Arachidonic acid (AA) regulates intracellular calcium concentration in a variety of cell types. In the present study, the effects of serine/threonine phosphatases and myosin light chain kinase (MLCK) on AA-induced Ca²⁺ signaling in mouse parotid acinar cells were investigated.

Treatment of acinar cells with MLCK inhibitors, ML9 and wortmannin, thapsigargin-induced Ca²⁺ entry (capacitative Ca²⁺ entry: CCE) was partially blocked. In contrast, AA-induced Ca²⁺ entry (noncapacitative Ca²⁺ entry: NCCE) was attenuated by wortmannin but increased by ML9. Calyculin A, a protein phosphatase (PP) inhibitor, resulted in an enhancement of AA-induced Ca²⁺ entry. Our previous study suggested that this inhibition of PP resulted in an enhancement of AA-induced Ca²⁺ entry via PKA. In the presence of ML9, AA-induced Ca²⁺ influx enhanced by calyculin A was further increased. However, even when the order of ML9 administration was changed, the enhancement of AA-induced Ca²⁺ entry by calyculin A was not changed. Taking everything into consideration, MLCK partially enhances CCE, but suppresses AA-induced Ca²⁺ entry, i.e. NCCE. MLCK is probably present in the vicinity of PKA and is presumed to have a cooerative effect on the PKA response against CCE and NCCE.

Congenital tooth agenesis often shows a simple Mendelian inheritance, but recent large surveys of the affected families have located mutations in previously reported loci only in half of the cases, suggesting many causative genes to be identified. We conducted whole-exome analysis of the two affected family members using a next-generation sequencer to identify novel causative mutations. Genomic DNA of the two patients who were diagnosed with tooth agenesis at Iwate Medical University Hospital Dental Care Center Orthodontics, as well as unaffected members of the respective families was extracted from the blood and subjected to the whole-exome sequencing. No candidate mutations that agreed with the inheritance pattern were identified at the previously reported loci in either case. Forty-five nonsynonymous mutations at 40 loci were extracted as candidates in a family in which agenesis is thought to occur through dominant inheritance of a causative mutation. In the other family in which agenesis is thought to be caused by either a de novo dominant mutation or an inheritance of recessive mutations, mutations at three loci （ATAD3A, FBRSL1 （assuming de novo dominant mutation）, and ZDHHC11B （assuming recessive mutation）were identified as candidates. To narrow down the candidates may require development of an assay system, further understanding of tooth development as well as accumulation of mutation data at the candidate loci in other congenital tooth agenesis families.

Epithelial-to-mesenchymal transition（ EMT） is closely related to the development of malignancy in human oral squamous cell carcinoma（ hOSCC） cells. “Cadherin switch” refers to expression changes from E-cadherin to N-cadherin that occur during the malignant transformation of a cancer cell accompanying the EMT. The molecular mechanisms underlying the cadherin switch in hOSCC have not yet been fully elucidated. The Hippo pathway is known to transmit extracellular mechanical cues induced by changes in cell density, which in turn affect gene expression. Here, we found that the cadherin switch is induced at a low but not high cell density in the hOSCC cell line HSC-4. We also found that knockdown of YAP/TAZ, which is part of the Hippo pathway, increased E-cadherin expression but decreased N-cadherin expression in HSC-4 cells. The nuclear translocation of EMT-related transcription factor Slug was observed in HSC-4 cells at low cell density. In addition, low cell density-induced Slug nuclear translocation was inhibited by knockdown of YAP. Moreover, disruption of cell-to-cell adhesion with anti E-cadherin antibody promoted nuclear translocation of YAP, resulting in the cadherin switch in HSC-4 cells. These results suggested that YAP/TAZ mediates the low cell density-induced cadherin switch through nuclear translocation of Slug and that intercellular contact mediated by E-cadherin inhibits the cadherin switch through negative modulation of the Hippo pathway. Thus, disruption of E-cadherin mediated cell-to-cell contact-induced YAP/TAZ activation promotes cadherin switch through promotion of EMT-related transcription factor Slug nuclear translocation in hOSCC HSC-4 cells.

Mesenchymal stem cells （MSCs） have been reported to modulate the properties in various types of immunocompetent cells. In this study, MSC-derived cytokine-like peptide, scrapie responsive gene 1 （SCRG1）, was investigated for paracrine activity in macrophages. C-C chemokine receptor type 7 （CCR7） was identified as a gene whose expression increases in mouse macrophagelike Raw264.7 cells upon stimulation with SCRG1 and co-culture with bone marrow-derived MSC, SG2 cells. Raw264.7 cells stimulated with SCRG1 were significantly increased the expression of CCR7. CCR7 is a receptor for CC-chemokine ligand （CCL） 19 and CCL21, which is an essential receptor for recruitment of T cells and dendritic cells to lymph nodes. Raw264.7 was pretreated with SCRG1, and then chemotaxis to CCL19 and CCL21 was investigated using trans-well migration assay. As a result, Raw264.7 cells pretreated with SCRG1 were significantly increased chemotaxis to CCL19; untreated with SCRG1 did not chemotaxis. Therefore, it was indicated that macrophages whose CCR7 expression was enhanced by SCRG1 stimulation specifically acquired chemotaxis to CCL19. It was suggested that SCRG1 secreted from MSCs in inflammatory tissue induces chemotaxis to CCL19 by paracrine activity in macrophages, and it will be involved in the mechanism by which macrophages exit the inflammatory tissue. The finding that MSC-derived SCRG1 promotes chemotaxis in macrophages may be available for cell therapy using MSC.

Osteoarthritis( OA)-related fibrosis is a plausible cause of temporomandibular joint( TMJ) stiffness. We previously demonstrated that receptor tyrosine kinase( RTK) ligands, fibroblast growth factor( FGF)-1, and epidermal growth factor( EGF), suppressed fibrogenic activity in a fibroblast-like synoviocytes( FLS) cell line, FLS1, derived from mouse TMJ. However, the molecular mechanisms underlying the suppression of fibrogenic activity in FLS1 cells by RTK ligands remained to be clarified.

In particular, in inflamed TMJ, it remained unclear how extracellular adenosine 5′-triphosphate( eATP), which is a danger associated molecular pattern( DAMP), affected the RTK-ligand-induced suppression of fibrogenic activity in FLS1 cells. Here, we found that FLS1 cells vigorously expressed the purinergic receptors, P2X₃, P2X₇, P2Y₂, P2Y₄, P2Y₁₂, and P2Y₁₃. We found that ATP significantly decreased the expression of the fibrogenic marker, alpha-smooth muscle actin( α-SMA), in FLS1 cells. In addition, ATP strengthened the RTK-ligand-induced suppression of α-SMA expression. Since ATP typically binds to P2X₃, P2X₇, P2Y₂, P2Y₄, and P2Y₁₃, and to determine which purinergic receptor mediates the suppressive effects of ATP on the fibrogenic marker expression, we examined the effects of P2Y₁₂ and P2Y₁₃ agonist, adenosine 5 ′-diphosphate( ADP), and P2Y₂ and P2Y₄ agonist, uridine 5 ′-triphosphate( UTP), and ligand-induced suppression of α-SMA expression. Importantly, P2X₃ antagonist, RO-3, and P2X₇ antagonists, A-438079 and A-804598, did not reverse the ATP-induced suppressive effects on the α-SMA expression. Taken together, these results strongly suggested that eATP strengthened the RTK-ligandinduced suppression of fibrogenic activity in FLSs possibly through P2Y₂, P2Y₄, or P2Y₁₃.

Our findings partially clarify the molecular mechanisms underlying the development of OA-related fibrosis in TMJ and may aid in identifying therapeutic targets for this condition. In addition, FGF-1 and EGF could be utilized as drugs to prevent OA-related fibrosis around inflammatory TMJ.