Kumi Sumiyoshi

Identification of miR-1 as a micro RNA that supports late-stage differentiation of growth cartilage cells.

The process of endochondral ossification is strictly regulated by a variety of extracellular and intracellular factors. Recently, it has become recognized that specific miRNAs are involved in this process by regulating the expression of the relevant genes at the post-transcriptional level. In this present study we obtained the first evidence of the involvement of a specific micro RNA (miRNA) in the regulation of the chondrocyte phenotype during late stages of differentiation. By use of the microarray technique, miR-1 was identified as this miRNA, the expression of which was most repressed upon hypertrophic differentiation. Transfection of human chondrocytic HCS-2/8 cells and chicken normal chondrocytes with miR-1 led to repressed expression of aggrecan, the major cartilaginous proteoglycan gene. Therefore, miR-1 was found to be involved in the regulation of the chondrocytic phenotype and thus to play an important role in chondrocytes during the late stage of the differentiation process, maintaining the integrity of the cartilage tissue.

Posttranscriptional Regulation of Chicken ccn2 Gene Expression by Nucleophosmin/B23 during Chondrocyte Differentiation

ABSTRACT CCN2/CTGF is a multifunctional factor that plays a crucial role in the growth and differentiation of chondrocytes. The chicken ccn2 gene is regulated not only at the transcriptional level but also by the interaction between a posttranscriptional element in the 3′ untranslated region (3′-UTR) and a cofactor. In the present study, we identified a nucleophosmin (NPM) (also called B23) as this cofactor. Binding of NPM to the element was confirmed, and subsequent analysis revealed a significant correlation between the decrease in cytosolic NPM and the increased stability of the ccn2 mRNA during chondrocyte differentiation in vivo. Furthermore, recombinant chicken NPM enhanced the degradation of chimeric RNAs containing the posttranscriptional cis elements in a chicken embryonic fibroblast extract in vitro. It is noteworthy that the RNA destabilization effect by NPM was far more prominent in the cytosolic extract of chondrocytes than in that of fibroblasts, representing a chondrocyte-specific action of NPM. Stimulation by growth factors to promote differentiation changed the subcellular distribution of NPM in chondrocytes, which followed the expected patterns from the resultant change in the ccn2 mRNA stability. Therefore, the present study reveals a novel aspect of NPM as a key player in the posttranscriptional regulation of ccn2 mRNA during the differentiation of chondrocytes.

Novel role of miR‐181a in cartilage metabolism

Micro RNA (miRNA) is a small non‐coding post‐transcriptional RNA regulator that is involved in a variety of biological events. In order to specify the role of miRNAs in cartilage metabolism, we comparatively analyzed the expression profile of known miRNAs in chicken sternum chondrocytes representing early and late differentiation stages. Interestingly, none of the miRNAs displaying strong expression levels showed remarkable changes along with differentiation, suggesting their roles in maintaining the homeostasis rather than cytodifferentiation of chondrocytes. Among these miRNAs, miR‐181a, which is known to play critical roles in a number of tissues, was selected and was further characterized. Human microarray analysis revealed remarkably stronger expression of miR‐181a in human HCS‐2/8 cells, which strongly maintained a chondrocytic phenotype, than in HeLa cells, indicating its significant role in chondrocytes. Indeed, subsequent investigation indicated that miR‐181a repressed the expression of two genes involved in cartilage development. One was CCN family member 1 (CCN1), which promotes chondrogenesis; and the other, the gene encoding the core protein of aggrecan, a major cartilaginous proteoglycan, aggrecan. Based on these findings, negative feedback system via miR‐181a to conserve the integrity of the cartilaginous phenotype may be proposed. J. Cell. Biochem. 114: 2094–2100, 2013.

Distribution, gene expression, and functional role of EphA4 during ossification

EphA4 receptor tyrosine kinase has been shown to be critically involved in neural tissue development. Here, we found EphA4 was also distributed among hypertrophic chondrocytes and osteoblasts in the growth plate of developing mouse long bones. In vitro evaluation revealed that ephA4 expression was elevated upon hypertrophic differentiation of chondrocytes and that markedly stronger expression was observed in osteoblastic SaOS-2 than chondrocytic HCS-2/8 cells. Of note, RNAi-mediated silencing of ephA4 in SaOS-2 cells resulted in the repression of osteocalcin gene expression and alkaline phosphatase activity. Interestingly, confocal laser-scanning microscopic analysis revealed the presence of EphA4 molecules in the nucleus as well as on the surface of SaOS-2 cells. These findings are the first indication of a critical role of EphA4 in ossification, especially at the final stage in which osteoblasts and hypertrophic chondrocytes play major roles.

Thrombopoietic-mesenchymal interaction that may facilitate both endochondral ossification and platelet maturation via CCN2.

CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2 in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets. To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts endochondral ossification.

A coding RNA segment that enhances the ribosomal recruitment of chicken ccn1 mRNA.

CCN1, a member of the CCN family of proteins, plays important physiological or pathological roles in a variety of tissues. In the present study, we initially found a highly guanine–cytosine (GC)‐rich region of approximately 200 bp near the 5′‐end of the open reading frame, which was always truncated by amplification of the corresponding cDNA region through the conventional polymerase chain reaction. An RNA in vitro folding assay and selective ribonuclease digestion of the corresponding segment of the ccn1 mRNA confirmed the involvement of a stable secondary structure. Subsequent RNA electromobility‐shift assays demonstrated the specific binding of some cytoplasmic factor(s) in chicken embryo fibroblasts to the RNA segment. Moreover, the corresponding cDNA fragment strongly enhanced the expression of the reporter gene in cis at the 5′‐end, but did not do so at the 3′‐end. According to the results of a ribosomal assembly test, the effect of the mRNA segment can predominantly be ascribed to the enhancement of transport and/or entry of the mRNA into the ribosome. Finally, the minimal GC‐rich mRNA segment that was predicted and demonstrated to form a secondary structure was confirmed to be a functional regulatory element. Thus, we here uncover a novel dual‐functionality of the mRNA segment in the ccn1 open reading frame, which segment acts as a cis‐element that mediates posttranscriptional gene regulation, while retaining the information for the amino acid sequence of the resultant protein. J. Cell. Biochem. 111: 1607–1618, 2010.

Regulation of CCN1 via the 3′-untranslated region

The 3′-untranslated region (UTR) is known to be a critical regulator of post-transcriptional events that determine the gene expression at the RNA level. The gene CCN1 is one of the classical members of the matricellular CCN family and is involved in a number of biological processes during mammalian development. In the present study, the 600-bp 3′-UTR of CCN1 was functionally characterized. Reporter gene analysis revealed that the entire 3′-UTR profoundly repressed gene expression in cis in different types of the cells, to which both the proximal and distal-halves of the 3′-UTR segments contributed almost equally. Deletion analysis of the 3′-UTR indicated a distinct functional element in the proximal half, whereas a putative target for microRNA-181s was predicted in silico in the distal half. Of note, the repressive RNA element in the proximal half was shown to be capable of forming a stable secondary structure. However, unexpectedly, a reporter construct with a tandem repeat of the predicted miR-181 targets failed to respond to miR-181a. In addition, the other major structured element predicted in the distal half was similarly characterized. To our surprise, the second element rather enhanced the reporter gene expression in cis. These results indicate the involvement of multiple regulatory elements in the CCN1 3′-UTR and suggest the complexity of the miRNA action as well as the 3′-UTR-mediated gene regulation.

Extraction of the lateral incisors to treat maxillary protrusion: Quantitative evaluation of the stomatognathic functions

To treat morphological abnormalities, impaction, and severe malposition of the teeth, the lateral incisors are sometimes extracted, followed by orthodontic space closure. This procedure often requires special consideration, not only with regard to esthetics but also for functional issues. However, thus far, few reports that have performed a functional evaluation in such cases. The purpose of this article is to report the successful treatment of an adult patient with a Class II division 1 malocclusion who was treated with extraction of the upper lateral incisors. The female patient, aged 23 years and 6 months, had a chief complaint of maxillary incisal protrusion and crooked teeth. In this patient, the upper lateral incisors were extracted to fulfill the patients strong request, followed by orthodontic treatment using edgewise appliances. A high-pull J-hook headgear on the lower dental arch was used to prevent further labial inclination of the lower incisors. The total active treatment period was 37 months. The resulting occlusion and a satisfactory facial profile were maintained during a 4-year retention period. Additionally, this treatment did not affect the stomatognathic functions as assessed by the following criteria: range of the incisal path or condylar motion during maximal open-close movement, protrusive excursion, lateral excursion, and the chewing test. In conclusion, extraction of the upper lateral incisors can be an effective treatment choice when the upper lateral incisors are dwarfed, are nonvital, or demonstrate severe malposition.

Runx/Cbfb signaling regulates postnatal development of granular convoluted tubule in the mouse submandibular gland

Background: The rodent salivary gland is not fully developed at birth and the cellular definitive differentiation takes place postnatally. However, little is known about its molecular mechanism. Results: Here we provide the loss‐of‐function genetic evidence that Runx signaling affects postnatal development of the submandibular gland (SMG). Core binding factor β (Cbfb) is a cotranscription factor which forms a heterodimer with Runx proteins. Cbfb was specifically expressed in the duct epithelium, specifically in the SMG. Epithelial Cbfb deficiency resulted in decrease in the size of the SMG and in the saliva secretion on postnatal day 35. The Cbfb mutant SMG specifically exhibited involution of the granular convoluted tubules (GCT), with a down‐regulated expression of its marker genes, such as Klk1, Ngf, and Egf. The induction of GCT is under the control of androgens, and the Cbfb mutant SMG demonstrated down‐regulated expression of Crisp3, an androgen‐dependent transcript. Because the circulating testosterone or tissue dihydrotestosterone levels were not affected in the Cbfb mutants, it appears that Runx/Cbfb signaling regulate androgen receptor pathway, but does not affect the circulating testosterone levels or the enzymatic conversion to DHT. Conclusions: Runx signaling is important in the postnatal development of androgen‐dependent GCT in the SMG. Developmental Dynamics 244:488–496, 2015.

Gap-junction-mediated Communication in Human Periodontal Ligament Cells

The human periodontal ligament (hPDL) senses localized changes in the extracellular environment including mechanical stress, and influences bone remodeling in the surrounding tissue. Gap junctions ...