Effects of connective tissue growth factor (CTGF/CCN2) on condylar chondrocyte proliferation, migration, maturation, differentiation and signalling pathway.

CCN2, also known as connective tissue growth factor (CTGF), is a 38 kDa cysteine-rich extracellular matrix protein that regulates a sequence of cellular functions and participates in multiple complex biological processes, such as chondrogenesis and osteogenesis. In the present study, we provided the first evidence describing the physiological role of CCN2 in condylar chondrocyte proliferation, migration, maturation and differentiation. CCN2 was widely expressed throughout the whole layers of condylar cartilage and predominantly distributed in the proliferative zone. Recombinant CCN2 promoted the proliferation, migration, proteoglycan synthesis and differentiation capacity of isolated condylar chondrocytes. The stimulatory effect of CCN2 on chondrocyte proliferation was associated with the activation of phosphatidylinositol 3-kinase/Akt signalling pathway. The blocking of this pathway by its inhibitor LY294002 impaired the proliferative effect of CCN2 on chondrocytes. These results suggested a novel physiological role of CCN2 in the development of condylar cartilage.

The CMT4B disease-causing phosphatases Mtmr2 and Mtmr13 localize to the Schwann cell cytoplasm and endomembrane compartments, where they depend upon each other to achieve wild-type levels of protein expression.

The demyelinating peripheral neuropathy Charcot-Marie-Tooth type 4B (CMT4B) is characterized by axonal degeneration and myelin outfoldings. CMT4B results from mutations in either myotubularin-related protein 2 (MTMR2; CMT4B1) or MTMR13 (CMT4B2), phosphoinositide (PI) 3-phosphatases that dephosphorylate phosphatidylinositol 3-phosphate (PtdIns3P) and PtdIns(3,5)P2, lipids which regulate endo-lysosomal membrane traffic. The catalytically active MTMR2 and catalytically inactive MTMR13 physically associate, although the significance of this association is not well understood. Here we show that Mtmr13 loss leads to axonal degeneration in sciatic nerves of older mice. In addition, CMT4B2-like myelin outfoldings are present in Mtmr13(-/-) nerves at postnatal day 3. Thus, Mtmr13(-/-) mice show both the initial dysmyelination and later degenerative pathology of CMT4B2. Given the key role of PI 3-kinase-Akt signaling in myelination, we investigated the state of the pathway in nerves of CMT4B models. We found that Akt activation is unaltered in Mtmr13(-/-) and Mtmr2(-/-) mice. Mtmr2 and Mtmr13 are found within the Schwann cell cytoplasm, where the proteins are partially localized to punctate compartments, suggesting that Mtmr2-Mtmr13 may dephosphorylate their substrates on specific intracellular compartments. Mtmr2-Mtmr13 substrates play essential roles in endo-lysosomal membrane traffic. However, endosomes and lysosomes of Mtmr13(-/-) and Mtmr2(-/-) Schwann cells are morphologically indistinguishable from those of controls, indicating that loss of these proteins does not cause wholesale dysregulation of the endo-lysosomal system. Notably, Mtmr2 and Mtmr13 depend upon each other to achieve wild-type levels of protein expression. Mtmr2 stabilizes Mtmr13 on membranes, indicating that the Mtmr13 pseudophosphatase is regulated by its catalytically active binding partner.

Fusobacterium nucleatum Facilitates Apoptosis, ROS Generation, and Inflammatory Cytokine Production by Activating AKT/MAPK and NF-κB Signaling Pathways in Human Gingival Fibroblasts.

Fusobacterium nucleatum (F. nucleatum) plays key roles in the initiation and progression of periodontitis. However, the pathogenic effect of F. nucleatum on human oral tissues and cells has not been fully evaluated. In this study, we aimed to analyze the pathogenic effects of F. nucleatum on human gingival fibroblasts (GFs) and clarify the potential mechanisms. RNA-sequencing analysis confirmed that F. nucleatum significantly altered the gene expression of GF as the stimulation time increased. Cell counting and EdU-labeling assays indicated that F. nucleatum inhibited GF proliferation and promoted cell apoptosis in a time- and dose-dependent manner. In addition, cell apoptosis, intracellular reactive oxygen species (ROS) generation, and proinflammatory cytokine production were dramatically elevated after F. nucleatum stimulation. Furthermore, we found that the AKT/MAPK and NF-κB signaling pathways were significantly activated by F. nucleatum infection and that a large number of genes related to cellular proliferation, apoptosis, ROS, and inflammatory cytokine production downstream of AKT/MAPK and NF-κB signaling pathways were significantly altered in F. nucleatum-stimulated GFs. These findings suggest that F. nucleatum inhibits GF proliferation and promotes cell apoptosis, ROS generation, and inflammatory cytokine production partly by activating the AKT/MAPK and NF-κB signaling pathways. Our study opens a new window for understanding the pathogenic effects of periodontal pathogens on the host oral system.

Liposomal delivery of MicroRNA-7-expressing plasmid overcomes epidermal growth factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have been strikingly effective in lung cancers harboring activating EGFR mutations. Unfortunately, the cancer cells eventually acquire resistance to EGFR-TKI. Approximately 50% of the acquired resistance involves a secondary T790M mutation. To overcome the resistance, we focused on EGFR suppression using microRNA-7 (miR-7), targeting multiple sites in the 3'-untranslated region of EGFR mRNA. Two EGFR-TKI-sensitive cell lines (PC-9 and H3255) and two EGFR-TKI-resistant cell lines harboring T790M (RPC-9 and H1975) were used. We constructed miR-7-2 containing miR-7-expressing plasmid. After transfection of the miR-7-expressing plasmid, using cationic liposomes, a quantitative PCR and dual luciferase assay were conducted to examine the efficacy. The antiproliferative effect was evaluated using a cell count assay and xenograft model. Protein expression was examined by Western blotting. The miR-7 expression level of the transfectants was approximately 30-fold higher, and the luciferase activity was ablated by 92%. miR-7 significantly inhibited cell growth not only in PC-9 and H3255 but also in RPC-9 and H1975. Expression of insulin receptor substrate-1 (IRS-1), RAF-1, and EGFR was suppressed in the four cell lines. Injection of the miR-7-expressing plasmid revealed marked tumor regression in a mouse xenograft model using RPC-9 and H1975. EGFR, RAF-1, and IRS-1 were suppressed in the residual tumors. These findings indicate promising therapeutic applications of miR-7-expressing plasmids against EGFR oncogene-addicted lung cancers including T790M resistance by liposomal delivery.

The 15-deoxy-Delta12,14-prostaglandin J2 inhibits LPS-stimulated AKT and NF-kappaB activation and suppresses interleukin-6 in osteoblast-like cells MC3T3E-1.

AIMS: Periodontitis is a chronic inflammatory disease that results in gingival inflammation and periodontal tissue destruction and is accompanied by alveolar bone resorption and eventual tooth loss. We examined the effect of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) on periodontitis by inhibiting the production of interleukin-6 (IL-6). MAIN METHODS: Osteoblast-like cells MC3T3E-1 were pretreated with 15d-PGJ(2) before being incubated with lipopolysaccharide (LPS), the effect of 15d-PGJ(2) on IL-6 production, expression and its regulatory mechanisms were studied by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, electrophoretic mobility shift assay (EMSA), and confocal laser scanning microscopy study. KEY FINDINGS: 15d-PGJ(2) inhibits LPS-stimulated IL-6 production in a concentration-dependent manner in osteoblast-like cells MC3T3E-1, without appreciable cytotoxicity. To further examine the mechanism responsible for the inhibition of IL-6 production by 15d-PGJ(2), we examined the effect of 15d-PGJ(2) on nuclear factor-kappaB (NF-kappaB) activation and the phosphorylation of protein kinase B (Akt). 15d-PGJ(2) treatment clearly reduced the DNA binding activity of NF-kappaB in LPS-stimulated osteoblast-like cells MC3T3E-1, an effect that was mediated by inhibiting the degradation of inhibitor kappaB (IkappaB) and nuclear translocation of NF-kappaB p65 subunit. In addition, 15d-PGJ(2) attenuated the LPS-mediated Akt pathway. These effects of 15d-PGJ(2) were not abrogated by the PPARgamma antagonist, GW9662, indicating that they are PPARgamma-independent actions. SIGNIFICANCE: These results suggest that 15d-PGJ(2) possess a potent suppressive effect on inflammatory responses of osteoblast-like cells MC3T3E-1 via the Akt and NF-kappaB pathways independent of PPARgamma and suggest that this compound may offer some insight into the development of a new therapeutic approach to the prevention and treatment of periodontal diseases.