, to investigate the role of amelogenins in chondrogenesis. MATERIALS AND METHODS: Recombinant mouse M180- or LRAP-protein-containing medium or control medium was mixed with a chondrogenesis-stimulating medium, and changes in the phenotype, gene expression levels and cell proliferation of cultured ATDC5 cells were analysed. RESULTS: The addition of amelogenins increased alkaline phosphatase activity and glycosaminoglycan secretion at 14 and 21 days of culture, respectively, as compared with the control. Quantitative PCR (Q-PCR) analysis revealed that LRAP increased the gene expression levels of Runx2, Col2a1 and Aggrecan at 7 days of differentiation. Moreover, both M180 and LRAP significantly increased the gene expression levels of ALP, Aggrecan, Col10a1 and osteopontin at 28 days of culture. Bromodeoxyuridine assay and Q-PCR analysis for Wnt signalling indicated that both M180 and LRAP reduced proliferation, but induced the cell differentiation possibly through altered non-canonical Wnt signalling. CONCLUSION: M180 and LRAP accelerate chondrogenic differentiation and maturation of ATDC5 cells.

Synergistic roles of amelogenin and ameloblastin.

Amelogenin and ameloblastin, the major enamel matrix proteins, are important for enamel mineralization. To identify their synergistic roles in enamel development, we generated Amel X(-/-)/Ambn(-/-) mice. These mice showed additional enamel defects in comparison with Amel X(-/-) or Ambn(-/-) mice. In 7-day-old Amel X(-/-)/Ambn(-/-) mice, not only was the ameloblast layer irregular and detached from the enamel surface, as in Ambn(-/-), but also, the enamel width was significantly reduced in the double-null mice as compared with Amel X(-/-) or Ambn(-/-) mice. Proteomic analysis of the double-null teeth revealed increased levels of RhoGDI (Arhgdia), a Rho-family-specific guanine nucleotide dissociation inhibitor, which is involved in important cellular processes, such as cell attachment. Both Amel X(-/-)/Ambn(-/-) mice and Ambn(-/-) mice displayed positive staining with RhoGDI antibody in the irregularly shaped ameloblasts detached from the matrix. Ameloblastin-regulated expression of RhoGDI suggests that Rho-mediated signaling pathway might play a role in enamel formation.

Micro-CT analysis of alveolar bone healing using a rat experimental model of critical-size defects.

OBJECTIVE: This study was designed to establish a rat model of a critical size alveolar bone defect. MATERIALS AND METHODS: Standardized buccal or mesiobuccal alveolar bone defects were made around the right first mandibular molar of 12-week-old rats, and the left was used as a control. Alveolar bone healing was examined quantitatively by three-dimensional micro-computed tomographic imaging. Bone matrix production of osteoblasts and osteocytes during repair of alveolar bone defects was examined with in situ hybridization for type I collagen. RESULTS: Buccal defects were repaired significantly and the volume decreased by 88.3% in week 24, whereas mesiobuccal defects were repaired little. Osteoblasts and osteocytes expressed type I collagen in both defects in week 3 but showed little expression by week 6 and thereafter, leaving the mesiobuccal defects largely unrepaired. CONCLUSION: The mesiobuccal defect is a critical-size defect that is not ultimately repaired with bone. It may be an appropriate experimental model for investigating the effectiveness of bone regenerative agents in human alveolar bone loss.

Mutual induction of noncollagenous bone proteins at the interface between epithelial cells and fibroblasts from human periodontal ligament.

BACKGROUND AND OBJECTIVE: Epithelial-mesenchymal interactions are responsible for cell differentiation during periodontal regeneration. The present study was undertaken to examine the expression of alkaline phosphatase and noncollagenous bone proteins, such as osteopontin, osteocalcin and bone sialoprotein, with respect to interaction between the cells of the epithelial rests of Malassez and fibroblasts from human periodontal ligament. MATERIAL AND METHODS: Explants of human periodontal ligament tissues produced outgrowths containing both putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts in a modified serum-free medium. Putative epithelial rests of Malassez cells cultured alone, and human periodontal ligament fibroblasts cultured alone, were used as controls. The expression levels of amelogenin were analyzed by in situ hybridization. The expression and distribution of alkaline phosphatase and noncollagenous bone proteins in both cell populations at the interface between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts were analyzed by immunohistochemistry, in situ hybridization and reverse transcription-polymerase chain reaction. RESULTS: Amelogenin mRNA was detected at high levels only in putative epithelial rests of Malassez cells at the interface. Alkaline phosphatase and bone sialoprotein mRNAs were detected significantly at the interface between putative epithelial rests of Malassez cells and human periodontal ligament fibroblast cells. In particular, bone sialoprotein and its mRNA were expressed significantly in human periodontal ligament fibroblasts at the interface between putative epithelial rests of Malassez cells and human periodontal ligament fibroblast cells. The expressions of osteopontin and its mRNA were not different between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts at the interface. Osteocalcin and its mRNA were expressed strongly in putative epithelial rests of Malassez cells at the interface between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts. CONCLUSION: These findings indicate that the epithelial-mesenchymal interaction modulates the expression of alkaline phosphatase, osteocalcin and bone sialoprotein in putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts, suggesting that epithelial-mesenchymal interactions play a role in the maintenance of periodontal ligament.

In vitro differentiation of epithelial cells cultured from human periodontal ligament.

BACKGROUND AND OBJECTIVE: Alkaline phosphatase and noncollagenous bone proteins are produced prior to cementum formation. While it has been suggested that epithelial rests of Malassez are involved in cementum formation, little is known about the relationship between epithelial rests of Malassez and cementum formation. The purpose of the present study was to determine whether the epithelial rests of Malassez cells cultured from human periodontal ligament can produce alkaline phosphatase and noncollagenous bone proteins, such as osteopontin, osteocalcin and bone sialoprotein. MATERIAL AND METHODS: An outgrowth of putative epithelial rests of Malassez cells was produced from periodontal ligament explant, and second passage cultures were used in the experiments. Human gingival epithelial cells and periodontal ligament fibroblasts were used as controls. The expression levels of amelogenin were analyzed by immunostaining and in situ hybridization. Furthermore, the expression levels of alkaline phosphatase and noncollagenous bone proteins were assessed by immunostaining and reverse transcription-polymerase chain reaction. RESULTS: Amelogenin, alkaline phosphatase and osteopontin proteins and their corresponding mRNAs were detected at high levels in putative epithelial rests of Malassez cells. Osteocalcin and bone sialoprotein were not expressed in putative epithelial rests of Malassez cells. Alkaline phosphatase and noncollagenous bone proteins were seen in periodontal ligament fibroblasts, but not in gingival epithelial cells. CONCLUSION: Our results suggest that putative epithelial rests of Malassez cells cultured alone do not transform into maturing cells to form the cementum, but may play a potential role in the mineralization process.

Proliferation and adhesion of periodontal ligament cells on synthetic biominerals.

OBJECTIVE: Hydroxiapatite (HA) has been suggested as a useful biomaterial to support the regeneration of tissues. In this study, we investigated the adhesion of periodontal ligament (PDL) cells on octacalcium phosphate (OCP) and its hydrolyzed apatitic product (HL), which are known precursors of HA. METHODS: Rat PDL cells were cultured on OCP or HL-coated dishes. Cell proliferation and adhesion and mRNA expression of collagen I, fibronectin integrin subunits were examined. Cell adhesion inhibition assays were carried out by GRGDSPK (Gly-Arg-Gly-Asp-Ser-Pro-Lys). RESULTS: In early culture period, the cell number of PDL cells was lower on OCP and HL than that on control without any coating. However, the cell number on OCP or HL caught up with control later period. mRNA expression level of collagen I and fibronectin on OCP and HL were similar among OCP HL and control, although they differed early in the culture period. Integrin subunits were expressed on both OCP and HL as well as on control. Cell adhesion was inhibited by RGD inhibitor peptide. CONCLUSION: Our findings indicated that rat PDL cells produce collagen I and fibronectin on OCP and HL, and then show increased cell numbers depending on adhesion to the matrices through integrins.

Amelogenin-mediated regulation of osteoclastogenesis, and periodontal cell proliferation and migration.

We previously reported that amelogenin isoforms M180 and leucine-rich amelogenin peptide (LRAP) are expressed in the periodontal region, and that their absence is associated with increased cementum defects in amelogenin-knockout (KO) mice. The aim of the present study was to characterize the functions of these isoforms in osteoclastogenesis and in the proliferation and migration of cementoblast/periodontal ligament cells. The co-cultures of wild-type (WT) osteoclast progenitor and KO cementoblast/periodontal ligament cells displayed more tartrate-resistant acid phosphatase (TRAP)-positive cells than the co-cultures of WT cells. The addition of LRAP to both co-cultures significantly reduced RANKL expression and the TRAP-positive cells. Proliferation and migration rates of the KO cementoblast/periodontal ligament cells were lower than those of WT cells and increased with the addition of either LRAP or P172 (a porcine homolog of mouse M180). Thus, we demonstrate the regulation of osteoclastogenesis by LRAP, and the proliferation and migration of cementoblast/periodontal ligament cells by LRAP and P172.

Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell-surface components through the CD14/Toll-like receptor system.

We compared human periodontal ligament fibroblasts with human gingival fibroblasts isolated from the same donor to examine interleukin-8 (IL-8) responses of the cells to Salmonella lipopolysaccharide, a water-soluble peptidoglycan from Staphylococcus epidermidis and the synthetic muramyldipeptide, with special reference to the possible involvement of the CD14/Toll-like receptor (TLR) system of the cells in the responses. Human gingival fibroblasts expressed CD14 on their surfaces and strongly expressed CD14 mRNA, while human periodontal ligament fibroblasts showed considerably lower levels of expression in both respects. Both cells expressed mRNA of TLR-related molecules, i.e. TLR2, TLR4, MD-2 and MyD88, although human periodontal ligament fibroblasts expressed TLR2 more strongly than human gingival fibroblasts. Human gingival fibroblasts exhibited a stronger IL-8 response than human periodontal ligament fibroblasts to lipopolysaccharide, while human periodontal ligament fibroblasts exhibited a response comparable to, or slightly stronger than, that of human gingival fibroblasts to S. epidermidis peptidoglycan and muramyldipeptide. The IL-8 responses of both cells to lipopolysaccharide and S. epidermidis peptidoglycan were completely inhibited by antihuman CD14 monoclonal antibody (MAb). The responses of both cells to lipopolysaccaride were significantly inhibited by antihuman TLR4 MAb, while those to S. epidermidis peptidoglycan were inhibited by antihuman TLR2 MAb. In contrast, muramyldipeptide activated both types of cells in a TLR2- and TLR4-independent manner, although the activities of muramyldipeptide on human gingival fibroblasts, but not human periodontal ligament fibroblasts, were significantly inhibited by anti-CD14 MAb.

Monocytic cell activation by Nonendotoxic glycoprotein from Prevotella intermedia ATCC 25611 is mediated by toll-like receptor 2.

Lipopolysaccharide (LPS) preparations from gram-negative black-pigmented bacteria such as Porphyromonas gingivalis and Prevotella intermedia activate cells from non-LPS-responsive C3H/HeJ mice, but it is still unclear whether this activity is due to the unique structure of LPS or to a minor component(s) responsible for the activity in the preparation. A nonendotoxic glycoprotein with bioactivity against cells from C3H/HeJ mice was purified from a hot phenol-water extract of P. intermedia ATCC 25611 and designated Prevotella glycoprotein (PGP). Treatment of human monocytic THP-1 cells with 22-oxyacalcitriol (OCT) induced maturation and marked expression of CD14 on the cells, but the cells constitutively expressed Toll-like receptor 2 (TLR2) and TLR4 on the cells irrespective of the treatment. PGP induced a high level of interleukin-8 production at doses of 100 ng/ml and higher in OCT-treated THP-1 cells compared with Salmonella LPS, and the production was significantly inhibited by anti-CD14 and anti-TLR2 but not anti-TLR4 antibodies. Consistent with this, TLR2-deficient murine macrophages did not respond to PGP. It was also shown that PGP activity on the THP-1 cells was LPS-binding protein dependent and was inhibited by a synthetic lipid A precursor IV(A). These results indicate that PGP activates monocytic cells in a CD14- and TLR2-dependent manner.

Roles of homeobox and bHLH genes in specification of a retinal cell type.

Previous analysis of mutant mice has revealed that the bHLH genes Mash1 and Math3, and the homeobox gene Chx10 are essential for generation of bipolar cells, the interneurons present in the inner nuclear layer of the retina. Thus, a combination of the bHLH and homeobox genes should be important for bipolar cell genesis, but the exact functions of each gene remain largely unknown. We have found that in Mash1-Math3 double-mutant retina, which exhibits a complete loss of bipolar cells, Chx10 expression did not disappear but remained in Müller glial cells, suggesting that Chx10 expression per se is compatible with gliogenesis. In agreement with this, misexpression of Chx10 alone with retrovirus in the retinal explant cultures induced generation of the inner nuclear layer cells, including Müller glia, but few of them were mature bipolar cells. Misexpression of Mash1 or Math3 alone did not promote bipolar cell genesis either, but inhibited Müller gliogenesis. In contrast, misexpression of Mash1 or Math3 together with Chx10 increased the population of mature bipolar cells and decreased that of Müller glia. Thus, the homeobox gene provides the inner nuclear layer-specific identity while the bHLH genes regulate the neuronal versus glial fate determination, and these two classes of genes together specify the bipolar cell fate. Moreover, Mash1 and Math3 promoted the bipolar cell fate, but not the other inner nuclear layer-specific neuronal subtypes in the presence of Chx10, raising the possibility that the bHLH genes may be involved in neuronal subtype specification, in addition to simply making the neuronal versus glial fate choice.

Local muscimol disinhibits mesolimbic dopaminergic activity as examined by brain microdialysis and Fos immunohistochemistry.

Infusion of muscimol (5 X 10[-5] M, 60 min) into the nucleus accumbens (NAC) through a dialysis membrane caused a significant increase in extracellular dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC). Fos-like immunoreactivity induced by intra-NAC infusion of muscimol was seen ipsilaterally in many accumbofugal target areas, but no Fos-positive neurons were seen in the vicinity of the dialysis membrane in the NAC. Sequential staining of Fos and tyrosine hydroxylase (TH) immunoreactivities revealed that a portion of A10 dopaminergic neurons were double-labelled. These results suggest that muscimol in the NAC disinhibits mesolimbic DA neuronal activity possibly through activity of the accumbofugal GABA neuron system.

[A case of primary pulmonary cryptococcosis].

A 53-year-old male was admitted because of an abnormal chest roentgenogram showing a solitary tumor shadow in the right upper lung field. Laboratory data was normal. Although examination with the bronchoscope and sputum cytology showed no abnormal findings, a partial resection of the right upper lobe was performed. Microscopic examination of the lesion was cryptococcosis. As another focus was not found by systemic examination, the lesion was disclosed primary pulmonary cryptococcosis. Cryptococcosis is rare but we must think of it when we meet a solitary tumor shadow on the chest roentgenogram.