CCN3 and bone marrow cells

CCN3 expression was observed in a broad variety of tissues from the early stage of development. However, a kind of loss of function in mice (CCN3 del VWC domain -/-) demonstrated mild abnormality, which indicates that CCN3 may not be critical for the normal embryogenesis as a single gene. The importance of CCN3 in bone marrow environment becomes to be recognized by the studies of hematopoietic stem cells and Chronic Myeloid Leukemia cells. CCN3 expression in bone marrow has been denied by several investigations, but we found CCN3 positive stromal and hematopoietic cells at bone extremities with a new antibody although they are a very few populations. We investigated the expression pattern of CCN3 in the cultured bone marrow derived mesenchymal stem cells and found its preference for osteogenic differentiation. From the analyses of in vitro experiment using an osteogenic mesenchymal stem cell line, Kusa-A1, we found that CCN3 downregulates osteogenesis by two different pathways; suppression of BMP and stimulation of Notch. Secreted CCN3 from Kusa cells inhibited the differentiation of osteoblasts in separate culture, which indicates the paracrine manner of CCN3 activity. CCN3 may also affect the extracellular environment of the niche for hematopoietic stem cells

MAML1 Enhances the Transcriptional Activity of Runx2 and Plays a Role in Bone Development

Mastermind-like 1 (MAML1) is a transcriptional co-activator in the Notch signaling pathway. Recently, however, several reports revealed novel and unique roles for MAML1 that are independent of the Notch signaling pathway. We found that MAML1 enhances the transcriptional activity of runt-related transcription factor 2 (Runx2), a transcription factor essential for osteoblastic differentiation and chondrocyte proliferation and maturation. MAML1 significantly enhanced the Runx2-mediated transcription of the p6OSE2-Luc reporter, in which luciferase expression was controlled by six copies of the osteoblast specific element 2 (OSE2) from the Runx2-regulated osteocalcin gene promoter. Interestingly, a deletion mutant of MAML1 lacking the N-terminal Notch-binding domain also enhanced Runx2-mediated transcription. Moreover, inhibition of Notch signaling did not affect the action of MAML1 on Runx2, suggesting that the activation of Runx2 by MAML1 may be caused in a Notch-independent manner. Overexpression of MAML1 transiently enhanced the Runx2-mediated expression of alkaline phosphatase, an early marker of osteoblast differentiation, in the murine pluripotent mesenchymal cell line C3H10T1/2. MAML1-/- embryos at embryonic day 16.5 (E16.5) had shorter bone lengths than wild-type embryos. The area of primary spongiosa of the femoral diaphysis was narrowed. At E14.5, extended zone of collagen type II alpha 1 (Col2a1) and Sox9 expression, markers of chondrocyte differentiation, and decreased zone of collagen type X alpha 1 (Col10a1) expression, a marker of hypertrophic chondrocyte, were observed. These observations suggest that chondrocyte maturation was impaired in MAML1-/- mice. MAML1 enhances the transcriptional activity of Runx2 and plays a role in bone development

Integrated Genotypic Analysis of Hedgehog-Related Genes Identifies Subgroups of Keratocystic Odontogenic Tumor with Distinct Clinicopathological Features

<div><p>Keratocystic odontogenic tumor (KCOT) arises as part of Gorlin syndrome (GS) or as a sporadic lesion. Gene mutations and loss of heterozygosity (LOH) of the hedgehog receptor PTCH1 plays an essential role in the pathogenesis of KCOT. However, some KCOT cases lack evidence for gene alteration of <i>PTCH1</i>, suggesting that other genes in the hedgehog pathway may be affected. PTCH2 and SUFU participate in the occurrence of GS-associated tumors, but their roles in KCOT development are unknown. To elucidate the roles of these genes, we enrolled 36 KCOT patients in a study to sequence their entire coding regions of <i>PTCH1</i>, <i>PTCH2</i> and <i>SUFU</i>. LOH and immunohistochemical expression of these genes, as well as the downstream targets of hedgehog signaling, were examined using surgically-excised KCOT tissues. <i>PTCH1</i> mutations, including four novel ones, were found in 9 hereditary KCOT patients, but not in sporadic KCOT patients. A pathogenic mutation of <i>PTCH2</i> or <i>SUFU</i> was not found in any patients. LOH at <i>PTCH1</i> and <i>SUFU</i> loci correlated with the presence of epithelial budding. KCOT harboring a germline mutation (Type 1) showed nuclear localization of GLI2 and frequent histological findings such as budding and epithelial islands, as well as the highest recurrence rate. KCOT with LOH but without a germline mutation (Type 2) less frequently showed these histological features, and the recurrence rate was lower. KCOT with neither germline mutation nor LOH (Type 3) consisted of two subgroups, Type 3A and 3B, which were characterized by nuclear and cytoplasmic GLI2 localization, respectively. Type 3B rarely exhibited budding and recurrence, behaving as the most amicable entity. The expression patterns of CCND1 and BCL2 tended to correlate with these subgroups. Our data indicates a significant role of <i>PTCH1</i> and <i>SUFU</i> in the pathogenesis of KCOT, and the genotype-oriented subgroups constitute entities with different potential aggressiveness.</p></div

Correlation between the presence of each LOH and the histological parameters.

<p>LOHs at the <i>PTCH1</i> and <i>SUFU</i> loci were significantly associated with the presence of epithelial budding, while <i>PTCH2</i> LOH was not.</p><p>No correlation was observed between the other histological features and LOH at any loci.</p

Schematic summary of this study.

<p>KCOT can be divided into three groups by genotype. Type 1 is KCOT with a germline mutation. Type 2 is KCOT with LOH but without a germline mutation. Type 3 is KCOT with neither a germline mutation nor LOH. Type 3 consists of two subtypes: Type 3A, with nuclear localization of GLI2; and Type 3B, with cytoplasmic localization of GLI2. Epithelial budding and recurrence are noted most frequently in Type 1 and least frequently in Type 3B.</p

Immunohistochemical expression of CCND1 and BCL2 in KCOT.

<p>A) CCND1. Each case was classified either as the basal and suprabasal (BS) pattern or the suprabasal-dominant (S) pattern. The basal-dominant (B) pattern was not observed. The dotted line indicates a basement membrane. Scale bars = 30 µm. B) BCL2. Each case was classified either as the basal and suprabasal (BS) pattern or the basal-dominant (B) pattern. The suprabasal-dominant (S) pattern was not observed. Scale bars = 30 µm. C) Schematic table of the CCND1 and BCL2 expression patterns in each subgroup. Number of cases with the designated expression patterns are depicted (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070995#pone-0070995-t001" target="_blank">Table 1</a>). The cases in which the examiners’ evaluation split were excluded.</p

Immunohistochemical expression of GLI2 in KCOT.

<p>A) In hereditary KCOT and sporadic KCOT with LOH, GLI2 was detected in the nuclei, whereas it was detected mainly in the cytoplasm in about a half of sporadic KCOT without LOH. Scale bars = 30 µm B) Schematic table of the GLI2 expression patterns in each subgroup. Number of cases with the designated expression patterns are depicted (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070995#pone-0070995-t001" target="_blank">Table 1</a>).</p

Periostin associates with Notch1 precursor to maintain Notch1 expression under a stress condition in mouse cells.

BACKGROUND: Matricellular proteins, including periostin, modulate cell-matrix interactions and cell functions by acting outside of cells. METHODS AND FINDINGS: In this study, however, we reported that periostin physically associates with the Notch1 precursor at its EGF repeats in the inside of cells. Moreover, by using the periodontal ligament of molar from periostin-deficient adult mice (Pn-/- molar PDL), which is a constitutively mechanically stressed tissue, we found that periostin maintained the site-1 cleaved 120-kDa transmembrane domain of Notch1 (N1) level without regulating Notch1 mRNA expression. N1 maintenance in vitro was also observed under such a stress condition as heat and H(2)O(2) treatment in periostin overexpressed cells. Furthermore, we found that the expression of a downstream effector of Notch signaling, Bcl-xL was decreased in the Pn-/- molar PDL, and in the molar movement, cell death was enhanced in the pressure side of Pn-/- molar PDL. CONCLUSION: These results suggest the possibility that periostin inhibits cell death through up-regulation of Bcl-xL expression by maintaining the Notch1 protein level under the stress condition, which is caused by its physical association with the Notch1 precursor

Keratin 17 Is Induced in Oral Cancer and Facilitates Tumor Growth

<div><p>Keratin subtypes are selectively expressed depending on the cell type. They not only provide structural support, but regulate the metabolic processes and signaling pathways that control the growth of the epithelium. KRT17 (keratin 17) is induced in the regenerative epithelium and acts on diverse signaling pathways. Here, we demonstrate that KRT17 is invariably and permanently induced in oral squamous cell carcinoma (OSCC), as revealed by immunohistochemistry and cDNA microarray analysis. Two representative OSCC cell lines; KRT17-weakly expressing Ca9-22 and KRT17-highly expressing HSC3 were used to establish KRT17-overexpressing Ca9-22 and KRT17-knockdown HSC3 cells. Analysis of these cells revealed that KRT17 promoted cell proliferation and migration by stimulating the Akt/mTOR pathway. KRT17 also upregulated the expression of SLC2A1 (solute carrier family 2 member 1/Glut1) and glucose uptake. To further investigate the effect of KRT17 on tumorigenesis, KRT17-knockout HSC3 cells were established and were transplanted to the cephalic skin of nude mice. The tumors that developed from KRT17-knockout HSC3 cells had a lower Ki-67 labeling index and were significantly smaller compared to the controls. These results indicate that KRT17 stimulates the Akt/mTOR pathway and glucose uptake, thereby facilitating tumor growth. We could not confirm the relationship between KRT17 and SFN (stratifin) in the cells examined in this study. However, our study reinforces the concept that the cellular properties of cancer are regulated by a series of molecules similar to those found in wound healing. In OSCC, KRT17 acts as a pathogenic keratin that facilitates tumor growth through the stimulation of multiple signaling pathways, highlighting the importance of KRT17 as a multifunctional promoter of tumorigenesis.</p></div