New Functions of Classical Compounds against Orofacial Inflammatory Lesions.

Anti-inflammatory agents have been widely used to ameliorate severe inflammatory symptoms of a number of diseases, and such therapeutics are particularly useful for diseases with intolerable pain without significant mortality. A typical example of this is a disease known as stomatitis; although stomatitis itself is not a life-threatening disease, it severely impairs the individual's quality of life, and thus a standard therapeutic strategy for it has already been established. The topical application of a bioactive agent is quite easy, and a strong anti-inflammatory agent can be used without significant adverse effects. In contrast, natural products with relatively mild bioactivity are used for systemic intervention. However, new aspects of classical drugs used in these established therapeutic methods have recently been discovered, which is expanding the utility of these compounds to other oral diseases such as osteoarthritis of temporomandibular joints (TMJ-OA). In this review article, after summarizing the general concept and pathobiology of stomatitis, its established therapeutics are explained. Thereafter, recent advances in the research into related compounds, which is uncovering new biological functions of the agents used therein, are introduced. Indeed, regenerative therapeutics for TMJ-OA may be developed with the classical compounds currently being used.

A Tumor Suppressor Gene Product, Platelet-Derived Growth Factor Receptor-Like Protein Controls Chondrocyte Proliferation and Differentiation.

The platelet-derived growth factor receptor-like (PDGFRL) gene is regarded as a tumor suppressor gene. However, nothing is known about the molecular function of PDGFRL. In this study, we initially clarified its function in chondrocytes. Among all cell lines examined, the PDGFRL mRNA level was the highest in chondrocytic HCS-2/8 cells. Interestingly, the proliferation of chondrocytic HCS-2/8 cells was promoted by PDGFRL overexpression, whereas that of the breast cancer-derived MDA-MB-231 cells was inhibited. Of note, in PDGFRL-overexpressing HCS-2/8 cells, the expression of chondrocyte differentiation marker genes, SOX9, ACAN, COL2A1, COL10A1, and ALP, was decreased. Moreover, we confirmed the expression of PDGFRL mRNA in normal cartilage tissue and chondrocytes. Eventually, the expression of PDGFRL mRNA in condrocytes except in the case of hypertrophic chondrocytes was demonstrated in vivo and in vitro. These findings suggest that PDGFRL plays the different roles, depending upon cell types. Particularly, in chondrocytes, PDGFRL may play a new and important role which is distinct from the function previously reported. J. Cell. Biochem. 118: 4033-4044, 2017. © 2017 Wiley Periodicals, Inc.

Role of LRP1 in transport of CCN2 protein in chondrocytes.

Low-density lipoprotein receptor-related protein 1 (LRP1) is known to be a receptor for signal transmission and endocytosis. We have previously reported that LRP1 regulates WNT-ß-catenin and protein kinase C signaling in chondrocytes, represses the hypertrophy of chondrocytes during endochondral ossification and that LRP1 is colocalized with a ligand, CCN family member 2 (CCN2; also known as connective tissue growth factor, CTGF), which conducts endochondral ossification, in chondrocytes. However, the role of LRP1 in the endocytic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytic trafficking. Small interfering RNA (siRNA)-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding and/or incorporation was decreased in the LRP1 downregulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin, and internalizated CCN2 was colocalized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the crucial involvement of LRP1 during experimental transcytosis. Moreover, under hypoxic conditions that mimic the cartilaginous microenvironment, the level of LRP1 and the amount of transcytosed CCN2 increased, and these increases were neutralized by treatment with the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 in this tissue, which is produced by and transported by LRP1 from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which might be a crucial event that determines the distribution of CCN2 in cartilage.

Role of the low-density lipoprotein receptor-related protein-1 in regulation of chondrocyte differentiation.

The low-density lipoprotein receptor-related protein 1 (LRP1) is known as an endocytic and signal transmission receptor. We formerly reported the gene expression and the localization of LRP1 in cartilage tissue and chondrocytes, but its roles in the differentiation of chondrocytes remained to be investigated. Here, in order to address this issue, we employed RNAi strategy to knockdown lrp1 in chondrocytic cells and obtained findings indicating a critical role therein. As a result of lrp1 knockdown, aggrecan and col2a1 mRNA levels were decreased. However, that of col10a1 or mmp13 mRNA was rather increased. Under this condition, we performed a promoter assay for Axin2, which is known to be induced by activation of the WNT/beta-catenin (betacat) signaling pathway. Thereby, we found that Axin2 promoter activity was enhanced in the lrp1 knockdown cells. Furthermore, when the WNT/beta-catenin pathway was activated in chondrocytic cells by WNT3a or SB216763, which inhibits the phosphorylation of GSK3beta, the mRNA levels of aggrecan and col2a1 were decreased, whereas that of mmp13 was increased. Additionally, the level of phosphorylated protein kinase C (PKC) zeta was also decreased in the lrp1 knockdown cells. When the phosphorylation of PKCzeta was selectively inhibited, aggrecan and col2a1 mRNA levels decreased, whereas the mmp13 mRNA level increased. These data demonstrate that LRP1 exerts remarkable effects to retain the mature phenotype of chondrocytes as a critical mediator of cell signaling. Our findings also indicate that the onset of hypertrophy during endochondral ossification appears to be particularly dependent on the WNT and PKC signaling initiated by LRP1.

Translational repression by the cis-acting element of structure-anchored repression (CAESAR) of human ctgf/ccn2 mRNA.

The cis-acting element of structure-anchored repression (CAESAR) is a post-transcriptional regulatory element of gene expression, which is located in the 3'-untranslated region (UTR) of the human ccn2 gene (ctgf/ccn2). In this report, the repression mechanism of CAESAR, as well as the structural requirement, was investigated. Removal of minor stem-loops from CAESAR resulted in proportional attenuation of the repressive function, whereas removal of the single bulge or modification of primary nucleotide sequence did not affect its functionality. In light of functional mechanism, CAESAR exerted no significant effects on stability or nuclear export of the cis-linked mRNA. However, this element significantly interfered with the association of such mRNA on ribosome and slowed down the translation process thereafter in vitro. A translation repression mechanism by RNA secondary structure to determine the basal ctgf/ccn2 expression level was uncovered herein.

Comparable response of ccn1 with ccn2 genes upon arthritis: An in vitro evaluation with a human chondrocytic cell line stimulated by a set of cytokines.

BACKGROUND: The chondrosarcoma-derived HCS-2/8 has been known to be an excellent model of human articular chondrocytes. By mimicking the arthritic conditions through the treatment of HCS-2/8 cells with cytokines, we estimated the gene expression response of ccn1 and ccn2 during the course of joint inflammation in vitro. RESULTS: In order to mimic the initiation of inflammation, HCS-2/8 cells were treated with tumor necrosis factor (TNF)-alpha. To induce pro-inflammatory or reparative responses, TGF-beta was employed. Effects of an anti-inflammatory glucocorticoid were also evaluated. After stimulation, expression levels of ccn1 and ccn2 were quantitatively analyzed. Surprisingly, not only ccn2, but also ccn1 expression was repressed upon TNF-alpha stimulation, whereas both mRNAs were uniformly induced by transforming growth factor (TGF)-beta and a glucocorticoid. CONCLUSION: These results describing the same response during the course of inflammation suggest similar and co-operative roles of these 2 ccn family members in the course of arthritis.

Novel enzyme-linked immunosorbent assay systems for the quantitative analysis of connective tissue growth factor (CTGF/Hcs24/CCN2): detection of HTLV-I tax-induced CTGF from a human carcinoma cell line.

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24/CCN2) is known as a multifunctional growth factor. It stimulates proliferation, migration, and extracellular matrix production of mesenchymal cells, and is highly expressed in hypertrophic chondrocytes. In this study, we constructed useful ELISA systems for the analysis of CTGF and its modular fragments. For this objective we prepared four different antihuman CTGF monoclonal antibodies. One, specific for the VWC module, was utilized as the detecting antibody, and the other three, recognizing CT, IGFBP, and VWC modules, respectively, were employed as capture antibodies. Then we established three novel quantitative analysis systems for CTGF. The first system recognizing CT and VWC modules was useful to measure full-length CTGF with improved sensitivity. Utilizing this system, we found significant enhancement of CTGF production from a human carcinoma cell line transduced by HTLV-I tax gene, where the finding indicates the possible involvement of Tax in carcinogenesis. The second system, seeing IGFBP and VWC modules, could quantify not only CTGF, but also may be useful to analyze processed N-terminal fragments. The third system, utilizing capture and detection antibodies against the VWC module, was able to quantify the VWC module only, while it did not recognize full-length CTGF. Since CTGF is actually processed into subfragments, and functional assignment of each module is of interest, these systems are expected to contribute to the progress of CTGF investigations.

Transcriptional induction of connective tissue growth factor/hypertrophic chondrocyte-specific 24 gene by dexamethasone in human chondrocytic cells.

Connective tissue growth factor (CTGF/Hcs24) is a critical growth factor for chondrocytic growth and differentiation. In this report, we describe for the first time glucocorticoid-mediated induction of the CTGF/Hcs24 gene in a chondrocytic cell line, HCS-2/8. Steady-state mRNA levels of CTGF/Hcs24 were remarkably increased after treatment with 50 nM dexamethasone, as confirmed by Northern blotting and quantitative real-time polymerase chain reaction (PCR) analysis. Corresponding to the increase in mRNA, production of CTGF/Hcs24 protein was remarkably enhanced, following a time course of up to 6 h. The observed increase in mRNA can be ascribed to transcriptional enhancement, since the stability of CTGF/Hcs24 mRNA was not affected by the same concentration of dexamethasone, which was indicated by the results of an mRNA degradation assay. However, unexpectedly, the prototypic ctgf/hcs24 promoter was not responsible for the dexamethasone stimulation, suggesting the glucocorticoid receptor binding site(s) to be elsewhere in the CTGF/Hcs24 gene. Enhancement of the prototypic promoter activity by dexamethasone was observed in murine fibroblastic cells, demonstrating the complexity of the regulatory mechanism of ctgf/hcs24 gene expression. Of importance, dexamethasone at the same concentration significantly stimulated proteoglycan synthesis in HCS-2/8 cells up to the same levels as exogenously added CTGF/Hcs24. These findings represent a novel effect of glucocorticoid on the production of CTGF/Hcs24 by chondrocytic cells, and indicate that CTGF/Hcs24 may mediate the stimulative effect of dexamethasone on chondrocytic phenotypes. Also, our results shed light on the complex mechanism of CTGF/Hcs24 induction by glucocorticoids.

Interaction of AP-1 and the ctgf gene: a possible driver of chondrocyte hypertrophy in growth cartilage.

The expression of the connective tissue growth factor ( ctgf) gene increases along with the differentiation of growth cartilage cells, and the highest expression is observed in the hypertrophic stage. Similarly, recent reports demonstrated c- fos expression in chondrocytes in the early hypertrophic zone of growth cartilage, and suggested that the c- fos gene may play a crucial role in the regulation of hypertrophic differentiation. A chondrocytic human cell line, HCS-2/8, is known to retain a variety of chondrocytic phenotypes. When such cells were kept overconfluent, they expressed increasing levels of c- fos transcripts along a time course phenotypically similar to that of hypertrophic differentiation. Moreover, by using a competitive electromobility-shift assay, we found that AP-1, a Fos/Jun heterodimer, in HCS-2/8 was capable of binding not only to a typical AP-1-binding DNA fragment but also to the enhancer fragment of the ctgf gene. Based on the findings above, we hypothesize that, prior to hypertrophic differentiation, AP-1-related oncogenes are activated and that their gene products subsequently activate ctgf gene expression, which might eventually induce hypertrophy.

Suppressive effect of overexpressed connective tissue growth factor on tumor cell growth in a human oral squamous cell carcinoma-derived cell line.

Connective tissue growth factor (CTGF) is known to be a multifunctional growth factor that is overexpressed in several types of malignancies. In this study, effects of CTGF gene overexpression on the phenotypes of oral squamous cell carcinoma cells were investigated by using a cell line with undetectable endogenous CTGF expression. Surprisingly, our results indicated that CTGF-overexpressed clones were characterized by attenuated cell growth and less potent tumorigenicity, with coincidental downregulation of prothymosin alpha gene. Although CTGF is known to promote cell proliferation in mesenchymal cells, our present results suggest that CTGF acts as a negative regulator of the cell growth in oral squamous cell carcinoma possibly through its interaction with growth modifiers inside the cell.